Novel bacteriocin

Abstract

Described herein are bacteriocin peptides and peptidomimetics as well as compositions comprising same. Aspects and embodiments described herein may be used in medical treatment and disinfection of surfaces.

Description

[0001] Novel bacteriocin

[0002] Field

[0003] Aspects herein generally pertain to the field of antimicrobial compounds, more particularly to bacteriocin peptides and peptidomimetics as well as compositions comprising same. Also encompassed are uses of bacteriocin peptides, peptidomimetics, and / or compositions comprising bacteriocin peptides and / or peptidomimetics in medical treatment and disinfection of surfaces.

[0004] Background

[0005] Extensive and widespread use of antibiotics to reduce or eliminate or neutralize undesired microbial organisms has led to the emergence of resistant strains. As a result, these resistant microbial organisms are no longer susceptible to the currently available antibiotic-based treatment or disinfection methods. Accordingly, there is a need for improved antimicrobial compounds and disinfection methods compared to the ones currently available in the art. In the case of infections and / or diseases caused by bacteria, such as infections and / or diseases caused by bacteria of the phylum Firmicutes, existing treatments against multiple clinically relevant strains are inadequate, especially against multiple-drug resistant (MDR) and extensively-drug resistant (XDR) strains that have emerged. Accordingly, there is a need for improved treatments against such infections and diseases, as well as for improved disinfection methods for surfaces contaminated with such bacteria.

[0006] Summary

[0007] The present inventors have identified novel bacteriocins that exhibit a surprisingly strong antibacterial effect against a broad spectrum of bacterial strains. Particularly, as elaborated in the experimental part, the present inventors have found that the novel bacteriocins of the present invention demonstrate the following advantages:

[0008] • The bacteriocin peptides and peptidomimetics as described herein demonstrate a widespread antibacterial effect (Examples 1 , 2, 4, 6). This is true for both bacteriocin peptides and peptidomimetics produced synthetically (Example 4) and produced cellularly (Example 6).

[0009] • The bacteriocin peptides and peptidomimetics as described herein demonstrate a potent antibacterial effect (Examples 2, 3, 4). This is true for both bacteriocin peptides and peptidomimetics produced synthetically (Example 4) and produced cellularly (Example 6).

[0010] Accordingly, the aspects and embodiments of this invention solve at least some of the problems and needs as discussed herein.

[0011] In a first aspect, this invention relates to a bacteriocin peptide or peptidomimetic, wherein said peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-10.

[0012] In some embodiments, a bacteriocin peptide or peptidomimetic according to this invention is such that the bacteriocin is a class II bacteriocin, preferably a class Ila bacteriocin or a class lid bacteriocin.

[0013] In some embodiments, a bacteriocin peptide or peptidomimetic according to this invention is such that the peptide or peptidomimetic comprises the amino acid sequence motif X1X2X3X4X5GG (SEQ ID NO: 11 ), preferably wherein said peptide or peptidomimetic comprises the amino acid sequence motif LX2X3X4 EGG (SEQ ID NO: 12).

[0014] In some embodiments, a bacteriocin peptide or peptidomimetic according to this invention is such that the combined percentage of comprised glycine and alanine residues with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic is less than 60%, preferably less than 55%. In some embodiments, a bacteriocin peptide or peptidomimetic according to this invention is such that the combined percentage of comprised lysine and arginine residues with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic is less than 6%.

[0015] Another aspect of the invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding a bacteriocin peptide or peptidomimetic according to this invention, preferably wherein said nucleic acid molecule is a cDNA molecule. In some embodiments, a nucleic acid construct comprising a nucleic acid molecule encoding a bacteriocin peptide or peptidomimetic according to this invention, is such that the nucleic acid construct is recombinant, preferably wherein said nucleic acid construct comprises a promoter sequence operably linked with the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic.

[0016] Another aspect of the invention relates to a vector comprising a nucleic acid construct according to this invention.

[0017] Another aspect of the invention relates to a cell expressing a bacteriocin peptide according to this invention, or comprising a nucleic acid construct according to this invention, or a vector according to this invention, preferably wherein said cell is an engineered cell.

[0018] Another aspect of the invention relates to a composition comprising a bacteriocin peptide or peptidomimetic according to this invention, a nucleic acid construct according to this invention, a vector according to this invention, or a cell according to this invention. In some embodiments, a composition according to this invention is such that the composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and / or pharmaceutically acceptable ingredients.

[0019] In some embodiments, a composition according to this invention is such that the composition further comprises one or more further bacteriocins, preferably one or more Streptococcus bacteriocins, more preferably one or more bacteriocins selected from the group consisting of SEQ ID NOs: 25-30 and SEQ ID NOs: 33-34.

[0020] Another aspect of this invention relates to a bacteriocin peptide or peptidomimetic according to this invention, a nucleic acid construct according to this invention, a vector according to this invention, a cell according to this invention, or a composition according to this invention, for use as a medicament. In some embodiments, a bacteriocin peptide or peptidomimetic for use according to this invention, a nucleic acid construct for use according to this invention, a vector for use according to this invention, a cell for use according to this invention, or a composition for use according to this invention is for use in the treatment, prevention, and / or delaying of an infection and / or disease, preferably wherein said infection and / or disease is caused by a bacterium of the phylum Firmicutes.

[0021] Another aspect of this invention relates to a composition according to this invention, wherein the composition is suitable for disinfecting a surface contaminated with a bacterium of the phylum Firmicutes, optionally further comprising one or more antimicrobial compounds and / or a solvent.

[0022] Description

[0023] The present inventors have surprisingly found that the novel bacteriocins described herein are able to effectively inhibit bacteria, such as bacteria of the phylum Firmicutes. Particularly, and as elaborated in the experimental part, the present inventors have surprisingly found that said bacteriocins are able to inhibit clinically relevant bacteria, such as bacteria of the genus Lactococcus, Staphylococcus, Enterococcus, Listeria, Streptococcus, and others. Additionally, said bacteriocins exhibit strong synergistic antibacterial effects when combined with other antimicrobial compounds, e.g., other bacteriocins. Accordingly, the aspects and embodiments described herein solve at least some of the problems and needs discussed herein.

[0024] Bacteriocin

[0025] Bacteriocins are antimicrobial compounds. A “bacteriocin” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to proteinaceous (peptidic) toxins produced by bacteria. The biological activity of bacteriocins is the inhibition of microbial cells other than the host cell by which the peptide is made. Said biological activity may also be referred to as antimicrobial activity. A bacteriocin may inhibit at least one cell and / or strain other than the host cell and / or strain in which the peptide is made, including cells and / or strains clonally related to the host cell and other microbial cells. Detailed descriptions of bacteriocins, including methods and compositions for using bacteriocins to control the growth of microbial cells can be found, for example, in U.S. Patent No. 9,333,227, which is hereby incorporated by reference in its entirety.

[0026] Bacteriocins are typically produced by both Gram-positive and Gram-negative bacteria. Examples of Gram-positive bacteriocin-producing bacteria are bacteria of the genus Streptococcus e.g., Streptococcus salivarius. Bacteriocin production may be strain-specific, e.g. it may be produced predominantly by a specific strain such as a Streptococcus salivarius strain.

[0027] Bacteriocins produced by Gram-positive bacteria are typically classified into two main groups (described in Alvarez- Sieiro et al. (2016) Appl Microbiol Biotechnol 100:2939-2951 , incorporated herein by reference in its entirety): class I comprises peptides that undergo a range of post-translational modifications, while class II comprises peptides that are unmodified or contain only minor modifications (e.g., disulfide bridges). Class II bacteriocins are typically subdivided in four subclasses: classes Ila, lib, lie, and lid that correspond to pediocin-like, two-peptide, leaderless, and non-pediocin-like single peptide bacteriocins, respectively. Except for class lie bacteriocins, class II bacteriocins are characterized by the presence of a leader sequence comprising a conserved GG motif (X1X2X3X4X5GG, wherein Xi is M or L or V, and X2-X5 can be any amino acid (SEQ ID NO: 11 ), described in Yount et al. (2020) Front Immunol 11 :1873, incorporated herein by reference in its entirety. This sequence plays a role in the secretion of the bacteriocin outside the producing cell and maintains the peptide in an inactive form to avoid intracellular toxicity. The leader sequence is typically cleaved from the bacteriocin and is absent in the mature peptide.

[0028] Thus, a bacteriocin peptide or peptidomimetic described herein can correspond to a precursor sequence or to a mature sequence. A precursor sequence comprises a leader sequence that is absent from the mature sequence. Accordingly, a mature bacteriocin sequence refers to a bacteriocin with no leader sequence.

[0029] Novel bacteriocins may be identified using modern bioinformatics tools according to standard methods available in the art, such as the BAGEL4 software (van Heel et al. Nucleic Acids Res 46(W1 ): W278-W281 , incorporated by reference herein in its entirety), available at http: / / bagel4.molgenrug.nl / , which enable researchers to mine bacterial (meta-)genomic DNA for bacteriocin-encoding genes.

[0030] Alternatively, novel bacteriocins may be discovered using conserved motifs to mine the bacterial genomes. In the genus Streptococcus, bacteriocin production is controlled by a cell-to-cell communication strategy known as quorum sensing. The regulation of class I and class II bacteriocin production is different and depends on the species. Class I bacteriocins as such act as stimulating pheromones and typically induce their own production through a phosphorylation cascade involving a dedicated two-component system. In contrast, the production of class II bacteriocins is regulated by unmodified peptide pheromones that do not exhibit toxic effects. Two regulation systems (i.e., ComRS and BlpRH) for the production of class II bacteriocins were described in streptococci (e.g., in Garcia-Curiel et al. (2021 ) World J Microbiol Biotechnol 37:15, incorporated herein by reference in its entirety).

[0031] In S. salivarius, the ComRS system synchronizes competence and predation through upregulation of comX and bacteriocin genes, respectively. The precursor ComS is produced, secreted, and matured in the pheromone XIP (comX-inducing peptide). At a threshold concentration, XIP is internalized and interacts with the cytoplasmic sensor ComR. Then, the complex ComR-XIP activates transcription of target genes through its binding to a well-conserved DNA sequence called ComR-box, which is a palindromic sequence oriented at the 3’ end by a T-rich stretch (T- track) (described in e.g., Fontaine et al. (2015) Infect Genet Evol 33:343-360, Fontaine et al (2013) Mol Microbiol 87: 1113-1132, and Mignolet et al. (2018) Cell Rep 22: 1627-1638, all of which incorporated herein by reference in their entireties). This predation-competence coupling allows the bacteria to ensure the presence of free DNA in the environment before entering the competence state. In other streptococcal species, the production of bacteriocins is generally under the control of the BIpRH system, which is absent or inactive in S. salivarius (Mignolet et al. (supra). The pheromone precursor BIpC is produced, processed at a double-glycine motif, and exported by the dedicated transporter BIpAB. When the mature pheromone BIpC* reaches a threshold concentration, it activates the two-component system BIpRH. When activated, the trans-membrane histidine kinase BipH phosphorylates the cytoplasmic response regulator BIpR, which in turn upregulates genes of the blp locus, including bacteriocin and self-immunity genes.

[0032] The present inventors exploited the presence of the conserved ComR-box in order to identify novel bacteriocins produced by Streptococcus salivarius using bioinformatics tools. Specifically, 100 genomes of S. salivarius strains were scouted for the presence of open reading frames (ORFs) comprising a ComR-box in their upstream genomic region. For the analysis, the search was restricted to ORFs downstream of the T-track at a maximal distance of about 4 kb. The search was further narrowed by selecting of sequences encoding precursor peptides ranging from about 40 to about 100 amino acids in length. Sequence that did not include the leader sequence motif represented by SEQ ID NO: 7 were discarded. Lastly, sequences devoid of a ribosome binding site upstream of the start codon were removed. Specific focus was placed on peptide sequences with specific lengths for leader sequences (length of from 14 to 28 amino acids) and mature peptide sequences (from 25 to 70 amino acids).

[0033] Following the abovementioned approach, the present inventors identified five novel bacteriocins that exhibit a surprisingly strong antibacterial effect against a broad spectrum of bacterial strains, particularly against bacteria of the phylum Firmicutes (alternatively referred to herein as Bacillota). Their antibacterial effect is experimentally demonstrated in the Examples section later herein.

[0034] Within the context of the disclosure, a bacteriocin may be a peptide or a peptidomimetic, preferably is a peptide. A "peptide” as described herein also encompasses polypeptides, as well as variants of peptides and polypeptides as described later herein. A definition of "peptidomimetic” is provided later herein. A "peptidomimetic” as described herein also encompasses variants of peptidomimetics, as described later herein. A bacteriocin peptide or peptidomimetic as described herein may be comprised in a composition, a description of which is provided later herein.

[0035] Accordingly, in an aspect, there is provided a bacteriocin peptide or peptidomimetic, preferably peptide, wherein said peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-10. In some embodiments, the bacteriocin is a Streptococcus salivarius bacteriocin.

[0036] SEQ ID NO: 1 and SEQ ID NO: 6 correspond to a bacteriocin denoted herein as Ssal, with SEQ ID NO: 1 representing the precursor sequence and SEQ ID NO: 6 representing the mature sequence. SEQ ID NO: 2 and SEQ ID NO: 7 correspond to a bacteriocin denoted herein as SsaL, with SEQ ID NO: 2 representing the precursor sequence and SEQ ID NO: 7 representing the mature sequence. SEQ ID NO: 3 and SEQ ID NO: 8 correspond to a bacteriocin denoted herein as SsaM, with SEQ ID NO: 3 representing the precursor sequence and SEQ ID NO: 6 representing the mature sequence. SEQ ID NO: 4 and SEQ ID NO: 9 correspond to a bacteriocin denoted herein as SsaJ, with SEQ ID NO: 4 representing the precursor sequence and SEQ ID NO: 9 representing the mature sequence. SEQ ID NO: 5 and SEQ ID NO: 10 correspond to a bacteriocin denoted herein as SsaK, with SEQ ID NO: 5 representing the precursor sequence and SEQ ID NO: 10 representing the mature sequence.

[0037] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 1 or SEQ ID NO: 6.

[0038] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 2 or SEQ ID NO: 7.

[0039] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 3 or SEQ ID NO: 8. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 4 or SEQ ID NO: 9.

[0040] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 5 or SEQ ID NO: 10.

[0041] In preferred embodiments, the bacteriocin is a class II bacteriocin, preferably a class lid bacteriocin.

[0042] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises the amino acid sequence motif X1X2X3X4X5GG (SEQ ID NO: 11 ). In SEQ ID NO: 11 , X1-X5 are denoted as follows:

[0043] • Xi is M or L or V

[0044] • X2 can be any amino acid

[0045] • X3 can be any amino acid

[0046] • X4 can be any amino acid

[0047] • X5 can be any amino acid

[0048] Preferably, "any amino acid” refers to proteinogenic amino acids as known to the skilled person.

[0049] In preferred embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises the amino acid sequence motif LX2X3X4 EGG (SEQ ID NO: 12). In SEQ ID NO: 12, Xz-X^ are denoted as follows:

[0050] • X2' is A (alanine or Ala) or S (serine or Ser)

[0051] • X3 is S (serine or Ser) or N (asparagine or Asn) or I (isoleucine or lie)

[0052] • X41is V (valine or Vai) or I (isoleucine or lie)

[0053] The skilled person understands that the amino acid sequence motif represented by SEQ ID NO: 11 or SEQ ID NO: 12 is comprised by a leader sequence of a bacteriocin peptide or peptidomimetic. Thus, in such embodiments, the bacteriocin peptide or peptidomimetic corresponds to a precursor peptide or peptidomimetic.

[0054] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises a pediocin-like motif. Preferably, in such embodiments, the bacteriocin peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising sequence identity or similarity with SEQ ID NO: 5 or SEQ ID NO: 10 as described earlier herein. In some embodiments, the pediocin-like motif is represented by a sequence comprising at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity or similarity with SEQ ID NO: 18.

[0055] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises a leader sequence having a length of from 14 to 28 amino acids, preferably from 16 to 26 amino acids, more preferably from 18 to 24 amino acids, even more preferably from 20 to 24 amino acids, most preferably of 23 amino acids.

[0056] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises a leader sequence represented by an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 13- 17.

[0057] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is a precursor peptide or peptidomimetic comprising a sequence having a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 59, 74, 75, 78, or 80 amino acids.

[0058] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is a mature peptide or peptidomimetic comprising a sequence having a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 36, 51 , 52, 55, or 57 amino acids.

[0059] As described above, within the context of a specific bacteriocin peptide or peptidomimetic, preferably peptide, "precursor” and "mature” corresponds to different forms of the same bacteriocin peptide or peptidomimetic (with the "mature” peptide or peptidomimetic lacking the leader sequence). Thus, it is understood that a bacteriocin precursor peptide or peptidomimetic represented by an amino acid sequence having a certain length corresponds to a mature peptide or peptidomimetic represented by an amino acid sequence having a shorter length by virtue of it lacking the leader sequence, with the remainder of the sequence being the same as the precursor sequence.

[0060] As a non-limiting example, SEQ ID NO: 1 (corresponding to precursor Ssal), having a length of 78 amino acids, comprises a leader sequence having a length of 23 amino acids (SEQ ID NO: 13). Its mature form (represented by SEQ ID NO: 6) has a length of 55 amino acids and lacks the leader sequence but is otherwise the same as SEQ ID NO: 1.

[0061] As another non-limiting example, SEQ ID NO: 2 (corresponding to precursor SsaL), having a length of 59 amino acids, comprises a leader sequence having a length of 23 amino acids (SEQ ID NO: 14). Its mature form (represented by SEQ ID NO: 7) has a length of 36 amino acids and lacks the leader sequence but is otherwise the same as SEQ ID NO: 2.

[0062] As another non-limiting example, SEQ ID NO: 3 (corresponding to precursor SsaM), having a length of 80 amino acids, comprises a leader sequence having a length of 23 amino acids (SEQ ID NO: 15). Its mature form (represented by SEQ ID NO: 8) has a length of 57 amino acids and lacks the leader sequence but is otherwise the same as SEQ ID NO: 3.

[0063] As another non-limiting example, SEQ ID NO: 4 (corresponding to precursor SsaJ), having a length of 75 amino acids, comprises a leader sequence having a length of 23 amino acids (SEQ ID NO: 16). Its mature form (represented by SEQ ID NO: 9) has a length of 52 amino acids and lacks the leader sequence but is otherwise the same as SEQ ID NO: 4.

[0064] As another non-limiting example, SEQ ID NO: 5 (corresponding to precursor SsaK), having a length of 74 amino acids, comprises a leader sequence having a length of 23 amino acids (SEQ ID NO: 17). Its mature form (represented by SEQ ID NO: 10) has a length of 51 amino acids and lacks the leader sequence but is otherwise the same as SEQ ID NO: 5.

[0065] Accordingly, in some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-5 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 59, 74, 75, 78, or 80 amino acids.

[0066] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 1 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 78 amino acids.

[0067] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 2 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 59 amino acids.

[0068] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 3 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 80 amino acids.

[0069] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 4 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 75 amino acids.

[0070] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 5 and has a length of from 43 to 98 amino acids, preferably of from 46 to 91 amino acids, more preferably from 53 to 84 amino acids, even more preferably from 59 to 80 amino acids, most preferably of 74 amino acids.

[0071] Accordingly, in some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 6-10 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 36, 51 , 52, 55, or 57 amino acids. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 6 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 55 amino acids.

[0072] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 7 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 36 amino acids.

[0073] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 8 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 57 amino acids.

[0074] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 9 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 52 amino acids.

[0075] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with SEQ ID NO: 10 and has a length of from 25 to 70 amino acids, preferably from 30 to 65 amino acids, more preferably from 35 to 60 amino acids, even more preferably from 36 to 57 amino acids, most preferably of 51 amino acids.

[0076] A bacteriocin peptide or peptidomimetic, preferably peptide, described herein may comprise a specific percentage of a particular amino acid, or of a combination of amino acids, with respect to the overall sum of amino acid residues it comprises. In other words, an exemplary bacteriocin peptide or peptidomimetic that has a length of 100 amino acids, out of which 50 amino acid residues are amino acid X, is to be understood as comprising 50% of amino acid X. Similar considerations apply to peptides or peptidomimetics of other lengths.

[0077] In some embodiments, the combined percentage of comprised glycine and alanine residues (Gly-Ala) with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic, preferably peptide, is less than 70%, preferably less than 60%, more preferably less than 55%.

[0078] In some embodiments, the combined percentage of comprised lysine and arginine residues (Lys-Arg) with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic, preferably peptide, is less than 7%, preferably less than 5%, more preferably less than 3%.

[0079] In such embodiments, the combined percentage of comprised Gly-Ala and Lys-Arg residues is preferably calculated with respect to the mature peptide or peptidomimetic sequence, e.g., with respect to a sequence comprising identity or similarity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 as described earlier herein.

[0080] A bacteriocin peptide or peptidomimetic, preferably peptide, described herein may have a specific molecular weight (MW), measured in kDa. In some embodiments, the bacteriocin peptide or peptidomimetic has a MW of from 3.0 kDa to 6.0 kDa, preferably from 3.2 kDa to 5.8 kDa, more preferably from 3.5 kDa to 5.5 kDa or from about 3.5 kDa to 5.5 kDa. Preferably, the MW value corresponds to the mature form of the bacteriocin peptide or peptidomimetic.

[0081] A bacteriocin peptide or peptidomimetic, preferably peptide, described herein may have a specific grand average of hydropathy index (GRAVY) value. The GRAVY value represents the average hydropathy value of a peptide or peptidomimetic. A positive GRAVY value indicates that a peptide or peptidomimetic is hydrophobic, and a negative GRAVY value indicates that a peptide or peptidomimetic is hydrophilic. The GRAVY value of a peptide or peptidomimetic can be calculated using standard methods in the art, for example as described in Kyte and Doolittle (1982) J. Mol. Biol. 157, 105-132 (incorporated herein by reference in its entirety). GRAVY values may be calculated using bioinformatic calculator tools available in the art, such as the https: / / www.gravy-calculator.de / calculator. Preferably, the GRAVY value corresponds to the mature form of the bacteriocin peptide or peptidomimetic.

[0082] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is hydrophobic. In some embodiments, the bacteriocin peptide or peptidomimetic has a grand average of hydropathy (GRAVY) value of from 0.2 to 1.5, preferably from 0.5 to 1.3, more preferably from 0.6 to 1.2 or from about 0.6 to about 1.2. In preferred embodiments wherein the bacteriocin peptide or peptidomimetic is hydrophobic, the peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-4 and SEQ ID NOs: 6-9 as described earlier herein. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, is hydrophilic. In some embodiments, the bacteriocin peptide or peptidomimetic has a grand average of hydropathy (GRAVY) value of from -1.5 to -0.1 , preferably from -1.3 to -0.5, more preferably from -1.0 to -0.3 or from about -1.0 to about -0.3, most preferably has a value of -0.3 or about -0.3. In preferred embodiments wherein the bacteriocin peptide or peptidomimetic is hydrophilic, the peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising sequence identity or similarity with SEQ ID NO: 5 or SEQ ID NO: 10 as described earlier herein.

[0083] A bacteriocin peptide or peptidomimetic, preferably peptide, described herein may have a specific isoelectric point (pl) value, preferably measured under IUPAC established standard temperature and pressure (STP). "Isoelectric point” is used herein with its standard meaning in the art, and can be calculated using standard methods such as from the mean of the pKas of the peptide or peptidomimetic. pl values may be calculated using bioinformatic calculator tools available in the art, such as the https: / / www.ebi.ac.uk / jdispatcher / seqstats / emboss_pepstats / calculator.

[0084] In some embodiments, the bacteriocin peptide or peptidomimetic has a pl value of from 5 to 9, preferably from 5.5 to 8.5, more preferably from 5.8 to 8.2 or from about 5.8 to about 8.2.

[0085] Preferably, the pl value corresponds to the mature form of the bacteriocin peptide or peptidomimetic.

[0086] "Inhibition”, "neutralization”, and variations of the terms as used herein have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure. They include any form of inhibition or arrest of microbial growth and / or division (bacteriostatic effect), as well as any cytotoxic or bactericidal effect (killing). Inhibition and / or neutralization may be full or partial, meaning a whole microbial cell population, such as a target bacterial population, or only a part thereof may be growth-inhibited or killed. Partial inhibition may mean that at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 99% of an initial microbial population, such as a target microbial population, is not growth-inhibited or killed. Optionally, inhibition or partial inhibition are determined after a defined timeframe following contacting and incubation of the microbial cell population with the bacteriocin peptide or peptidomimetic, for example after at least 30 min, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least at least 48 hours, at least 60 hours, at least 72 hours, or longer.

[0087] The ability of an antimicrobial compound such as a bacteriocin peptide or peptidomimetic to inhibit and / or neutralize a microbial cell (i.e. its biological activity) such as a bacterium of the phylum Firmicutes (for example bacteria of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus) may be determined using standard methods in the art, for example utilizing standard commercial in vitro tests such as ASTM E2149-20 or ASTM E1054-08 (ASTM, PA, USA), and the like, as well as methods such as spot-on-lawn assays. In a typical spot-on-lawn assay, a feeding layer of agar may be cast on a plate, on top of which a second layer agar containing a population of a target cell may be poured. Such a second layer can e.g., obtained by mixing a grown culture of the target cell with agar before solidification. Subsequently, a specific amount of bacteriocin peptide or peptidomimetic (or of a population of cells producing a bacteriocin peptide) can be spotted and the inhibition zone can be determined and measured by eye or other suitable methods. As long as an inhibition zone is formed upon application of the bacteriocin peptide or peptidomimetic (or of a population of cells producing a bacteriocin peptide), the bacteriocin peptide or peptidomimetic can be considered to inhibit and / or neutralize the target cell. An example of a spot-on-lawn assay is provided in the experimental section herein. Spot-on-lawn assays are also compatible with serial dilutions of the bacteriocin (e.g. assaying progressively lower concentrations in order to accurately pinpoint the relevant lowest concentrations wherein antimicrobial activity is present) or of the populations of cells producing it, allowing for the determination of minimal inhibitory concentration (MIC) and / or minimal bactericidal concentration (MBC) values. Alternatively, MIC and MBC may be determined utilizing liquid cultures of target cells to which the bacteriocin peptide or peptidomimetic is added. 'Minimal inhibitory concentration” as used herein refers to the lowest concentration of a bacteriocin peptide or peptidomimetic which inhibits the growth of the target cell. Alternatively, or in addition, the minimum bactericidal concentration (MBC), also known as minimum lethal concentration (MLC) may be used to assess biological activity, which refers to the lowest concentration of a bacteriocin peptide or peptidomimetic which is able to kill 99.9% of the cells present in a liquid culture of a target cell.

[0088] The MIC and / or MBC values for different target microbial cells may also be determined according to methods commonly used in the art, such as discussed in standard handbooks such as Schwalbe R. et al., Antimicrobial susceptibility testing protocols, Boca Raton: CRC Press (2007) (incorporated herein by reference in its entirety), and / or protocols (CLSI Clinical and Laboratory Standards Institute, 2018; NCCLS, 1999; both of which incorporated herein by reference in their entireties) and / or commercially available kits such as ETEST® (Biomerieux, NC, USA).

[0089] Within the context of the invention, a bacteriocin peptide may be produced by a cell or be synthetic. Production of a peptide by a cell can be endogenous or exogenous. Endogenous production refers to production of a peptide by a cell that is natively able to produce it (i.e. a cell that comprises the required genetic information for its production). In some embodiments, endogenous production refers to production by Streptococcus salivarius, for example production of a bacteriocin peptide represented by a sequence selected from the group consisting of SEQ ID NO: 1-10 by Streptococcus salivarius. An exemplary S. salivarius strain is strain HSISS4.

[0090] Exogenous production typically refers to production of the peptide by a different organism and / or cell (i.e. a host), by which said peptide is not natively produced, the capability of which having been introduced via means of recombinant DNA technology. Within the context of the invention, the term exogenous production also encompasses cases wherein the native production of a peptide, preferably Streptococcus salivarius, is increased via means of recombinant DNA technology using standard molecular toolbox techniques as compared to the corresponding endogenous production. It also encompasses cases wherein a variant peptide is produced, as described later herein. Said increase may be achieved by modification of any of the steps of bacteriocin production, including transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Said increase may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% compared to the corresponding endogenous production. A definition of peptide production, alternatively referred to herein as peptide expression, is provided in the section titled "general information”.

[0091] Exogenous production can be achieved by introduction of a nucleotide sequence comprising a bacteriocin encoding sequence (ORF) to a host organism and / or cell, e.g. E. coll or Streptococcus salivarius (for example S. salivarius strain HSISS4) . Recombinant DNA techniques, suitable host organisms and / or cells for exogenous protein production, and culturing methods are well-known in the art and are described in standard handbooks such as Ausubel et al., Current Protocols in Molecular Biology, 3rd edition, John Wiley & Sons Inc (2003) and in Sambrook and Green, Molecular Cloning. A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (2012); both of which are incorporated herein by reference in their entireties. To achieve exogenous protein production, a bacteriocin ORF, operably linked to (i. e. in a functional relationship with) a suitable transcription initiation sequence such as a promoter, will typically be introduced to a suitable host cell according to standard techniques. A promoter may be constitutive i.e. allowing constant expression of a bacteriocin peptide, or inducible i.e. only allowing expression of a bacteriocin peptide under specific culture conditions or upon induction with chemical compounds. Optionally, other regulatory sequences such as transcription terminators, enhancers, kozak sequences, polyA sequences, leader sequences, and the like may be operably linked to the bacteriocin ORF. The choice of a particular regulatory sequence will depend on the choice of the host cell and is well within the capabilities of the skilled person. Exemplary regulatory sequences are described later herein.

[0092] The bacteriocin ORF may be stably integrated in a suitable host cell’s genome or may be introduced in a selfreplicating vector. The bacteriocin ORF may be codon-optimized for expression in a particular host cell, e.g. Escherichia coli or Streptococcus salivarius, using commonly used computer algorithms. A definition of codon optimization is given in the section titled "general information”. Suitable host cells may be selected from mammalian, insect, plant, or microbial cells, preferably are selected from microbial cells. Examples of suitable microbial cells include eukaryotes such as yeasts, filamentous fungi, and algae, and prokaryotes such as bacteria and archaea, of which bacteria is preferred. Bacterial host cells include both Gram-negative and Gram-positive bacteria and can be selected from suitable groups known in the art such as Bacillus species (for example Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis), Paenibacillus species, Streptomyces species, Staphylococcus species, Streptococcus species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species, Pseudomonas species, Lactobacillus species, Lactococcus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus, Pseudomonas stutzeri, Clostridium thermocellus, Escherichia coli, including strains thereof, of which Streptococcus species are preferred with Streptococcus salivarius being more preferred.

[0093] Algae host cells may be selected from suitable groups known in the art such as Botryococcus braunii, Chlorella species, Dunaliella tertiolecta, Gracilaria species, Pleurochrysis carterae, and Sargassum species. Yeast host cells may be selected from suitable groups known in the art such as Saccharomyces species (for example, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (for example, Candida utilis, Candida krusei), Schizosaccharomyces species (for example Schizosaccharomyces pombe, Schizosaccharomyces japonicus), Pichia or Hansenula species (for example, Pichia pastoris or Hansenula polymorpha) species, and Brettanomyces species (for example, Brettanomyces claussenii). Filamentous fungal host cells may be selected from suitable groups known in the art such as Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Peniciffium, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria. Species include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Penicillium chrysogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0094] A bacteriocin peptide may be isolated and / or purified from its producing cell. Suitable downstream processing methods for isolation and / or purification of products from cell cultures are well-known in the art and are described in standard handbooks such as Wesselingh, J. A and Krijgsman, J., 1stedition, Downstream Processing in Biotechnology, Delft Academic Press (2013), incorporated herein by reference in its entirety. Examples of suitable isolation and / or purification techniques are chromatographic methods such as high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, immunoprecipitation via the use of tags, and the like. Accordingly, in some embodiments the bacteriocin peptide is an isolated and / or purified peptide.

[0095] A cell to which bacteriocin production has been introduced may be called an engineered cell. Accordingly, in an aspect, there is provided a cell, preferably a bacterium, expressing a bacteriocin peptide as described herein, preferably wherein said cell is an engineered cell.

[0096] Within the context of the invention, a bacteriocin peptide may be produced in vitro, using isolated and / or purified cellular components (cell-free extracts) comprising the necessary transcription and translation machinery. In vitro protein production typically comprises transcription and translation of isolated circular or linear DNA, or only translation when isolated mRNA is used as a template, said DNA or mRNA comprising a bacteriocin encoding sequence optionally operably linked to regulatory sequences as discussed elsewhere herein. The corresponding cellular components may be isolated / purified and the reaction conditions can be chosen according to standard methods, such as for example described in Gregorio et al., Methods Protoc 2(1 ):24 (2019), incorporated herein by reference in its entirety. Alternatively, commercial in vitro protein synthesis kits such as PURExpress® (New England Biolabs, MA, USA) may be used. Depending the on the cell used for isolation / purification of the cellular components, e.g. Escherichia coll or Streptococcus salivarius, and / or the commercial kit used, the bacteriocin ORF may be codon optimized for expression in that particular cell and / or commercial kit. As a non-limiting example, a nucleotide sequence encoding a bacteriocin peptide represented by SEQ ID NO: 6, 7, 8, 9, 10 (or a sequence comprising identity or similarity to SEQ ID NO: 6, 7, 8, 9, or 10 as described earlier herein) may be used in conjunction with PURExpress® according to the manufacturer’s protocol to produce a bacteriocin. The nucleotide sequence may be comprised in an nucleic acid construct or a vector, described later herein. Accordingly, in some embodiments the bacteriocin peptide is an in vitro produced peptide.

[0097] Within the context of the invention, a bacteriocin peptide may be synthetic. The term “synthetic peptide” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a peptide which is generated by means of chemical peptide synthesis. A synthetic bacteriocin peptide according to the invention may be prepared or synthesized using conventional methods that are well-known in the art. For instance, peptides can be synthesized by commonly used solid-phase synthesis methods such as those that involve a tert-butyloxycarbonyl-protecting group (t-BOC) or fluorenylmethyloxycarbonyl-protecting group (FMOC) for protection of alpha-amino groups. In such methods, amino acids are added sequentially to a growing amino acid chain. Such methods are, for instance, described in Merrifield, J. Am. Chem. Soc. 85(14):2149-2154 (1963), and Atherton & Sheppard, Solid Phase Peptide Synthesis: A practical Approach, IRL Press, Oxford, UK (1999), both of which are incorporated herein by reference in their entireties. Accordingly, in some embodiments, the bacteriocin peptide is a synthetic peptide. Within the context of the invention, a bacteriocin may be a peptidomimetic. As used herein, a “peptidomimetic” (alternatively referred to as "mimetic”) is understood to encompass all compounds whose essential elements mimic a natural peptide and which retain the ability to interact with the biological target and exert the natural peptide’s biological activity. The biological activity of a bacteriocin peptidomimetic may be the same, decreased, or increased as compared to a bacteriocin peptide. Decreased biological activity of a bacteriocin peptidomimetic may mean that the peptidomimetic exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the biological activity of the corresponding bacteriocin peptide. Increased biological activity of a bacteriocin peptidomimetic may mean that the peptidomimetic exhibits an increase of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in biological activity as compared to the corresponding bacteriocin peptide. A definition of "biological activity” and measurement methods thereof are provided elsewhere herein.

[0098] In some embodiments, the peptidomimetic comprises, consists essentially of, or consists of, preferably comprises, a non-naturally occurring amino acid sequence. In some embodiments, the peptidomimetic does not occur in nature and is considered to be man-made. Peptidomimetics typically arise either from modification of an existing peptide, or by designing similar systems that mimic peptides, such as peptoids and p-peptides. Structures and synthesis of peptidomimetics are for instance described in William D. Lubell (ed.), Peptidomimetics I and II, Topics in Heterocyclic Chemistry (Book 48), Springer 1sted., XVI, 310 p (2017); Trabocchi A. Chapter 6 - Principles and applications of small molecule peptidomimetics, Small Molecule Drug Discovery Methods, Molecules and Applications, pp 163-195, Elsevier (2020); Vagner et al., Curr Opin Chem Biol. 12(3): 292-296 (2008), all of which incorporated herein by reference in their entireties. A bacteriocin peptidomimetic may be a structural mimetic of a bacteriocin peptide described herein. Structural mimetics, also known as type I mimetics, have analogous structural features to the bacteriocin peptide they mimic. A bacteriocin peptidomimetic may be a functional mimetic of a bacteriocin peptide described herein. Functional mimetics, also known as type II mimetics, retain the ability to interact with the biological target and exert the natural peptide’s biological activity without apparent structural analogy to the peptide. A bacteriocin peptidomimetic may be a functional-structural mimetic of a bacteriocin peptide described herein. Functional-structural mimetics, also known as type III mimetics, generally comprise a scaffold having a structure different from the bacteriocin peptide that they mimic, in which all the functional groups needed for the biological activity are mounted in a well-defined spatial orientation.

[0099] In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which a modification has been introduced, for example to the backbone and / or the side chains.

[0100] In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which a non-natural amino acid has been introduced. Examples of non-natural amino acids are provided later herein. In some embodiments, a natural amino acid is substituted by a non-natural amino acid or a D-amino acid, which may, for example, be corresponding as described later herein.

[0101] In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which the peptide backbone has been replaced completely, for example by a heterocycle, a sugar, or other scaffolds. Examples of suitable scaffolds are known to the skilled person and discussed, for example, in Pelay-Gimeno et al., Angew Chem Int Ed Engl; 54(31 ): 8896-8927 (2015), incorporated herein by reference in its entirety. In some embodiments, a bacteriocin peptidomimetic corresponds to a peptoid. In some embodiments, a bacteriocin peptidomimetic corresponds to a p-peptide.

[0102] Modification of an existing peptide may be the result of natural processes, such as post-translational processing, or chemical modification techniques. In some embodiments, a peptidomimetic refers to a compound containing non- peptidic structural elements. Typical but non-limiting examples of non-peptidic structural elements are modifications of one or more existing amino acids, conformational restraints, cyclization of the polypeptide, isosteric replacement or other modifications. In some embodiments, a peptidomimetic may contain one or more or all substitutions of an amino acid by the corresponding D-amino acid. As used herein, “corresponding D-amino acid” denotes the D-amino acid counterpart of an L-amino acid. A peptidomimetic may also optionally contain non-natural amino acids. As used herein, “non-natural amino acid” has its customary and ordinary meaning as understood by one of skill in the art in view of this invention. It refers to non-genetically encoded amino acids, irrespective of whether they appear in nature or not. Non-natural amino acids that can be present in a peptidomimetic as described herein include: p- amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl-L-tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine; 4-tert-butyl hydrogen 2-azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid; 2,4-diamino butyric acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2- aminopimelic acid; p-aminophenylalanine; 2,3-diaminobutyric acid; 2,3-diamino propionic acid; 2,2’-diaminopimelic acid; p-amino-L-phenylalanine; p-azido-L- phenylalanine; D-allyl glycine; p-benzoyl-L- phenylalanine; 3-benzothienyl alanine p-bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosine; epsilon-amino hexanoic acid; N- ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxy lysine; alio-hydroxy lysine; 3-(3-methyl-4-nitrobenzyl)-L- histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3- methyl-phenylalanine; N- methylglycine; N-methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; 17e sulfoxide; 2- napthylalanine; L-3-(2-naphthyl)alanine; isoserine; 3-phenylserine; norvaline; norleucine; 5,5,5-trifluoro-DL-leucine; ornithine; 3-chloro-tyrosine; N5-carbamoylornithine; penicillamine; phenylglycine; piperidinic acid; pyridylalanine; 1 ,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid; beta-2-thienylalanine; y-carboxy-DL-glutamic acid; 4-fluoro-DL- glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy-tryptophan; 5-fluoro-tryptophan; 3- fluoro-valine. In some embodiments, a natural amino acid of a bacteriocin peptide or peptidomimetic according to the invention is substituted by a corresponding non-natural amino acid. As used herein, a “corresponding non- natural amino acid” refers to a non-natural amino acid that is a derivative of the reference natural amino acid. For instance, a natural amino acid can be substituted by the corresponding p-amino acid, which has its amino group bonded to the p-carbon rather than the a-carbon. In some embodiments, a peptide or peptidomimetic of the invention may further be provided with a targeting moiety. It is known that peptidomimetics are able to circumvent some of the disadvantages associated with natural peptides: e.g. stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved.

[0103] Within the context of the invention, a bacteriocin peptide or peptidomimetic, preferably peptide, may further be modified by natural processes, such as post-translational processing, or by chemical modification techniques. Such modifications may be inserted in the peptide at any location, including in the backbone, amino acid side-chains and at the N- or C-terminus. Multiple types of modifications may occur in a single peptide, or a peptide may comprise several modifications of a single type. Types of modifications and modification techniques are well-known in the art and described in standard handbooks such as Peptide Modifications to Increase Metabolic Stability and Activity, 1stedition, Ed. Predrag Cudic, Humana Press (2013), incorporated herein by reference in its entirety. Accordingly, in some embodiments, the bacteriocin peptide or peptidomimetic comprises at least one amino acid modification selected from the group consisting of alkylation, acetylation, amidation, acylation, phosphorylation, methylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitination, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation, succinylation, and sulfation, preferably selected from methylation or glycosylation. Within the context of the invention, a bacteriocin peptide or peptidomimetic, preferably peptide, may comprise one or more modifications in its sequence, resulting in bacteriocin peptide or peptidomimetic variants (alternatively referred to herein as mutants). Said sequence modifications may include amino acid substitutions, deletions and / or insertions.

[0104] Variant peptides or peptidomimetics can, for example, be synthetically made or made by cellular (or in vitro) production as described elsewhere herein, after modifying the nucleotide sequence encoding for said peptides using mutagenesis techniques known to the skilled person, such as, random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, CRISPR / Cas-mediated mutagenesis and the like, so that the resulting nucleotide sequence encodes a peptide that differs by at least one amino acid from the non-modified peptide or peptidomimetic, i.e. wherein at least one amino acid is substituted by a different amino acid and / or at least one amino acid is deleted and / or at least one amino acid is inserted. Amino acid substitutions may be conservative. A definition of a "conservative” substitution is provided in the section titled "general information”. Variant peptides and / or peptidomimetics according to the invention may retain decreased, but still detectable, or increased biological activity as compared to the corresponding non-modified peptide or peptidomimetic. Biological activity (i.e. antimicrobial activity) against a target microbial cell such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis, may be assessed as described elsewhere herein. Decreased biological activity of a variant may mean that the variant exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the biological activity of the corresponding non-modified bacteriocin peptide or peptidomimetic. Increased biological activity of a variant may mean that the variant exhibits an increase of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in biological activity as compared to the corresponding non-modified bacteriocin peptide or peptidomimetic.

[0105] Within the context of the invention, the physicochemical properties of a bacteriocin peptide or peptidomimetic, preferably peptide, may be the same or differ as compared to a corresponding naturally-occurring (wild-type) peptide. The skilled person is aware of such properties, non-limiting examples of which include susceptibility to enzymatic degradation (e.g. by proteinases, peptidases, aminopeptidases, carboxypeptidases, Rnases, phospholipases, amylases, and the like), susceptibility to degradation by organic solvents (e.g. to acetone, chloroform, acetonitrile, ethanol, 2-propanol, butanol, methanol, and the like), susceptibility to degradation by surfactants (e.g. anionic, non-ionic, cationic, amphoteric, silicon-based, fluorinated, polymeric, and the like), susceptibility to reducing agents (e.g. DTT, p-mercaptoethanol, and the like), susceptibility to heat degradation, pH optimum, and the like. In some embodiments, a bacteriocin peptide or peptidomimetic exhibits at least one improved physicochemical property as compared to a corresponding naturally-occurring (wild-type) peptide. Physicochemical properties of peptides or peptidomimetics may be assessed by commonly used methods in the art, such as discussed in standard handbooks like Hansen, P. R., Antimicrobial Peptides: Methods and Protocols, 1stEdition, Humana Press, US, (2017) and Remington: The Science and Practice of Pharmacy, 23rded., Ed. Adejare A., Academic Press, US (2021 ), both of which are incorporated herein by reference in their entireties. A non-limiting examples of a variant bacteriocin peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-10 as described earlier herein.

[0106] Within the context of the invention, a bacteriocin peptide or peptidomimetic, preferably peptide, may be in salt form. Suitable salts forms of peptides and peptidomimetics and their preparation are known in the art and discussed in standard handbooks, such as Remington: The Science and Practice of Pharmacy (supra) and Koutsopoulos, Peptide Applications in Biomedicine, Biotechnology and Bioengineering’ 1 st Edition, Woodhead Publishing, UK (2017), incorporated herein by reference in their entireties. Preparation of peptide salts generally involves mixing of the peptide or peptidomimetic with an acid or base, for instance, by reacting the free acid or free base forms of the peptide or peptidomimetic with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed by vacuum or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin. In some embodiments, the solvent is DMF or DMSO, preferably is DMSO. Table 1. Exemplary sequences

[0107] Nucleic acids

[0108] In a further aspect, there is provided a nucleic acid molecule encoding a bacteriocin peptide or peptidomimetic, preferably peptide, as described herein. Preferably, the nucleic molecule is a cDNA molecule. A "cDNA” molecule refers to complementary DNA as commonly used in the art. In some embodiments, the nucleic acid molecule is codon-optimized for expression in a particular host cell, e.g., E. coli or S. salivarius. A definition of codonoptimization is provided later herein. In a further aspect, there is provided a nucleic acid construct comprising a nucleic acid molecule, preferably a cDNA molecule, described herein. A nucleic acid construct may be alternatively referred to herein as an "expression construct”. In preferred embodiments, the nucleic acid construct is recombinant, i.e., it comprises one or more nucleotide sequences that are not naturally associated with the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic, and preferably comprises a promoter sequence operably linked with the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic.

[0109] In some embodiments, the promoter is inducible. In some embodiments, the promoter is constitutive. Non-limiting examples of suitable promoters include the XIP-inducible blpK promoter (SEQ ID NO: 35) and the T7 promoter (SEQ ID NO: 37). In some embodiments, the blpK promoter comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO: 35, or a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.

[0110] A nucleic acid construct may further comprise one or more other regulatory sequences operably linked with the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic, e.g., a transcription terminator, enhancer, kozak sequence, polyA sequence, leader sequence, and the like. In some embodiments, the nucleic acid construct comprises a blpK leader sequence (SEQ ID NO: 36). The use of a blpK leader sequence is particularly advantageous for cellular expression in host cells comprising the ComA bacteriocin exporter (described in Mignolet et al. (2018) Cell Rep 22:1627-1638, incorporated herein by reference in its entirety), such as Streptococcus salivarius.

[0111] In some embodiments, the nucleic acid construct comprises the upstream and / or downstream region of tRNASeroperably linked to the nucleic acid encoding the bacteriocin peptide or peptidomimetic.

[0112] A nucleic acid construct may further comprise a nucleic acid molecule encoding a selection marker. Non limiting examples of selection markers include antibiotic resistance markers, e.g., genes conferring resistance to ampicillin, kanamycin, puromycin, neomycin, hygromycin B, spectinomycin, blasticidin, and the like, of which the spec gene (conferring spectinomycin resistance) is preferred. The nucleic acid molecule encoding the selection marker may be operably linked to the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic.

[0113] In some embodiments, the nucleic acid construct as described herein has the nucleotide sequence of SEQ ID NO: 38, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , or a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity therewith.

[0114] A nucleic acid molecule or nucleic acid construct described herein may be comprised in a vector. Accordingly, in a further aspect, there is provided a vector comprising a nucleic acid molecule or nucleic acid construct as described herein. A vector may alternatively be called an expression vector. A vector may be an integration vector (i.e., a vector that can integrate in the host cell’s genome) or a vector that can independently replicate within the host cell. The skilled person understands that a suitable nucleotide sequence allowing for independent replication of the vector will vary depending on the host, and many "backbone” vectors optimized for expression in particular hosts (to which the nucleic acid molecule or construct encoding the bacteriocin may be inserted) are commercially available. Preferably, the vector is suitable for replication in a bacterium, more preferably is a plasmid. A non-limiting example of a suitable vector is the pUC57 plasmid and derivatives thereof (e.g., as provided by GenScript, Piscataway, NJ, USA). In a further aspect, there is provided a cell, preferably a bacterium, comprising a nucleic acid molecule, a nucleic acid construct, or a vector encoding a bacteriocin peptide as described herein, preferably wherein said cell is an engineered cell. Suitable cells are described earlier herein, of which E. coll and S. salivarius are preferred.

[0115] Compositions

[0116] A bacteriocin peptide or peptidomimetic as described herein exhibits a number of activities that can be advantageously used in a wide range of applications, including therapeutic applications and applications in disinfection of surfaces (including surfaces of chemically fragile medical devices), biotechnology, biofermentation processes, and food preservation.

[0117] Accordingly, in a further aspect, there is provided a composition comprising a bacteriocin peptide or peptidomimetic, preferably peptide, a nucleic acid molecule, a nucleic acid construct, a vector, or a cell as described earlier herein. Optionally, said composition further comprises an acceptable ingredient, such as a carrier, diluent, and / or excipient as discussed later herein. The skilled person is aware that each of the acceptable ingredients will be suitable for the intended use or application, for example pharmaceutical application or application in the disinfection of surfaces. In embodiments wherein the compositions comprise bacteriocin peptide or peptidomimetic salts as described earlier herein, the skilled person is aware that said salts will be suitable for the intended use or application, for example pharmaceutical application or application in the disinfection of surfaces. Non-limiting examples of pharmaceutically and cosmetically acceptable acids or bases suitable for the preparation of a bacteriocin or peptidomimetic salt as described earlier herein include organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid, and thiocyanic acid, which form ammonium salts with free amino groups of polypeptides, and bases that form carboxylate salts with free carboxylic groups of polypeptides, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di-and trialkylamines, and arylamines.

[0118] In some embodiments, the composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and / or pharmaceutically acceptable ingredients.

[0119] In some embodiments, the composition is suitable for disinfecting a surface contaminated with a bacterium of the phylum Firmicutes, optionally further comprising one or more antimicrobial compounds and / or a solvent. The skilled person understands that "suitable for disinfecting” in the context of the invention refers to the composition being capable of inhibiting and / or neutralizing a bacterium of the phylum Firmicutes, such as, but not limited to bacteria of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, when said composition is applied on a surface contaminated with said bacterium. A definition of a "contaminated surface” is provided later herein.

[0120] In some embodiments, the concentration value of the bacteriocin peptide or peptidomimetic in the compositions or pharmaceutical compositions described herein is at least 0.01 pg / ml, at least 0.1 pg / ml, at least 1 pg / ml, at least 2 pg / ml, at least 3 pg / ml, at least 4 pg / ml, at least 5 pg / ml, at least 6 pg / ml, at least 7 pg / ml, at least 8 pg / ml, at least 9 pg / ml, at least 10 pg / ml, at least 11 pg / ml, at least 12 pg / ml, at least 13 pg / ml, at least 14 pg / ml, at least 15 pg / ml, at least 16 pg / ml, at least 17 pg / ml, at least 18 pg / ml, at least 19 pg / ml, at least 20 pg / ml, at least 21 pg / ml, at least 22 pg / ml, at least 23 pg / ml, at least 24 pg / ml, at least 25 pg / ml, at least 26 pg / ml, at least 27 pg / ml, at least 28 pg / ml, at least 29 pg / ml, at least 30 pg / ml, at least 31 pg / ml, at least 32 pg / ml, at least 33 pg / ml, at least 34 pg / ml, at least 35 pg / ml, at least 36 pg / ml, at least 37 pg / ml, at least 38 pg / ml, at least 39 pg / ml, at least 40 pg / ml, at least 41 pg / ml, at least 42 pg / ml, at least 43 pg / ml, at least 44 pg / ml, at least 45 pg / ml, at least 46 pg / ml, at least 47 pg / ml, at least 48 pg / ml, at least 49 pg / ml, at least 50 pg / ml, at least 51 pg / ml, at least 52 pg / ml, at least 53 pg / ml, at least 54 pg / ml, at least 55 pg / ml, at least 56 pg / ml, at least 57 pg / ml, at least 58 pg / ml, at least 59 pg / ml, at least 60 pg / ml, at least 61 pg / ml, at least 62 pg / ml, at least 63 pg / ml, at least 64 pg / ml, at least 65 pg / ml, at least 66 pg / ml, at least 67 pg / ml, at least 68 pg / ml, at least 69 pg / ml, at least 70 pg / ml, at least 71 pg / ml, at least 72 pg / ml, at least 73 pg / ml, at least 74 pg / ml, at least 75 pg / ml, at least 76 pg / ml, at least 77 pg / ml, at least 78 pg / ml, at least 79 pg / ml, at least 80 pg / ml, at least 81 pg / ml, at least 82 pg / ml, at least 83 pg / ml, at least 84 pg / ml, at least 85 pg / ml, at least 86 pg / ml, at least 87 pg / ml, at least 88 pg / ml, at least 89 pg / ml, at least 90 pg / ml, at least 91 pg / ml, at least 92 pg / ml, at least 93 pg / ml, at least 94 pg / ml, at least 95 pg / ml, at least 96 pg / ml, at least 97 pg / ml, at least 98 pg / ml, at least 99 pg / ml, at least 100 pg / ml, at least 110 pg / ml, at least 120 pg / ml, at least 130 pg / ml, at least 140 pg / ml, at least 150 pg / ml, at least 160 pg / ml, at least 170 pg / ml, at least 180 pg / ml, at least 190 pg / ml, at least

[0121] 200 pg / ml, at least 250 pg / ml, at least 300 pg / ml, at least 400 pg / ml, at least 500 pg / ml, at least 550 pg / ml, at least

[0122] 600 pg / ml, at least 650 pg / ml, at least 700 pg / ml, at least 750 pg / ml, at least 800 pg / ml, at least 850 pg / ml, at least

[0123] 900 pg / ml, at least 950 pg / ml, or at least 1000 pg / ml.

[0124] In some embodiments, the concentration value of the bacteriocin peptide or peptidomimetic in the compositions or pharmaceutical compositions described herein is at least 0.015 pM, at least 0.03 pM, at least 0.06 pM, at least 0.125 pM, at least 0.25 pM, at least 0.25 pM, at least 0.05 pM, or at least 1 pM.

[0125] Compositions and pharmaceutical compositions described herein may be in any form as commonly used in the art. The skilled person is aware that the form of the respective composition will be suitable for the intended use or application, for example pharmaceutical, cosmetic, or application in the disinfection of surfaces. Non-limiting examples of suitable forms include tablets, capsules, pills, lyophilized, liquids, creams, ointments, gels, pastes, powders, emulsions, lotions, suspensions, sticks, aerosols (i.e. sprays), and the like.

[0126] As used herein, a "solvent” includes any solvent or mixture of solvents in which a bacteriocin peptide or peptidomimetic as described herein can be dissolved at a suitable concentration. Generally, the number and types of ionic charges in the peptide determine its solubility in aqueous solutions. Generally, the more charged residues the peptide possesses, the more soluble it is in aqueous solutions. In addition, peptides generally have more charges at pH 6-8 than at pH 2-6. It is for this reason that peptides are generally better dissolved at near neutral pH. Among the many exceptions to the rule are peptide sequences that are very hydrophobic and those that tend to aggregate. While the hydrophobicity of the sequence is the primary cause of aggregation, peptides can also aggregate or "gel" through extensive hydrogen bonding network. Non-limiting examples of solvents that can be used in the context of the invention are water, ethanol, ammoniumhydroxide, dimethylsulfoxide (DMSO), acetic acid, acetonitrile and dimethylformamide (DMF). Dissolution can be enhanced by sonication. The skilled person is aware of which solvent will be applicable in each case depending on the hydrophobicity of the bacteriocin peptide or peptidomimetic, discussed earlier herein. In some embodiments, the solvent is DMF or DMSO, preferably is DMSO.

[0127] A "pharmaceutical composition” is a composition which is suitable for use in therapy. As used herein, “pharmaceutically acceptable ingredients” include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients. Accordingly, the one or more pharmaceutically acceptable ingredients may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients may be found in standard handbooks such as in Remington: The Science and Practice of Pharmacy (supra). Compositions and pharmaceutical compositions as described herein may optionally further comprise one or more antimicrobial compounds. Said antimicrobial compound may be selected from any antimicrobial compound such as, but not limited to, antifungal agents, antiviral agents, essential oils, other bacteriocins, and / or antibiotics. Non- limiting examples of suitable groups of bacteriocins are further given in Table 2.

[0128] Table 2. Non-limiting examples of suitable groups of bacteriocins

[0129] In some embodiments, a composition or pharmaceutical composition comprising a bacteriocin peptide or peptidomimetic, preferably peptide, described herein further comprises one or more (distinct) further bacteriocins (other than the bacteriocin peptide or peptidomimetic described herein). In some embodiments, the one or more further bacteriocins are peptide or peptidomimetics, preferably peptides. In some embodiments, the composition or pharmaceutical composition comprises a bacteriocin peptide or peptidomimetic, preferably peptide, described herein and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight (distinct) further bacteriocins (other than the bacteriocin peptide or peptidomimetic described herein). Such compositions may be alternatively referred to as comprising a "cocktail” of bacteriocins, and may be particularly advantageous by exhibiting a synergistic improvement in the exerted antimicrobial effect compared to compositions or pharmaceutical compositions comprising individual bacteriocin peptides or peptidomimetics. Preferably, the one or more further bacteriocins are one or more Streptococcus bacteriocins, for example one or more S. thermophilus or S. salivarius bacteriocins, of which BIpK, SIvV, SIvW, SIvX, SIvY, SIvZ, BIpE, and BipF are preferred. In some embodiments, the one or more further bacteriocins are peptides or peptidomimetics represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34, preferably from the group consisting of SEQ ID NOs: 25-30 and SEQ ID NOs: 33-34, more preferably selected from the group consisting of SEQ ID NOs: 25- 30.

[0130] A preferred composition (cocktail) comprises a bacteriocin peptide or peptidomimetic, preferably peptide, described herein, BIpK (preferably represented by SEQ ID NO: 25), SIvV (preferably represented by SEQ ID NO: 26), SIvW (preferably represented by SEQ ID NO: 27), SIvX (preferably represented by SEQ ID NO: 28), SIvY (preferably represented by SEQ ID NO: 29), and SIvZ (preferably represented by SEQ ID NO: 30).

[0131] Compositions and pharmaceutical compositions as described herein may optionally comprise additional compounds. Said compounds may help in delivery of the compositions. Suitable compounds in this context are: compounds capable of forming complexes, nanoparticles, micelles and / or liposomes that deliver each constituent as described herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these compounds are known in the art. Suitable compounds comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives; synthetic amphiphiles (SAINT-18); lipofectin, DOTAP. The skilled person will know which type of formulation is the most appropriate for a composition as described herein.

[0132] Therapeutic methods / uses

[0133] The bacteriocin peptides and peptidomimetics described herein, as well as cells expressing them, are able to exhibit an antimicrobial, preferably antibacterial effect, i.e., they are preferably able to inhibit and / or neutralize bacterial growth. Preferably, the antibacterial effect is against a bacterium of the phylum Firmicutes, more preferably against a clinically relevant (pathogenic) bacterium of the phylum Firmicutes. Thus, the bacteriocin peptides, peptidomimetics, nucleic acid molecules, nucleic acid constructs, vectors, cells, and compositions, all as described herein are suitable for use in therapy.

[0134] Accordingly, in a further aspect, there is provided a bacteriocin peptide or peptidomimetic, preferably peptide, a nucleic acid molecule, a nucleic acid construct, a vector, a cell, or a composition, preferably a pharmaceutical composition, as described herein, for use as a medicament (i.e., for use in medicine), preferably for use as a medicament against a bacterium of the phylum Firmicutes. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, nucleic acid molecule, nucleic acid construct, vector, cell, or composition, are for use in the treatment, prevention, and / or delaying of an infection and / or disease, preferably wherein said infection and / or disease is caused by a bacterium of the phylum Firmicutes.

[0135] In a further aspect, there is provided a use of a bacteriocin peptide or peptidomimetic, preferably peptide, a nucleic acid molecule, a nucleic acid construct, a vector, a cell, or a composition, preferably a pharmaceutical composition, as described herein, for the manufacture of a medicament, preferably for the manufacture of a medicament against a bacterium of the phylum Firmicutes. In some embodiments, the medicament is for treating, delaying, and / or preventing an infection and / or disease, preferably wherein said infection and / or disease is caused by a bacterium of the phylum Firmicutes.

[0136] In a further aspect, there is provided a method of treating, delaying, and / or preventing an infection and / or disease, particularly wherein said infection and / or disease is caused by a bacterium of the phylum Firmicutes, comprising administering a bacteriocin peptide or peptidomimetic, preferably peptide, a nucleic acid molecule, a nucleic acid construct, a vector, a cell, or a composition, preferably a pharmaceutical composition, as described herein, to a subject in need thereof.

[0137] In therapeutic methods and uses described herein, the bacteriocin peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-10. In some embodiments, the bacteriocin is a Streptococcus salivarius bacteriocin.

[0138] The terms "infection” and "disease” as used herein have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure. An infection refers to the invasion and growth of a microbial cell in a subject’s (host’s) body and / ortissues, which may be accompanied by spreading throughout the body and / orto other tissues. A disease refers to an abnormal condition that negatively affects the structure or function of all or part of an organism due to the unwanted growth of a microbial cell in a subject’s body and / or tissues. A disease may be an infectious disease. An infection and / or disease may result in injury to the affected body and / or tissue. The microbial cell may come into contact with the subject and / or tissue via the environment, such as physical contact with a contaminated surface, or may already be present in the subject and / or tissue as part of the microbiome of said tissue and / or subject. An infection and / or disease may be caused by a pathogenic or potentially pathogenic microbial cell, such as a bacterium of the phylum Firmicutes. "Pathogenicity” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to the infection- and / or disease-causing capacity of a microbial cell. A potentially pathogenic microbial cell will cause an infection and / or disease under certain conditions, for example in cases wherein the immune system of the host is compromised or a bodily wound allows for entry of said cell in the body.

[0139] The bacteriocin peptides and peptidomimetics described herein, as well as cells expressing them, are able to exhibit an antimicrobial effect against a wide range of pathogenic or potentially pathogenic bacteria. In preferred embodiments, the infection and / or disease is caused by a bacterium of the phylum Firmicutes.

[0140] In the context of this disclosure, the phylum Firmicutes (Bacillota) is to be understood with its common meaning in the art. The Firmicutes are a division of bacteria, most of which have Gram-positive cell wall structure. Certain members lack cell walls altogether and do not respond to Gram staining, but still lack the second membrane found in other Gram-negative forms. Other members have a porous pseudo-outer-membrane resulting in them staining as negative in Gram-staining. Typically, bacteria of the phylum Firmicutes are characterized as "low G+C content” bacteria; however, exceptions do exist (e.g., Geobacillus thermocatenulatus having a G+C content as high as 55%). Groups of Firmicutes are typically classified based on characteristics like their form (e.g., cocci or rods), type of cell envelope, endospore formation and aerotolerance (aerobic / anaerobic). Currently, there are seven recognized Classes of Firmicutes: the Erysipelotrichia, the Negativicutes, the Limnochordia, the Tissierellia, the Thermolithobacteria, the Clostridia and the Bacilli. Among bacteria of the phylum Firmicutes, the genera Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus are preferred.

[0141] Accordingly, in some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, nucleic acid molecule, nucleic acid construct, vector, cell, or composition, preferably pharmaceutical composition, is for use as a medicament against a bacterium selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, preferably against a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis, more preferably against a bacterium selected from the group consisting of S. pneumoniae, S. aureus, S. epidermidis, and L. monocytogenes.

[0142] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, nucleic acid molecule, nucleic acid construct, vector, cell, or composition, preferably pharmaceutical composition, is for use as a medicament against an antibiotic resistant bacterium. In the context of this disclosure, the term “antibiotic resistant” is to be understood with its common meaning in the art. Antibiotic resistance pertains to the capacity of bacteria and other microorganisms to withstand the bactericidal or bacteriostatic effects of antibiotics. This phenomenon emerges from genetic alterations, including point mutations, gene amplifications, or horizontal gene transfer events, leading to the acquisition or expression of resistance determinants. Mechanisms of resistance commonly involve enzymatic degradation or modification of antibiotics, alteration of antibiotic targets, or enhanced efflux of antibiotics from bacterial cells. Antibiotic resistance of bacteria can be assessed or determined using the standard methods as described in the art, for example as described in Balouiri et al. (2016) J Pharm Anal 6(2):71-79, incorporated herein by reference in its entirety. Typically, antibiotic resistance is measured through the Minimum Inhibitory Concentration (MIC), which represents the lowest concentration of an antibiotic required to inhibit visible growth of a bacterial strain within a standardized growth medium over a defined incubation period. The determination of MIC typically entails the utilization of standardized methodologies, conforming to established guidelines such as those delineated by the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In practice, this involves the preparation of serial dilutions of the antibiotic in a suitable growth medium, such as broth or agar, within microtiter plates or other standardized assay formats.

[0143] In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, nucleic acid molecule, nucleic acid construct, vector, cell, or composition, preferably pharmaceutical composition, is for use as a medicament against an antibiotic resistant bacterium, wherein the antibiotic resistant bacterium is resistant against amoxicillin and / or penicillin. In the context of this disclosure, a bacterium which is resistant against amoxicillin and / or penicillin displays a MIC value higher than 1 pg / mL. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, nucleic acid molecule, nucleic acid construct, vector, cell, or composition, preferably pharmaceutical composition, is for use as a medicament against S. pneumoniae bacterium, wherein said bacterium is antibiotic resistant, preferably amoxicillin and / or penicillin resistant. In some embodiment, the antibiotic resistant S. pneumoniae bacteria is bacteria from a S. pneumoniae 19A strain.

[0144] In preferred embodiments the infection and / or disease is caused by a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus. Among the bacteria of the genus Lactococcus, the species Lactococcus lactis is preferred. Among bacteria of the genus Staphylococcus, the species Staphylococcus aureus and Staphylococcus epidermidis are preferred. Among bacteria of the genus Enterococcus, the species Enterococcus faecium is preferred. Among bacteria of the genus Listeria, the species Listeria monocytogenes is preferred. Among bacteria of the genus Streptococcus, the species Streptococcus salivarius, Streptococcus pneumoniae, Streptococcus vestibularis, , Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis are preferred.

[0145] In further preferred embodiments, the infection and / or disease is caused by a bacterium selected from the group consisting of S. pneumoniae, S. aureus, S. epidermidis, and L. monocytogenes.

[0146] Infections and / or diseases caused by bacteria of the genus Staphylococcus, preferably by Staphylococcus aureus or Staphylococcus epidermidis, include, but are not limited to, skin infections, lung or respiratory tract infections (e.g., pneumonia), CNS infections (e.g., meningitis), bone infections (e.g., osteomyelitis), heart infections (e.g., endocarditis), toxic shock syndrome, joint infections (e.g., infectious arthritis), blood infections (e.g., bacteremia), sepsis, and the like. Infections and / or diseases caused by bacteria of the genus Enterococcus, preferably by Enterococcus faecium, include, but are not limited to, urinary tract infections, blood infections (e.g., bacteremia), heart infections (e.g., endocarditis), intra-abdominal or pelvic infections (e.g., peritonitis), CNS infections (e.g., meningitis), and the like. Infections and / or diseases caused by bacteria of the genus Listeria, preferably Listeria monocytogenes, include listeriosis, meningitis, and the like. Infections and / or diseases caused by bacteria of the genus Streptococcus, preferably by Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis Streptococcus pyogenes, Streptococcus oralis, or Streptococcus mitis, include, but are not limited to, lung or respiratory tract infections (e.g., pneumonia), sinusitis, otitis media, blood infections (e.g., bacteremia), joint infections (e.g., septic arthritis), bone infections (e.g., osteomyelitis), heart infections (e.g., endocarditis), meningitis, group A streptococcal disease (GAS), throat infections (e.g., pharyngitis), and the like.

[0147] Accordingly, in some embodiments of the therapeutic uses and method described herein, the infection and / or disease is selected from the group consisting of a skin infection, a lung or respiratory tract infection (e.g., pneumonia), a CNS infection (e.g., meningitis), a bone infection (e.g., osteomyelitis), a heart infection (e.g., endocarditis), toxic shock syndrome, a joint infection (e.g., infectious arthritis or septic arthritis), a blood infection (e.g., bacteremia), sepsis, a urinary tract infection, an intra-abdominal or pelvic infection (e.g., peritonitis), a group A streptococcal disease (GAS), a throat infection (e.g., pharyngitis), listeriosis, sinusitis, and otitis media.

[0148] A bacteriocin peptide or peptidomimetic described herein may exhibit an antibacterial effect against specific bacterial strains, for example strains that comprise a mutation decreasing or abolishing the activity of a certain protein. The skilled person is aware of how to pinpoint the relevant mutations in this regard using standard techniques in the art, for example by screening libraries of the relevant bacterial species comprising knockdowns and / or deletions of all individual operons / genes. As an example, CRISPRi libraries (libraries in which each individual operon / gene has been knocked down in a species) can be used together with spot-on-lawn assays or liquid cultures to which a bacteriocin peptide or peptidomimetic (or composition) as described herein are added, to identify mutations associated with bacteriocin susceptibility in the species. An example of the use of a CRISPRi library to identity particularly susceptible strains is provided in the experimental section herein. A "knockdown” as used herein refers to any mutation that results in decrease of the activity of a particular enzyme or protein compared to its wild type counterpart, and includes amino acid substitutions, insertions, deletions (incl. whole ORF deletions), mutations in the regulatory sequences of the gene / operon, epigenetic changes, and the like.

[0149] In some embodiments, the infection and / or disease is caused by a Streptococcus pneumoniae strain that comprises a knockdown or deletion in the ftsH gene (ATP-dependent zinc metalloprotease FtsH; Uniprot Ref: A0A0H2ZMA2). In some embodiments, the Streptococcus pneumoniae strain comprises an amino acid modification in the ftsH gene compared to wild type ftsH. In some embodiments, the amino acid modification is an amino acid substitution.

[0150] In some embodiments, the infection and / or disease is caused by a Streptococcus pneumoniae strain that comprises a knockdown or deletion in the lafA gene (glycosyltransferase; Uniprot Ref: A0A0H2ZMQ4). In some embodiments, the Streptococcus pneumoniae strain comprises an amino acid modification in the lafA gene compared to wild type lafA. In some embodiments, the amino acid modification is an amino acid substitution.

[0151] In therapeutic methods and uses described herein, the subject treated may be a vertebrate, preferably a mammal such as a cat, a mouse, a rat, a dog, or a human. In preferred embodiments, the subject treated is a human.

[0152] In therapeutic methods and uses described herein, administration of the bacteriocin peptides, peptidomimetics and compositions, preferably pharmaceutical compositions, may be performed to an individual, a cell, tissue, and / or an organ of an individual affected and / or at risk of developing an infection and / or disease as discussed herein. Administration may be performed directly or indirectly in vivo, ex vivo or in vitro, using suitable means known in the art. When administering a bacteriocin peptide, peptidomimetic, or composition as described herein, it is preferred that it is dissolved in a solution that is compatible with the delivery method. Such solutions are generally known in the art, see for example Remington: The Science and Practice of Pharmacy (supra). Improvements in means for providing an individual or a cell, tissue, and / or organ of said individual with the bacteriocin peptide, peptidomimetic and / or composition are anticipated, considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the invention. The skilled person understands that the type and frequency of administration will vary depending on the infection and / or disease. Administration may be one-time (single) or may involve multiple administrations over two, three, four, five, six, seven, eight, nine, ten days or more. Administration may be once daily or multiple times daily. Administration modes are generally known in the art. An administration mode may be topical, transdermal, intradermal, parenteral, intravenous, intramuscular, intraperitoneal, via inhalation, intraparenchymal, subcutaneous, intraarticular, intraadipose tissue, oral, intrahepatic, intrapulmonary, intrasplanchnic, intra-ear, intrathoracic, intracardial, or intratracheal administration. In some embodiments, administration is oral. Oral administration refers to administration via the mouth. In some embodiments, administration is parenteral. Parenteral administration refers to administration that bypasses the Gl tract (e.g., via intravenous injection). In some embodiments, administration is topical (i.e. , at the site of infection), transdermal, or intradermal.

[0153] In therapeutic methods and uses described herein, administration of the bacteriocin peptides, peptidomimetics and compositions, preferably pharmaceutical compositions, may result in the alleviation of at least one symptom and / or the improvement of at least one parameter associated with an infection and / or disease discussed herein. Alleviating a symptom of a disease and / or infection refers to said symptom being improved or decreased in intensity or to the progression of a typical symptom having been slowed down in an individual, in a cell, tissue or organ of said individual as assessed by a physician. A decrease or improvement of a typical symptom may mean a slowdown in progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus also a decrease in symptoms, can be assessed using a variety of methods, to a large extent the same methods as used in diagnosis of the relevant infection and / or disease, including clinical examination and routine laboratory tests. Laboratory tests may include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays or CT-scans, biochemical methods, immunohistochemical methods, culturing methods, and others. In this context, “decrease” (respectively “improvement”) means at least a detectable decrease (respectively a detectable improvement) using an assay known to a person of skill in the art. The decrease may be a decrease of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. The decrease may be seen after at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days or more of treatment using a bacteriocin peptide, peptidomimetic, and / or composition as described herein. Symptoms of infections and / or diseases discussed herein are known in the art.

[0154] In some embodiments, administration of the bacteriocin peptide, peptidomimetic, or composition results in the alleviation of at least one symptom associated with an infection and / or disease caused by a bacterium of the phylum Firmicutes, preferably by a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, more preferably by a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis. In some embodiments, administration of the bacteriocin peptide, peptidomimetic, or composition results in the alleviation of at least one symptom associated with an infection and / or disease selected from the group consisting of a skin infection, a lung or respiratory tract infection (e.g., pneumonia), a CNS infection (e.g., meningitis), a bone infection (e.g., osteomyelitis), a heart infection (e.g., endocarditis), toxic shock syndrome, a joint infection (e.g., infectious arthritis or septic arthritis), a blood infection (e.g., bacteremia), sepsis, a urinary tract infection, an intra-abdominal or pelvic infection (e.g., peritonitis), a group A streptococcal disease (GAS), a throat infection (e.g., pharyngitis), listeriosis, sinusitis, and otitis media.

[0155] Administration may involve a therapeutically effective amount of a peptide, peptidomimetic, or composition, preferably pharmaceutical composition, described herein. As used herein, an “effective amount” is an amount sufficient to exert beneficial or desired results. Accordingly, a “therapeutically effective amount” is an amount that, when administered to a subject such as a subject in need thereof, is sufficient to exert some therapeutic effect as described herein, such as, but not limited to, a reduction in the magnitude of at least one symptom and / or the improvement of at least one parameter associated with an infection and / or disease, as described earlier herein. An amount that is "therapeutically effective" will vary from subject to subject, depending on the age, the infection and / or disease type and its progression, and overall general condition of the individual. An appropriate "therapeutically effective" amount in any individual case may be determined by the skilled person using routine experimentation, such as discussed elsewhere herein. A "subject in need” may be any individual affected by, and / or at risk of developing an infection and / or disease.

[0156] Within the context of the therapeutic uses and methods described herein, the target microbial cell such as a bacterium of the genus Lactococcus, Staphylococcus, Enterococcus, Listeria, or Streptococcus, may be comprised in a biofilm. A "biofilm” comprises any syntrophic consortium of microbial cells, preferably bacteria of the genus Lactococcus, Staphylococcus, Enterococcus, Listeria, or Streptococcus, in which cells stick to each other and often also to a surface. Typically, biofilms are resistant to commonly used antimicrobials, such as antibiotics.

[0157] Non-therapeutic methods / uses

[0158] In a further aspect, there is provided an ex-vivo method of disinfecting a surface comprising contacting said surface with a bacteriocin peptide or peptidomimetic, preferably peptide, or a composition, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the phylum Firmicutes, optionally further comprising one or more antimicrobial compounds and / or a solvent, as described herein. In some embodiments, the surface is contaminated with a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, more preferably by a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus thermophilus, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis. A "surface” as used herein refers to any non-living, preferably solid, surface which may serve as scaffold (i.e. provide physical support) for microbial growth. The method is further applicable both to the disinfection of instruments, such as medical instruments, and articles placed in small disinfection chambers, biological safety cabinets, isolators, glove boxes, incubators, materials airlocks, and the like. The method is also applicable for disinfection of food containers, industrial equipment and the like. In some embodiments, the surface is a surface of a medical instrument. Non-limiting examples of medical instruments include bedpans, cannulas, cardioverters, defibrillators, catheters, dialysers, electrocardiograph machines, enema equipment, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control equipment such as masks, gowns, face shields, and goggles, instrument sterilizers, kidney dishes, nasogastric tubes, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiographers, sphygmomanometers, thermometers, tongue depressors, transfusion kits, tuning forks, ventilators, watches, and the like. In some embodiments, the surface is the surface of industrial equipment. Non-limiting examples of industrial equipment include fermentation equipment, such as fermenters, tubing, feeding vessels, spargers, mixers, compressors, and the like, freezers, fridges, cargo vehicles, storage vessels, rotor blades, mills, and the like. A surface is "contaminated” with a bacterium, when its presence can be detected on said surface, using standard methods in the art such as swab tests.

[0159] "Disinfection”, otherwise known as "decontamination” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It generally refers to the inhibition and / or killing of microbial cells on inert surfaces. Disinfection may be partial, i.e. a part of the target microbial cell population may not be neutralized and / or killed. Partial disinfection may mean that at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 99% of the targeted population is not inhibited and / or killed.

[0160] The ex-vivo method may result in at least a 2 log (a factor of 100), at least a 3 log (a factor of 1000), at least a 4 log (a factor of 10000), at least a 5 log (a factor of 100000), or at least a 6 log (a factor of 1000000) reduction of the non-inhibited and / or alive target microbial cell population, preferably a population of a bacterium of the phylum Firmicutes, more preferably a population of a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, more preferably by a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus thermophilus, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis.

[0161] Contacting with a surface may be followed by a waiting period, wherein the bacteriocin peptide, peptidomimetic, or composition is left in contact with the surface. Said period may last at least 15 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 25 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, or more. After contacting with a surface or after the waiting period, the bacteriocin peptide, peptidomimetic or composition may be removed from the surface, e.g. by rinsing with water or by wiping said surface with a clean cloth. Accordingly, in some embodiments, the invention provides an ex-v / vo method of disinfecting a surface, preferably a surface contaminated with a bacterium of the phylum Firmicutes, more preferably a surface contaminated with a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, more preferably by a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus thermophilus, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis, comprising contacting said surface with a bacteriocin peptide, peptidomimetic, or a composition, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the phylum Firmicutes, optionally further comprising one or more antimicrobial compounds and / or a solvent as described herein, followed by a waiting period of at least 15 seconds.

[0162] The antimicrobial effect of an ex-vivo method according to the invention may be assessed by standard methods in the art, such as commercial in vitro laboratory tests such as ASTM E2149-20 or ASTM E1054-08 (ASTM, PA, USA), and the like, or alternative methods discussed elsewhere herein. In some embodiments, the ex-vivo method results in a reduction of the target microbial cell population by at least 2-fold as assessed using the ASTM E1054-08 test in vitro.

[0163] Within the ex-vivo methods described herein, the target microbial cell such as a bacterium a bacterium of the phylum Firmicutes, preferably a bacterium of a genus selected from the group consisting of Lactococcus, Staphylococcus, Enterococcus, Listeria, and Streptococcus, more preferably a bacterium selected from the group consisting of Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus thermophilus, Streptococcus pyogenes, Streptococcus oralis, and Streptococcus mitis may be comprised in a biofilm as discussed earlier herein.

[0164] General information

[0165] Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs, and read in view of this disclosure.

[0166] Sequence identity

[0167] In the context of the invention, a nucleic acid encoding a bacteriocin peptide or peptidomimetic is represented by a nucleotide sequence. In the context of the invention, a bacteriocin peptide or peptidomimetic is represented by an amino acid sequence. It is to be understood that each nucleic acid molecule or peptide or peptidomimetic as identified herein by a given sequence identity number (SEQ ID NO) is not limited to said specific sequence as disclosed.

[0168] Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given protein fragment or polypeptide or peptide or derived peptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO: X; ii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. a nucleotide sequence that encodes an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

[0169] Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0170] Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0171] Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively.

[0172] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (peptide, polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given sequences (for example as represented by a SEQ ID NO herein) or on a part thereof, preferably based on the full length of two given sequences. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated by reference herein in its entirety.

[0173] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below). A global alignment is suitably used to determine sequence similarity or identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) 1 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) 1 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference in its entirety).

[0174] Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al., J. Mol. Biol. 215:403-10 (1990), incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402 (1997), incorporated herein by reference in its entirety. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov / . The above algorithms may also be used to determine corresponding nucleotide or amino acid residue positions between sequences being aligned. For example, an amino acid residue in sequence Y which corresponds to position 1 (or any other position) of sequence X may be determined.

[0175] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so- called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0176] Alternative conservative amino acid residue substitution classes :

[0177] Alternative physical and functional classifications of amino acid residues:

[0178] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

[0179] Codon optimization

[0180] “Codon optimization”, as used herein, refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host cell. For example, to suit the codon preference of mammalian, insect, plant, or microbial cells, preferably microbial cells. Examples of microbial cells include eukaryotes such as yeasts, filamentous fungi, and algae, and prokaryotes such as bacteria and archaea, for example E. coli and S. salivarius. Codon optimization also eliminates elements that potentially impact negatively RNA stability and / or translation (e. g. termination sequences, TATA boxes, splice sites, ribosomal entry sites, repetitive and / or GC rich sequences and RNA secondary structures or instability motifs). Proteins and amino acids

[0181] The terms "protein" or "peptide" or “amino acid sequence” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. In amino acid sequences as described herein, amino acids or “residues” are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to a person of skill in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non-proteinogenic amino acid such as D-amino acids and modified amino acids formed by post-translational modifications, and also any non-natural amino acid.

[0182] Peptide expression

[0183] Peptide "expression” or "production” by a cell may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of gene expression on the level of the mRNA or the peptide by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein -de rived peptides or ELISA.

[0184] General terms

[0185] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of or "to essentially consist of, meaning that a composition as described herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention. In addition, the verb “to consist” may be replaced by “to consist essentially of meaning that a method or use as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention. In addition, the verb “to consist” may be replaced by “to consist essentially of meaning that a nucleotide or amino acid sequence as described herein may comprise additional nucleotides or amino acids than the ones specifically identified, said additional nucleotides or amino acids not altering the unique characteristics of the invention.

[0186] Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0187] As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.

[0188] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 1 % of the value.

[0189] As used herein, the term "and / or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0190] Various embodiments are described herein. Each embodiment as identified herein may be combined together unless otherwise indicated.

[0191] All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0192] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0193] Description of the figures

[0194] Fig. 1. Spot-on-lawn activity assays performed with mature bacteriocins (SEQ ID NOs: 6-10) produced in vitro by a cell-free system against three indicator bacteria strains (Lactococcus I act is IL1403, Streptococcus thermophilus LMD-9, Streptococcus salivarius HSISS4). Activity is demonstrated by the presence of a lysis zone on the bacterial lawn following spotting of the bacteriocin peptides. BIpK (SEQ ID NO: 19) was used as positive control.

[0195] Fig. 2A-2B. In Fig. 2A, the activity of tested bacteriocins against 12 Gram-positive bacteria belonging to the Streptococcus, Lactococcus, Enterococcus, Staphylococcus, and Listeria genera using spot-on-lawn assays is shown in a schematic representation (grey box indicates high activity, striped box indicates weak activity, white box indicates no activity). In Fig. 2B, the spot-on-lawn assays used to generate Fig. 2A is shown. Strain HSISS4 Aslv5 was used as a negative control. Bacteriocin producing strains (SEQ ID NOs: 6-10) were compared with strains producing known bacteriocins (BIpK (SEQ ID NO: 25), SIvV (SEQ ID NO: 26), SlvW (SEQ ID NO: 27), SIvX (SEQ ID NO: 28), SIvY (SEQ ID NO: 29), SIvZ (SEQ ID NO: 30), BIpE (SEQ ID NO: 33), or BipF (SEQ ID NO: 34)).

[0196] Fig. 3. Spot-on-lawn assays of HSISS4 S. salivarius strains (wild-type and Aslv5) producing SsaJ (SEQ ID NO: 9), SsaK (SEQ ID NO: 10), or SsaL (SEQ ID NO: 7) against 4 Gram-positive bacteria (S. thermophilus, S. aureus, S. epidermidis, L. monocytogenes). Strains Aslv5 and HSISS4 (wild-type) were used as negative (Ctl-) and positive (Ctl+) control, respectively.

[0197] Fig. 4. Activity of SsaL (SEQ ID NO: 7) against S. aureus. 3 pL of SsaL at a concentration of 100 pM were spotted on the plate. DMSO was used as negative control.

[0198] Fig. 5. Spot-on-lawn assays performed against S. pneumoniae strains (D39V, R6, TIGR4, PMEN1 , PMEN31 , 19A) using SsaL (SEQ ID NO: 7) and Ssal (SEQ ID NO: 6) producing strains. The bacteriocin producing strains were cultivated to mid-log phase and were spotted (3 pl) on the top of the soft-agar layer. The S. salivarius strain HSISS4 Aslv5 was used as a negative control.

[0199] Fig. 6A-6C. In Fig. 6A, spot-on-lawn assays performed against S. pneumoniae strains using SsaL (SEQ ID NO: 7) dissolved either in DMF (left) or DMSO (right) are shown. 3 pl of bacteriocin solution were spotted at concentrations ranging from 100 to 3 pM (100, 50, 25, 12.5, 6, 3 pM; 0 pM indicates negative control). In Fig. 6B-6C, SsaL (SEQ ID NO: 7) was added to growing cultures of 10 S. pneumoniae strains at a 2:2 serial dilution (0.0625-1 pM) and growth was monitored for 24 hours by measuring OD595 every 10 min (Fig 6B: DMF; Fig. 6C: DMSO).

[0200] Fig. 7A-7B. In Fig. 7A, the estimated fitness score (Iog2 fold change between induced and uninduced conditions) following differential enrichment analysis of a CRISPRi S. pneumoniae D39V library incubated with SsaL (SEQ ID NO: 7) and induced or uninduced with IPTG is shown. Big circles indicate significantly affected genes (adjusted P value below 0.05). The analysis was performed on R with the DESeq2 package. In Fig. 7B, SsaL was added to a growing culture of S. pneumoniae D39V AftsH and its effect was compared to the one exhibited against the parental D39V strain by measuring OD595 every 10 min for 24 hours.

[0201] Examples

[0202] General materials and methods

[0203] Bacterial strain and growth conditions

[0204] Streptococcus salivarius HSISS4 Aslv5 (genotype: AslvX-HSISS4_01664..lox72 blpKl..lox72 AsIvY- HSISS4_01744 ::lox72 sivV ::lox72 sivW-blpG ::lox72), a non-bacteriocin producing strain described in Mignolet et al. (2018) Cell Rep 22:1627-1638 (incorporated herein by reference in its entirety) was grown without shaking in M17G (glucose 1% [w / v]) medium (Oxoid) or CDMG (chemically-defined medium with glucose 1% [w / v]) (described in Letort and Juillard (2001 ) J Appl Microbiol 91 :1023-1029, incorporated herein by reference in its entirety) at 37°C. Lactococcus lactis IL1403 (described in Chopin et al. (1984) Plasmid 11 :260-263, incorporated herein by reference in its entirety), Streptococcus vestibularis F0396 (Craig Venter Institute, Rockville, MD, USA), Streptococcus thermophilus LMD-9 (ATCC), Streptococcus pyogenes 4549 (described in Smeesters et al. (2006) PLoS One 1 :e10, incorporated herein by reference in its entirety), Streptococcus oralis Si0464, Streptococcus mitis LMG 14557 (BCCM / LMG collection, Laboratory of Microbiology, Ghent University, Belgium), Staphylococcus aureus ATCC 6838, Staphylococcus epidermidis LMG 10273 (BCCM / LMG collection, Laboratory of Microbiology, Ghent University, Belgium), Enterococcus faecium ATCC 19434, Enterococcus faecium Si0159, Listeria monocytogenes ATCC 51777, and Streptococcus pneumoniae (strains D39V, R6, TIGR4, PMEN1 , PMEN31 , and 19A), were all grown without shaking in M17G at 37°C, except L. lactis IL1403 that was grown at 30°C. Solid plates inoculated with S. salivarius were incubated anaerobically (AnaeroGen 2.5L, Thermo Scientific, Waltham, MA, USA) at 37°C. Spectinomycin was added when needed at 200 pg / ml. Synthetic peptide sXIP (purity of 95%) was supplied by Peptide 2.0 Inc. (Chantilly, VA, USA), resuspended in water, and used at a concentration of 250 nM or 1 pM, except if otherwise stated. All utilized strains are available and can be obtained from publicly available collections utilizing the indicated strain names.

[0205] Strain construction

[0206] S. salivarius HSISS4 Aslv5 (non-bacteriocin producing strain) or S. salivarius HSISS4 wild-type (naturally producing BIpK (SEQ ID NO: 25), SIvV (SEQ ID NO: 26), SIvW (SEQ ID NO: 27), SIvX (SEQ ID NO: 28), SIvY (SEQ ID NO: 29), and SIvZ (SEQ ID NO: 30) were transformed with Gibson assembly products composed of three PCR fragments: (i) the upstream region of tRNASerassociated with a blpK gene fragment containing expression signals (i.e., blpK promotor - SEQ ID NO: 35) and encoding the leader sequence of BlpK (SEQ ID NO: 36); (ii) the downstream region of tRNASerassociated with the spec cassette (spectinomycin resistance); and (iii) the mature sequence of a bacteriocin (SEQ ID NOs: 6-10, and SEQ ID NOs: 26-30 and 33-34).

[0207] PCR fragments were amplified with the Q5 polymerase (NEB, Ipswich, MA, USA), following the manufacturer’s recommended protocol. To induce natural transformation, an overnight CDMG preculture was diluted in 500 pl of fresh CDMG at a final OD600 of 0.05 and incubated for 135 min at 37°C. Then, 1 pM of sXIP (blpK inducer) and linear DNA (Gibson assembly product) were added to the culture, and cells were further incubated for 3 h at 37°C before plating on M17G agar supplemented with spectinomycin. After transformation, all constructions were verified by DNA sequencing.

[0208] Single-bacteriocin producing reference strains (expressing one of BlpK (SEQ ID NO: 25), SIvV (SEQ ID NO: 26), SIvW (SEQ ID NO: 27), SIvX (SEQ ID NO: 28), SIvY (SEQ ID NO: 29), SIvZ (SEQ ID NO: 30), BIpE (SEQ ID NO: 33), or BipF (SEQ ID NO: 34) were obtained by transformation of expression constructs generated in the same way as for SEQ ID NO: 6-10 expression, following by their introduction to S. salivarius HSISS4 Aslv5.

[0209] Spot-on-lawn assays for bacteriocin activity

[0210] To detect bacteriocin activity, 50 pl of overnight cultures of producer strains were diluted in 1 ml of M17G and grown for 3 hours at 37°C (final OD600 of 0.5). In parallel, a first feeding layer (15 ml) of M17G 1.5% agar supplemented with XIP (250 nM or 1 pM) was cast on a plate. Then, a second layer (7 ml) of M17G 0.3% agar containing the target bacterium was poured on top of it. This layer contained 300 pl of an overnight culture of the target bacterium. On the top of this last layer, 3 pl of bacteriocin producer strain cultures (mid-log phase) were spotted. Finally, the plate was incubated overnight at 37°C. S. salivarius HSISS4 Aslv5 was used as negative control. Spot-on-lawn assays utilizing in vitro or synthetically produced bacteriocin peptides were performed in the same way, with the difference that bacteriocin peptide solutions were spotted instead of bacteriocin producer strain cultures.

[0211] In vitro production of bacteriocins

[0212] Synthetic plasmids (pUC57 derivative) expressing the mature parts of bacteriocins (peptides represented by SEQ ID NOs: 5-10) under the control of the T7 promoter were provided by GenScript (Piscataway, NJ, USA). These plasmids in combination with the cell-free system PURExpress (NEB, MA, USA) were used to produce bacteriocins in vitro. To synthesize bacteriocins, the reaction mixture was assembled on ice in the following order: 4 pl of solution A, 3 pl of solution B, 2 pl of nuclease-free water, and 1 pl of template DNA (100 ng / pl). Then, the reaction mixture was incubated for 3 hours at 37°C, and the activity was measured using a spot-on-lawn assay against target bacteria.

[0213] Chemical synthesis of SsaL

[0214] The mature peptide form of SsaL (SEQ ID NO: 7) was chemically synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA). The peptide was solubilized in 100% DMF or DMSO and its activity was measured using a spot-on-lawn assay against target bacteria.

[0215] Liquid culture activity tests

[0216] S. pneumoniae liquid cultures (grown on C+Y medium) were inoculated and subsequently diluted after 5h of growth in fresh C+Y medium at a final OD595 of 0.01 . The liquid culture was then supplemented with 2:2 serial dilution of chemically synthesized SsaL (SEQ ID NO: 7) and OD595 was measured every 10 min for 24 hours.

[0217] Example 1. Antibacterial effect of bacteriocins produced in vitro

[0218] In Example 1 , the bacteriocins represented by SEQ ID NOs: 1-5 were obtained in their mature form (SEQ ID NOs: 6-10) using in vitro production as described in the general methods, and their antibacterial effect against Lactococcus lactis IL1403, Streptococcus thermophilus LMD-9, and Streptococcus salivarius HSISS4 using spot- on-lawn assays, with BIpK used as a positive control (SEQ ID NO: 25). As it can be seen in Fig. 1 , all five bacteriocins demonstrate an antibacterial effect (indicated by the presence of circular lysis zone on the bacterial lawn).

[0219] Example 2. Antibacterial effect of bacteriocins produced by engineered S. salivarius

[0220] In Example 2, the bacteriocins represented by SEQ ID NOs: 1-5 were obtained in their mature form (SEQ ID NOs: 6-10) by expression in a non-bacteriocin producing S. salivarius background (HSISS4 Aslv5). Their expression in a strain devoid of residual antibacterial activity allows for individual evaluation of their antibacterial effect when they are cellularly produced, and to assess whether cellular production has an impact on it since class II bacteriocins may undergo minor post-translational modifications that require enzymes present in the host. Their antibacterial effect against 12 Gram-positive bacteria belonging to the Streptococcus, Lactococcus, Enterococcus, Staphylococcus, and Listeria genera was assessed with spot-on-lawn assays by spotting the producer strains as described in the general materials and methods and compared to the one of strains producing previously known bacteriocins (BIpK (SEQ ID NO: 25), SIvV (SEQ ID NO: 26), SIvW (SEQ ID NO: 27), SIvX (SEQ ID NO: 28), SIvY (SEQ ID NO: 29), SIvZ (SEQ ID NO: 30), BIpE (SEQ ID NO: 33), or BipF (SEQ ID NO: 34). SIvY and SIvZ were co-expressed in the same strain due to their nature as two-peptide bacteriocins. As shown in Fig. 2A and Fig. 2B, the tested bacteriocins show a potent antibacterial effect. Additionally, Ssal (SEQ ID NO: 6), SsaL (SEQ ID NO: 7), and SsaM (SEQ ID NO: 8) have the broadest spectrum in terms of target bacteria inhibition. The bacteriocins are active against closely related species, but also against clin ically-relevant pathogenic bacteria that are more phylogenetically distant, such as Staphylococcus aureus, Enterococcus faecium, and Listeria monocytogenes. Particularly, SsaK (SEQ ID NO: 10) shows a potent antibacterial effect against S. salivarius and L. monocytogenes. SsaJ (SEQ ID NO: 9) is active against salivarius streptococci such as Streptococcus thermophilus, Streptococcus vestibularis and Streptococcus salivarius.

[0221] Example 3. Potentiated antibacterial effect of bacteriocin cocktails

[0222] In Example 3, the antibacterial effect of effect of SsaJ (SEQ ID NO: 9), SsaK (SEQ ID NO: 10), and SsaL (SEQ ID NO: 7), was tested in compositions comprising additional distinct bacteriocins ("cocktails”). Each cocktail comprised SsaJ, SsaK, or SsaL, and a combination of BIpK (SEQ ID NO: 25), SIvV (SEQ ID NO: 26), SIvW (SEQ ID NO: 27), SIvX (SEQ ID NO: 28), SIvY (SEQ ID NO: 29), and SIvZ (SEQ ID NO: 30).

[0223] These cocktails were obtained by transformation of expression constructs encoding SsaJ, SsaK, or SsaL to the wild-type S. salivarius HSISS4 strain as described in the materials and methods, which naturally produces BIpK, SIvV, SIvW, SIvX, SIvY, and SIvZ. The three resulting producer strains were tested for their antimicrobial activity against S. thermophilus, S. aureus, S. epidermidis, and L. monocytogenes by spot-on-lawn assays as performed in Example 2. Despite the native production of multiple bacteriocins of strain HSISS4, we observed that that the addition of a single bacteriocin described herein could potentiate antimicrobial activity against specific species (Fig. 3). Specifically, the addition of SsaK or SsaJ strongly increased activity against L. monocytogenes or S. thermophilus, respectively. Further, the addition of the broad-spectrum bacteriocin SsaL remarkably increased the cocktail efficiency against the human pathogens S. aureus, S. epidermidis, and L. monocytogenes.

[0224] Example 4. Synthetically produced SsaL displays antibacterial effect against S. aureus

[0225] In Example 4, a chemically synthesized SsaL peptide (SEQ ID NO: 7) was used to test its antibacterial effect against S. aureus using spot-on-lawn assays performed as described in the general materials and methods. As it can be seen in Fig. 4, SsaL displays a potent antibacterial effect against this bacterium.

[0226] Example 5. Cellularly produced SsaL and Ssal display antibacterial effect against S. pneumoniae

[0227] In Example 5, S. salivarius Aslv5 strains expressing either SsaL (SEQ ID NO: 7) or Ssal (SEQ ID NO: 6) were used to test the antibacterial effect of the bacteriocins against several strains of S. pneumoniae using spot-on-lawn assays performed as described in the general materials and methods.

[0228] As it can be seen in Fig. 5, both bacteriocins displayed a potent antibacterial effect against all tested S. pneumoniae strains.

[0229] Example 6. Synthetically produced SsaL displays antibacterial effect against S. pneumoniae

[0230] In Example 6, a chemically synthesized SsaL peptide (SEQ ID NO: 7) was used to test its antibacterial effect against S. pneumoniae using spot-on-lawn assays performed as described in the general materials and methods, with the difference that serial dilutions (2:2) of the bacteriocin were tested. Bacteriocin solutions in which SsaL was dissolved in DMF (Fig. 6A left) or DMSO (Fig. 6A right) were tested. As it can be seen in Fig. 6A, SsaL is active against all tested strains, with the DMSO solution displaying a further increase in its activity compared to DMF. In parallel, we performed growth inhibition tests against 10 strains of S. pneumoniae in liquid media (C+Y), supplemented with SsaL (dissolved in DMF). Growth of all strains was impacted when SsaL was added to the medium at a final concentration of 1 pM (Fig. 6B). We further carrier out the same growth inhibition utilizing SsaL dissolved in DMSO instead. Compared to the results obtained with DMF-dissolved SsaL (Fig. 6B-6C), we observed a further decrease in the growth of 2 of the tested strains (D39V and PMEN31 ) to a total growth inhibition, when the bacteriocin solution was added at a final concentration of 1 pM. Overall, growth of several tested strains is already impacted in liquid cultures at a bacteriocin concentration of 0.0625 pM.

[0231] Example 7. S. pneumoniae mutants with increased sensitivity to bacteriocins

[0232] In Example 7, we used a described CRISPRi library of S. pneumoniae D39V (such CRISPRi libraries are described in Liu et al. (2021 ) Cell Host Microbe 29(1 ): 107-120 e6 and in de Bakker et al. (2022) Nat Protoc 17(2):252-281 , both of which are incorporated herein by reference in their entireties) to assess the importance of different genes / operon in S. pneumoniae sensitivity to bacteriocin SsaL (SEQ ID NO: 7).

[0233] In brief, a nuclease-dead mutant of Cas9 (dCas9) in the CRISPRi library strains is conditionally produced from the IPTG-inducible P / ac. Each cell co-expresses the dCas9 with a single guide RNA (sgRNA) targeting a specific gene / operon, resulting in a conditional knockdown mutant of this gene / operon. The CRISPRi library was engineered to pool clones in a single experiment, where each clone encodes the inducible dCas9 with a constitutively expressed sgRNAs targeting one of the 1499 operons present in the S. pneumoniae D39V genome.

[0234] For the experiments, liquid culture (C+Y) growth assays were performed using synthetically produced SsaL dissolved in DMSO (Example 6), or DMSO alone as negative control, in conditions where dCas9 was produced (+IPTG) or repressed (-IPTG). Specifically, the CRISPRi library was cultivated in four replicates in C+Y medium for 2.5 hours at 37°C. As a next step, cultures were diluted 100x in fresh medium supplemented with IPTG (induced) or water (uninduced) and SsaL (treated) or DMSO (untreated) and incubated for 3.6 hours (ca. 7 generations) at 37°C before cells were harvested, washed in PBS and pelleted for downstream genomic extraction. The sgRNA libraries were PCR-amplified from genomic DNA to add NGS-compatible indexes and pooled for Illumina sequencing.

[0235] After sequencing of the treated libraries, data were computed to assess the fitness score of each gene / operon in IPTG-induced vs uninduced conditions. Data were collected, demultiplexed on the Illumina machine following the manufacturer’s instructions and processes with 2FAST2Q (described in Bravo et al. (2022) PeerJ 10:p.e14041 , incorporated herein by reference in its entirety) to provide an sgRNA count for each condition. The final differential enrichment analysis that estimated the fitness score was performed on R with the DESeq2 package. Scores were plotted for each gene (Iog2-fold change between induced vs uninduced conditions) and significantly affected genes were depicted in Fig. 7A as big circles (adjusted P values below 0.05).

[0236] Comparing them in SsaL-treated vs untreated conditions revealed that strain with knockdowns of two genes (ftsH and lafA, encoding a membrane protease and a glycosyltransferase, respectively) were significantly affected (Fig. 7A). Their fitness scores were significantly lower when SsaL was added to the cultures, indicating that decreased activity of these genes increases the susceptibility of S. pneumoniae to SsaL.

[0237] To confirm these results, we deleted ftsH 'm D39V and performed a liquid culture assay. C+Y medium was inoculated with S. pneumoniae (D39V or AftsH) and diluted 5 hours later in fresh C+Y to a final OD595 of 0.004. The medium was supplemented with SsaL (1 pM final concentration ain DMSO) and the experiment was run for 24 hours, with an OD595 measurement every 10 min. As it can be seen in Fig. 7B, deletion of AftsH makes S. pneumoniae more susceptible to the antibacterial effect of this bacteriocin.

Claims

Claims1. A bacteriocin peptide or peptidomimetic, wherein said peptide or peptidomimetic is represented by an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence comprising at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity or similarity with a sequence selected from the group consisting of SEQ ID NOs: 1-10.

2. A bacteriocin peptide or peptidomimetic according to claim 1 , wherein said bacteriocin is a class II bacteriocin, preferably a class Ila bacteriocin or a class lid bacteriocin.

3. A bacteriocin peptide or peptidomimetic according to claim 1 or 2, wherein said peptide or peptidomimetic comprises the amino acid sequence motif X1X2X3X4X5GG (SEQ ID NO: 11 ), preferably wherein said peptide or peptidomimetic comprises the amino acid sequence motif LX2X3X4 EGG (SEQ ID NO: 12).

4. A bacteriocin peptide or peptidomimetic according to claim 1 or 2, wherein the combined percentage of comprised glycine and alanine residues with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic is less than 60%, preferably less than 55%.

5. A bacteriocin peptide or peptidomimetic according to any one of claims 1 , 2, or 4, wherein the combined percentage of comprised lysine and arginine residues with respect to the overall sum of amino acid residues comprised by the peptide or peptidomimetic is less than 6%.

6. A nucleic acid construct comprising a nucleic acid molecule encoding a bacteriocin peptide or peptidomimetic as defined in any one of claims 1 to 5, preferably wherein said nucleic acid molecule is a cDNA molecule.

7. A nucleic acid construct according to claim 6, wherein said nucleic acid construct is recombinant, preferably wherein said nucleic acid construct comprises a promoter sequence operably linked with the nucleic acid molecule encoding the bacteriocin peptide or peptidomimetic.

8. A vector comprising a nucleic acid construct as defined in claim 6 or 7.

9. A cell expressing a bacteriocin peptide as defined in any one of claims 1 to 5, or comprising a nucleic acid construct as defined in claim 6 or 7 or a vector as defined in claim 8, preferably wherein said cell is an engineered cell.

10. A composition comprising a bacteriocin peptide or peptidomimetic as defined in any one of claims 1 to 5, a nucleic acid construct as defined in claim 6 or 7, a vector a defined in claim 8, or a cell as defined in claim 9.

11. A composition according to claim 10, wherein said composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and / or pharmaceutically acceptable ingredients.

12. A composition according to claim 10 or 11 , wherein said composition further comprises one or more further bacteriocins, preferably one or more Streptococcus bacteriocins, more preferably one or more bacteriocins selected from the group consisting of SEQ ID NOs: 25-30 and SEQ ID NOs: 33-34.

13. A bacteriocin peptide or peptidomimetic according to any one of claims 1 to 5, a nucleic acid construct according to claim 6 or 7, a vector according to claim 8, a cell according to claim 9, or a composition according to any one of claims 10 to 12, for use as a medicament.

14. A bacteriocin peptide or peptidomimetic according to any one of claims 1 to 5, a nucleic acid construct according to claim 6 or 7, a vector according to claim 8, a cell according to claim 9, or a composition according to any one of claims 10 to 13, for use in the treatment, prevention, and / or delaying of an infection and / or disease, preferably wherein said infection and / or disease is caused by a bacterium of the phylum Firmicutes.

15. A composition according to claim 10 or 12, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the phylum Firmicutes, optionally further comprising one or more antimicrobial compounds and / or a solvent.

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