Engineered streptococcus salivarius k12 strains and probiotic compositions containing them

Abstract

The invention concerns genetically engineered strains of Streptococcus salivarius K12, probiotic compositions containing them and their use for the treatment of infections caused by Gram-positive and Gram-negative bacteria.

Description

[0001] ENGINEERED STREPTOCOCCUS SALIVARIUS K12 STRAINS AND PROBIOTIC COMPOSITIONS CONTAINING THEM

[0002] The invention concerns engineered strains of Streptococcus salivarius K12 and probiotic compositions containing said strains. The compositions of the invention are effective both against Gram-positive oropharyngeal, skin and vaginal pathogens (5. pyogenes, S. pneumoniae, S. agalactiae, S. aureus) and against Gram-negative pathogenic bacteria potentially inhabiting oral (i.e. Aggregatibacter, Porphyromonas, Fusobacterium), vaginal (i.e. Gardnerella, Atopobium, Prevotella, Sneathia), fecal (i.e. Escherichia, Klebsiella, Pseudomonas, Enterobacter) and skin microbiota (generally belonging to the phylum Proteobacteria).

[0003] Background of the invention

[0004] The microbiota of the oral cavity hosts numerous bacterial taxa, including Streptococcus, Prevotella, Veillonella, Actinomyces, Rothia, Gemella, Granulicatella. Streptococcus salivarius is one of the most abundant species (Int J Oral Sci. 2022 Mar 2; 14(1): 14). A strain of Streptococcus salivarius (K12) capable of anti-5, pyogenes activity was isolated from the oral cavity of a 5-year-old child, apparently refractory to infections caused by S. pyogenes (Indian J Med Res. 2004 May: 119 Suppl: 13-6). The anti-5, pyogenes action was attributable to the presence of a DNA plasmid coding for two bacteriocins (lantibiotics) known as Salivaricin A2 and Salivaricin B. The gene clusters of these two lantibiotics were originally identified respectively as SalA2 and SboB (Appl Environ Microbiol. 2007 Feb;73(4):l 107-13). The 5. salivarius K12 strain, in addition to the anti-5, pyogenes action, is capable of a lantibiotic-related action also extended to other typical species of the oropharynx such as 5. pneumoniae (BMC Microbiol. 2016 Sep 29;16(1):225). Finally, the K12 strain is capable of non-lantibiotic-related competition against some respiratory viruses (Probiotics Antimicrob Proteins. 2024 Feb;16(l):93-98), including SARS-Cov-2 (Microorganisms. 2022 Sep 28; 10(10): 1926), and fungi like Candida (Biofouling. 2021 Aug; 37(7): 767 -776). Among the all mechanisms of action, the lantibiotic-mediated anti-5, pyogenes action demonstrated in vitro by the K12 strain, prompted clinicians to its prophylactic use aimed at preventing recurrent pharyngo-tonsillar streptococcal infections in children (Drug Health Patient Saf. 2016 Nov 21 :8:77-81). Despite the suggested prophylactic health benefits of K12, placebo-controlled clinical trials did not provide conclusive results in the treatment of streptococcal pharyngitis and / or otitis media (Probiotics Antimicrob Proteins. 2017 Jun;9(2): 102-110). This therapeutic limit has prompted to investigate the ecological relationship in the oropharyngeal niche between S. salivarius and S. pyogenes. Recent studies (Nat Microbiol. 2024 Feb;9(2):502-513) have led to the following conclusions:

[0005] 1) the complete removal of the plasmid from the K12 strain abolishes the in vitro action exerted against S. pyogenes, but the removal of the two salivaricins (A2 and B) genes does not abolishes this activity;

[0006] 2) a third gene cluster, named sar, coding for a different anti-bacterial substance, not of the lantibiotic type, known as salivabactin, is present in the plasmid of strain K12;

[0007] 3) a gene, named nip (NRPS-inducing peptide), also present in the plasmid of strain K12, codes for a protein which is released concomitant with salivaricins (A2 and B) and salivabactin;

[0008] 4) the NIP protein, through quorum sensing mechanisms, fuels the synthesis and release of the SpeB protein from S. pyogenes, SpeB is capable of hydrolyzing salivaricins (but not salivabactin);

[0009] 5) K12-released salivabactin, although not inactivated by SpeB, is not very efficient since it is released, as a consequence of its weak promoter, in insufficient quantities to exert a sustainable anti -A', pyogenes action.

[0010] Based on these information, a K12 strain named S. salivarius eSAL, was produced by recombinant DNA techniques (Nat Microbiol. 2024 Feb;9(2):502-513). The modifications of the eSAL strain (versus the K12 strain) consist in:

[0011] 1) inactivation of the nip sequence (as described above, NIP protein release makes S. salivarius "visible" to S. pyogenes and activates in the latter a salivaricins-hydrolyzing protein, SpeB);

[0012] 2) insertion of the minimal (150 base pairs) fragment of the promoter for salivabactin, named PtufA, capable of fusing with the existing promoter and the gene cluster sar (Di Pierro F., “Can we predict the natural evolution of probiotics?”, Minerva Medica 2024 Sep 12, DOI: 10.23736 / S0026-4806.24.09568-5).

[0013] Tests carried out on (i) human saliva, (ii) mouse nasal cavity and (iii) mouse vagina, have demonstrated that the engineered strain, eSAL, is more effective than the strain K12 in limiting the growth of S. pyogenes. These results are due to the longer action of salivaricins (not negatively affected by SpeB) and to the more abundant release of salivabactin. Purified salivabactin, exactly as it was previously demonstrated for salivaricins (A2 and B), is not effective on Gram-negative species. Therefore, the strain eSAL, as for the strain K12, is not effective on Gram-negative bacteria. Description of the invention

[0014] The present invention aims to provide improved genetically modified K12 strains having high effectiveness not only against S. pyogenes and other Gram-positive bacteria, especially those inhabiting the oral and nose mucosa and the skin as already demonstrated for the strain eSAL, but also against Gram-negative bacteria (both inhabiting the oral and nose mucosa as well as the gut, vagina and skin environments).

[0015] It was surprisingly found that by functionally coupling the PtufA promoter in the complete version (SEQ ID NO:1 - 235 base pairs, instead of the minimal one constituted by 150 base pairs) with the N / / -BGC gene cluster (SEQ ID NO:3), in addition to inactivating the nip gene (Figure 1), a genetically modified S. salivarius K12 strain (hereinafter referred to as S. salivarius eK12) is obtained which is characterised by strong activity against S. pyogenes and unexpectedly by a strong antibiotic action towards Gram-negative bacteria, including E. coli (Table 1). The invention accordingly provides an engineered strain of Streptococcus salivarius K12 characterized by the insertion of the complete 235 base pairs PtufA promoter, upstream of the Sar gene and by inactivation of the nip gene. The promoter is functionally linked to, and drives the expression of, the biosynthetic gene cluster N / / -BGC.

[0016] Insertion of the PtufA promoter to drive expression of the Sar gene may be carried out using standard molecular biology techniques. Suitable methods include: homologous recombination, including allelic exchange using donor DNA flanked by homology arms, whereby the promoter sequence is integrated upstream of the coding sequence;

[0017] CRISPR / Cas-mediated editing, whereby a targeted double-strand break is introduced upstream of the gene and repaired with a donor DNA carrying the promoter sequence; recombineering or site-directed mutagenesis, employing recombinase systems to replace the native regulatory region with the desired promoter;

[0018] Transposon- or integrase-mediated insertion, wherein site-specific recombinases or transposable elements insert the promoter sequence in proximity to the target gene.

[0019] In each case, the promoter is positioned in correct orientation and reading frame relative to the gene of interest, so that it is operatively linked and capable of directing transcription of the gene.

[0020] Methods for silencing or inactivating the nip gene are well known in the art and may be carried out by introducing modifications in the nucleotide sequence of the gene or in its regulatory regions, so as to abolish or significantly reduce the production of a functional Nip protein. Inactivation may be achieved through deletion, mutation, or insertion of one or more nucleotides within the gene structure, particularly within the coding region or its flanking sequences. Non-limiting examples include: deletion of all or part of the coding sequence, resulting in the absence of translation of a functional protein; point mutations or substitutions that alter essential codons and prevent the expression of a functional protein; insertion of one or more nucleotides causing a frameshift mutation and premature termination of translation; introduction of stop codons upstream of or within the coding region, leading to truncated and non-functional proteins; alteration of the start codon or of sequences necessary for proper transcription or translation initiation.

[0021] In addition to direct sequence modification, inactivation can be obtained by disrupting gene regulatory elements such as the promoter or ribosome-binding site, thereby preventing transcription or translation. These modifications can be introduced by standard molecular biology methods, including but not limited to homologous recombination, site-directed mutagenesis, CRISPR / Cas-mediated gene editing, transposon insertion, or other genome engineering technologies.

[0022] A preferred strain has been deposited under the Budapest Treaty on July 17, 2024, at the Belgian Coordinated Collections of Micro-organisms (BCCM), Laboratorium voor Microbiologie - Bacterienverzameling (LMG), Universiteit Gent, Belgium, deposit number LMG P-33696.

[0023] In another embodiment, the invention provides the eK12 strain, wherein said strain is viable, inactivated or dead.

[0024] In a further embodiment, the invention provides a culture of eK12 in a suitable medium, or a supernatant thereof.

[0025] In another embodiment, the invention provides a probiotic composition comprising the engineered Streptococcus salivarius eK12 as above defined, a culture or supernatant thereof, in combination with suitable carriers, excipients and optionally other ingredients, probiotics and / or prebiotics.

[0026] The compositions of the invention may be administered by oral, parenteral or topical administration and may further include one or more ingredients selected from vitamins, amino acids, trace elements, short-chain fatty acids, prebiotics, anti-oxidants, substances of herbal or fungal origin, bacterial supernatants (i. e. those deriving from cultures of E. faecium L3), antibiotics, antifungal or chemotherapeutic agents, checkpoints inhibitors, monoclonal antibodies with anti-TNF activity or anti-ILp activity.

[0027] The compositions of the invention may be in any form suited for the administration of the K12 strain, for instance tablets, capsules, granules, gels, ovules, suppositories, sterile solutions, toothpaste, candies, chewing gums, drinks, comprising effective cell counts of live, inactivated or dead S. salivarius eK12. Supernatants of cultures of eK12 strains may also be used in addition or in substitution of the live, inactivated or dead cells.

[0028] According to another invention embodiment, the probiotic composition is used in the treatment of infections of the oral, vaginal or gastro-intestinal tract sustained by Gram-negative bacteria or by Gram-negative and Gram-positive bacteria.

[0029] In particular, the compositions of the invention are active against the following taxa: S. pyogenes, S. pneumoniae, M. catarrhalis, H. influenzae, S. agalactiae, Prevotella spp., Mobiluncus spp., Sneathia spp., L. iners, Ureaplasma spp., Aggregatibacter spp., Fusobacterium spp. , Porphyromonas spp. , Escherichia spp. , Enter obacter spp. , Pseudomonas spp., Klebsiella spp., Salmonella spp., Listeria spp., S. aureus, S. intermedins, S. mutans, Actinomyces spp., Rothia spp., Olsenella spp., Gemella spp., Granulicatella spp.

[0030] Detailed description of the invention

[0031] As shown in Figure 1, along with the two well-known lantibiotics (Salivaricin A2 and Salivaricin B), S. salivarius K12 produces the antibiotic salivabactin, effective in inhibiting some Gram-positive pathogens, including S. pyogenes. Salivabactin synthesis is carried out by a 14-gene biosynthetic gene cluster (BGC) that is encoded in the megaplasmid of the strain K12. The BGC expression is under the control of a peptide signaling pathway. The signaling pathway triggers the production of salivabactin transiently and only during late exponential phase of the K12 bacterial growth. To maximize salivabactin production, we decoupled BGC expression from the peptide signaling pathway by introducing two modifications: 1) inactivating the NRPS-inducing peptide gene (nip gene) by replacing the start codon of peptide to stop codon (SEQ ID NO:2) and 2) by fusing the BGC operon with a highly active, constitutive promoter, PtufA (SEQ ID NO: 1), that controls the production of ribosomal elongation factor-Tu (Mol Microbiol. 2017 Oct;106(l):22-34). The 235 bp promoter region is the entire region between two genes, and precisely: FtsW family cell division protein (locus tag RSSL 01732) and Elongation factor Tu (locus tag RSSL 01731). The thus obtained strain was called eK12.

[0032] Inclusion of the entire promoter in eK12 increased salivabactin BGC expression by additional four-fold and resulted in increased salivabactin production compared to eSAL. Quite unexpectedly, the genetically modified eK12 strain, unlike the previously described eSAL, proved able to inhibit the growth of Gram-negative bacteria.

[0033] To compare the antimicrobial role played by the strains K12 (5. salivarius K12), eSAL (5. salivarius eSAL) and eK12 (5. salivarius eK12), against S. pyogenes and E. coli. overnight cultures of S. pyogenes were diluted 1 : 1000 in pH adjusted (pH=6) THY broth (Todd Hewitt yeast extract). Differently, overnight cultures of E. coli were diluted 1 : 1000 at the same pH in LB (Luria-Bertani) broth. eSAL or eK12 strains were introduced at 105CFU / ml. Cultures were then grown for 6 h at 37°C and cells were plated to enumerate CFUs. As shown in Table 1, untreated cultures grown properly (++++: > 108CFU / ml). Co-culture with S. salivarius KI 2, counteracts S. pyogenes (++: > 106CFU / ml) without affecting E. coli. Co-culture with S. salivarius eSAL furtherly counteracts S. pyogenes (+: > 105CFU / ml) without affecting E. coli growth. Co-culture with S. salivarius eK12, abolish completely the growth of both S. pyogenes and E. coli (ND: growth not detected).

[0034] Table 1. Comparison of eSAL and eK12 for anti-S. pyogenes and anti-E. coli activity.

[0035] Table 2 compares salivabactin production and E. coli growth depending on the length of the promoter used:

[0036] Table 2

[0037] Legend for salivabactin

[0038] +: < 0.01 pg / ml salivabactin is produced. ++: < 0.1 pg / ml salivabactin is produced. +++: > 1 pg / ml salivabactin is produced. Legend for E. coli

[0039] ++++: > 108CFU / ml.

[0040] ND: growth not detected. The results reported in Tables 1 and 2 show that salivabactin is not effective against Gram-negative bacteria as recently reported (Nat Microbiol. 2024 Feb;9(2):502-513). The effect cannot therefore be explained unless it is assumed that other, so far not observed and described, active substances are released by the strain only when the entire 235 bp promoter region of PtufA is used. The antibiotic action against E. coli is not observed using both purified salivaricins and purified salivabactin, even when used at concentrations higher than those used for normal antibiotics with anti-Gram-negative action (Front Cell Infect Microbiol. 2022 Mar 15: 12:823684). The activity is evident also using the whole supernatants of S. salivarius eK12 at doses 10 times lower than the active doses of S. salivarius eSAL supernatants against S. pyogenes. The same result is obtained also using freeze-dried S. salivarius eK12 supernatants.

[0041] The S. salivarius K12 to S. salivarius eK12 modification was done according to three different steps.

[0042] A) In the first step, a PCR fragment was generated in which the start codon of nip gene was replaced with stop codon (TAG, TAA, or TGA) to abort the translation of NIP by quick change mutagenesis.

[0043] B) In the second step, the sar gene was coupled with 235 bp long tiifA promoter that has high constitutive activity by overlapping PCR.

[0044] C) In the final step, the PCR products from steps 1 and 2 were fused to generate the complete fragment.

[0045] Subsequently, the fragment was cloned into the multi-cloning site of the temperaturesensitive plasmid pJL1005 (Nat Commun. 2018 Mar 23;9(l): 1203). The S. salivarius K12 was then transformed with the resultant plasmid by competence-peptide based DNA uptake and integration. The transformants with plasmid incorporated into the S. salivarius K12 were selected for subsequent plasmid curing. Through a series of passaging the transformants, the cured candidates were selected for PCR screening. The presence of the desired modifications was confirmed by DNA sequencing. Subsequently, whole genome sequencing of the confirmed candidates were performed to ensure that no spurious mutation is present in the genome of S. salivarius eK12. Figure 2 shows the scheme and sequence details (SEQ ID NO:4) of S. salivarius eK12.

[0046] Examples

[0047] Example 1 - Genetic modification of S. salivarius KI 2 to generate S. salivarius eK12

[0048] The genetic modification of S. salivarius K12 to generate S. salivarius eK12 was performed in four steps. In the first step, we replaced the start codon (ATG) of the nip gene with a stop codon (TAG) to abort NIP translation. To this end, we amplified the PCR fragment using mutagenic primers that introduced a stop codon in nip. In the second step, we amplified the intergenic region (235 bp) between SAL K12 chromosomal genes encoding FtsW family cell division protein (locus tag RSSL 1732) and elongation factor Tu (locus tag RSSL 01737), which contains the entire promoter region of elongation factor Tu (PtufA). In the third step, we amplified the 600 bp fragment of the first gene of sar-BGC with overlapping sequences at the 5' end with PtufA. Finally, in the fourth step, we fused all 3 fragments to generate a single fragment comprising inactivated nip (nip*), PtufA, and the first gene of sar-BGC. The final PCR product was then cloned into the multi-cloning site of the temperature-sensitive plasmid pJL1055 (1. https: / / www.pnas.org / doi / full / 10.1073 / pnas.94.24.13251). The resultant plasmid was introduced into SAL K12 cells via competence-based DNA uptake. Briefly, overnight SAL growth was diluted in 0.3 ml of chemically defined medium (Do, H. et al. Leaderless secreted peptide signaling molecule alters global gene expression and increases virulence of a human bacterial pathogen. roc. Natl Acad. Sci. USA 114, E8498-E8507 (2017) https: / / www.pnas.org / doi / abs / 10.1073 / pnas.17Q5972114 ) and incubated at 37 °C for 75 min. Subsequently, synthetic competence-stimulating (ComS) peptide with the amino acid sequence of LPYFAGCL and plasmid was added to the cells and incubated for 3 h at 37 °C. Incorporation of the vector into the SAL K12 chromosome was accomplished by growing the cells in THY with chloramphenicol at 28°C (permissive temperature), whereas insertion of the vector into the chromosome was accomplished by shifting the culture temperature to 37°C (non-permissive temperature) and continuing to grow the cells in THY with chloramphenicol. Plasmid curing was performed by growing the cells at 28°C in the absence of chloramphenicol. After 3 cycles of growth, cells were plated onto Todd-Hewitt with yeast extract (THY) broth, and individual colonies were checked for sensitivity to chloramphenicol by patching them on plates containing antibiotics as well as plates without antibiotics. Chloramphenicol-sensitive colonies were selected, and sequencing of 1,000 bp on either side of the insert in the SAL K12 chromosome was performed to ensure that the correct genetic construct was obtained. In the final step, wholegenome sequencing of the selected eK12 candidate was performed to ensure that no spurious mutations were present in the eK12 strain.

[0049] Example 2 - Cell free supernatants (CFS) preparations from S. salivarius strains.

[0050] CSF preparations were prepared according to the method described by Kaewchomphunuch T et coll. (BMC Vet. Res. 18, 60). Briefly, overnight culture broth of the selected probiotics was transferred into 1.5 mL microcentrifuge tube and centrifuged for 2 min at 5,000 rpm (Denville Micro 260D Microcentrifuge, Denville Scientific, Inc., Metuchen, USA). Supernatants were collected by pass through 0.22 pm sterile syringe filter (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou, China). The filtrated CFS was either used freshly in agar well diffusion assay or stored at -20 °C for further analysis. Overnight culture broth of the selected probiotics was transferred into 1.5 mL microcentrifuge tube and centrifuged for 2 min at 5,000 rpm. Supernatants were collected by pass through 0.22 pm sterile syringe filter. The filtrated CFS was either used freshly in agar well diffusion assay or stored at -20 °C for further tests. Bacterial suspension was initially diluted into 0.5 McFarland standard and performed spread plate method onto nutrient agar. Then, the inoculated nutrient agar was pierced with the sterile 8 mm diameter cork borer to create wells. The volume of 100 pL of CFS was loaded into wells and incubated at 37 °C for the time necessary to observe activity.

[0051] Example 3 - Orosoluble tablet with fructose

[0052] Ingredients mg / dose

[0053] Fructose 635.00

[0054] Maltodextrins 275.00

[0055] S. salivarius eK12 (1011CFU / g) 50.00

[0056] Silicon dioxide 15.00

[0057] Magnesium salts of fatty acids 15.00

[0058] Strawberry flavour 10.00

[0059] Total 1000.00

[0060] Example 4 - Orosoluble tablet

[0061] Ingredients mg / dose

[0062] Sorbitol 520.00

[0063] Maltodextrins 275.00

[0064] S. salivarius eK12 (1011CFU / g) 50.00

[0065] Silicon dioxide 15.00

[0066] Magnesium salts of fatty acids 15.00

[0067] Strawberry flavour 15.00

[0068] Milk flavour 10.00

[0069] Total 900.00

[0070] Example 5 - Orosoluble powder for newborns (contained in openable hydroxypropyl methylcellulose capsule shell)

[0071] Ingredients mg / dose

[0072] S. salivarius eK12 (1011CFU / g) 30.00

[0073] Maltodextrins 270.00

[0074] Capsule (empty) 95.00 Example 6 - Coated tablet

[0075] Ingredients mg / dose

[0076] Magnesium stearate 340.00

[0077] S. salivarius eK12 (1011CFU / g) 50.00

[0078] Silicon dioxide 60.00

[0079] Total 450.00

[0080] Example 7 - Coated tablet with eK12 inactivated or killed

[0081] Ingredients mg / dose

[0082] Magnesium stearate 340.00

[0083] S. salivarius eK12 100.00

[0084] Silicon dioxide 60.00

[0085] Total 500.00

[0086] Example 8 - Coated tablet with prebiotic fibers

[0087] Ingredients mg / dose

[0088] Magnesium stearate 300.00

[0089] S. salivarius eK12 (1011CFU / g) 50.00

[0090] Silicon dioxide 60.00

[0091] Prebiotic fibers 40.00

[0092] Total 450.00

[0093] Example 9 - Coated tablet with eK12 inactivated or killed and prebiotic fibers

[0094] Ingredients mg / dose

[0095] Magnesium stearate 300.00

[0096] S. salivarius eK12 100.00

[0097] Silicon dioxide 60.00

[0098] Prebiotic fibers 40.00

[0099] Total 450.00

[0100] Example 10 - Double sachet with prebiotic fibers

[0101] Ingredients of sachet A mg / dose

[0102] Maltodextrins 200.00

[0103] Magnesium stearate 400.00

[0104] S. salivarius eK12 (1011CFU / g) 50.00

[0105] Ingredients of sachet B mg / tab

[0106] Silicon dioxide 160.00 Prebiotic fibers 40.00

[0107] Edulcorant 20.00

[0108] Total 870.00

[0109] Example 11 - Double sachet with eK12 inactivated or killed and prebiotic fibers

[0110] Ingredients of sachet A mg / dose

[0111] Maltodextrins 200.00

[0112] Mg stearate 400.00

[0113] S. salivarius eK12 100.00

[0114] Ingredients of sachet B mg / tab

[0115] Silicon dioxide 160.00

[0116] Prebiotic fibers 40.00

[0117] Edulcorant 20.00

[0118] Total 920.00

[0119] Example 12 - Sachet with another probiotic

[0120] Ingredients of sachet mg / dose

[0121] Maltodextrins 200.00

[0122] Mg stearate 400.00

[0123] Isomalt 140.00

[0124] S. salivarius eK12 50.00

[0125] B. animalis lactis BB-12 50.00

[0126] Silicon dioxide 160.00

[0127] Total 1000.00

[0128] Example 13 - Orosoluble tablet with lyophilized supernatants from eK12

[0129] Ingredients mg / dose

[0130] Anhydrous dextrose 400.00

[0131] Rice starch 40.00

[0132] Supernatants 100.00

[0133] Total 540.00

[0134] Example 14 - Intravaginal gel with lyophilized supernatants from eK12

[0135] Ingredients % per dose

[0136] Hyaluronic acid 3

[0137] Noveon 2

[0138] Supernatants 5

[0139] Rice starch 10 Water 80

[0140] Total 100

[0141] Example 15 - Vaginal tablet with lyophilized supernatants from eK12

[0142] Ingredients mg / dose

[0143] Lactic acid (60%) 15.00

[0144] Supernatants 18.00

[0145] Mannitol 572.33

[0146] Starch 200.00

[0147] Carboxymethylcellulose 20.00

[0148] Magnesium stearate 8.00

[0149] Total 900.00

[0150] Example 16 - Vaginal capsule with lyophilized supernatants from eK12

[0151] Ingredients mg / dose

[0152] Pullulan capsule 122.00

[0153] Supernatants 18.00

[0154] Lactic acid (60%) 10.00

[0155] Magnesium stearate 10.00

[0156] Starch 10.00

[0157] Total 170.00

[0158] Example 17 - Vaginal capsule with faecium) L3 strains

[0159] Ingredients mg / dose

[0160] Pullulan capsule 122.00

[0161] Supernatants from eK12 20.00

[0162] Supernatants from L3 18.00

[0163] Lactic acid (60%) 10.00

[0164] Magnesium stearate 10.00

[0165] Starch 10.00

[0166] Total 170.00

[0167] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

Claims

CLAIMS1. An engineered strain of Streptococcus salivarius K12 wherein:- the nip gene is inactivated by deletion, mutation or insertion of nucleotides that abolish expression of a functional Nip protein;- the complete PtufA promoter of S. salivarius comprising or consisting of SEQ ID NO: 1 is inserted upstream of the Sar gene cluster (N / / -BGC).

2. A strain according to claim 1 deposited at the Belgian Coordinated Collections of Micro-organisms (BCCM), Laboratorium voor Microbiologie - Bacterienverzameling (LMG), Universiteit Gent, Belgium, deposit accession number LMG P-33696.

3. A strain according to claim 1 or 2, wherein the nip gene has been inactivated by substituting the start codon with a stop codon.

4. A strain according to claims 1-3, wherein said strain is viable, inactivated or dead.

5. A culture of the strain according to claims 1-3, or a supernatant thereof.

6. A probiotic composition comprising the engineered Streptococcus salivarius K12 of claims 1-5, a culture or supernatant thereof according to claim 5, in combination with suitable carriers, excipients and optionally other ingredients, probiotics and / or prebiotics.

7. A probiotic composition according to claim 6 for oral, parenteral or topical administration.

8. A probiotic composition according to claim 6 or 7 further including one or more ingredients selected from vitamins, amino acids, trace elements, short-chain fatty acids, prebiotics, anti-oxidants, substances of herbal or fungal origin, bacterial supernatants, antibiotics, antifungal or chemotherapeutic agents, checkpoints inhibitors, monoclonal antibodies with anti-TNF activity or anti-ILp activity.

9. A probiotic composition according to any one of claims 6-8 in form of tablets, capsules, granules, gels, ovules, suppositories, sterile solutions.

10. A probiotic composition according to claims 6-9 for use in the treatment of infections of the oral, vaginal or gastro-intestinal tract sustained by Gram-negative bacteria or by Gram-negative and Gram-positive bacteria.

Images