Compositions and methods for treating bacterial disease

Abstract

The present invention relates to compositions and methods for preventing and / or treating bacterial disease (e.g., disease caused by Neisseria spp. such as Neisseria gonorrhoeae or Neisseria meningitidis). In particular, the present invention provides compositions comprising an effective amount of a nucleic acid, wherein such compositions are capable of killing or inhibiting the growth of a Neisseria spp and sensitizing Neisseria spp to antibiotic treatment.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING BACTERIAL DISEASE CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to and the benefit of U.S. Provisional Application No. 5 63 / 669,984, filed July 11, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. R21AI1151117 10 awarded by National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION The present invention relates to compositions and methods for preventing and / or treating bacterial disease (e.g., disease caused by Neisseria psp. such as Neisseria gonorrhoeae or 15 Neisseria meningitidis). In particular, the present invention provides compositions comprising an effective amount of a nucleic acid, wherein such compositions are capable of killing or inhibiting the growth of a Neisseria spp and sensitizing Neisseria spp to antibiotic treatment. BACKGROUND 20 Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis (Nme) are pathogens that cause high impact diseases in humans. Ngo infects the urinary tract and oropharynx of males and females. Ngo causes over 160 million new infections each year, worldwide. There is currently no vaccine against Ngo. Ngo has developed resistance to all antibiotics used for its treatment, leading the NIH, CDC and WHO to place Ngo on their list of "superbugs". NIH has announced 25 initiatives to accelerate the development of novel antimicrobials and identify new targets for antimicrobials and antibiotics against these superbugs. Nme colonizes the upper respiratory tract, entering the bloodstream to cause septicemia and crossing the blood-brain barrier to cause meningitis. Crowded living conditions and large migrations encourage the spread of Nme, and are the main cause of epidemics and micro- 30 epidemics around the world. As the CDC does not require the reporting of Nme infections, there is no accurate information on their incidence. Vaccines have significantly reduced the incidence 1

[0002] of meningococcal disease in developed countries. However, they do not cover all Nme serogroups and are unaffordable in poor countries. Nme continues to cause occasional epidemics in parts of Africa and the Middle East. On occasion, Nme also causes infections in the genitals and anus. 5 Improved therapeutic options for preventing, and / or treating infection by Neisseria gonorrhoeae and Neisseria meningitidis are needed. SUMMARY OF THE INVENTION Experiments described herein identified discreet pieces of DNA whose nucleotide 10 sequences derive from Ngo chromosomal loci and whose nucleotides are methylated differently than those in Ngo DNA. These DNAs are an improvement over N. elongata chromosomal DNA because their sequences are in Ngo and absent from, or not commonly found in, commensal Neisseria, and because of this leave commensal Neisseria spp unharmed. Further experiments identified gene alleles involved in sensitizing a subset of Neisseria cells that survive DNA killing 15 to antibiotics. Accordingly, in some embodiments, provided herein is a composition comprising a nucleic acid comprising at least one DNA uptake sequence (DUS) and at least one nucleic acid encoding at least a portion (e.g., 10, 50, 100, 1000, 2000, or 5000 nucleotides up to the entire gene) of a protein essential for viability of a pathogenic Neisseria, wherein the nucleic acid is 20 selected from, for example, gyrA, gyrB or parC. In some embodiments, the nucleic acid has a methylation pattern different from that of a pathogenic Neisseria. In some embodiments, the nucleic acid further comprises a nucleic acid encoding one or more additional proteins essential for viability of a pathogenic Neisseria (e.g., Neisseria gonorrhoeae (Ngo) or Neisseria meningitidis (Nme)). In some embodiments, the one or more 25 additional nucleic acids encode DNA methyltransferases e.g., ngoAXIV, ngoAI, and ngoAXV) and / or DNA replication and maintenance proteins. In some embodiments, the one or more additional nucleic acids are tdfF, tdfH, or iga. Also provided is a method for the inhibition of bacterial growth and / or for the killing of a target bacterium, comprising the step of adding to the target bacterium a composition described 30 herein, thereby inhibiting bacterial growth and / or killing the bacteria. 2

[0003] These nucleic acid sequences can span the entire gene or a portion of the genes in question (e.g., 10, 50, 100, 1000, 2000, or 5000 nucleotides up to the entire gene) and include both coding and non-coding regulatory portions of the genes. 5 Yet other embodiments provide the use of a composition described herein to inhibit bacterial growth and / or kill a target bacterium. The present disclosure is not limited to particular nucleic acid delivery molecules. Examples include but are not limited to plasmid DNA, linear DNA, bacterial artificial 10 chromosomal DNA, or genomic DNA. In some embodiments, the one or more nucleic acids contain sequences within one, two or all three of the following loci: ngoAXIV, ngoAI, and ngoAXV (e.g., present as a concatemer). In some embodiments, the one or more nucleic acids contain sequences within one, two or all three of the following loci: tdfF, tdfH, and iga (e.g., present as a concatemer). These nucleic acid sequences can span the entire gene or a portion of 15 the genes and include both coding and non-coding sequences. In some embodiments, the nucleic acid is produced by another organism, e.g. E. coli, or synthesized in vitro. The present disclosure is not limited to a particular DUS. Examples include but are not limited to the sequence N1N2N3N4N5N6N7CTGN8A (SEQ ID NO:4), wherein N1 is A or T, N2 is T, G, or A, N3 is G or C, N4 is C or T, N5 is C, T, or A, N6 is G or A, N7 is T or C, and N8 is C or 20 A (e.g., A[T / G]GCCGTCTGAA (SEQ ID NO:5) or GCCGTCTGAA (SEQ ID NO:6)). In some embodiments, the composition is a pharmaceutical composition (e.g., a personal lubricant). In some embodiments, the target bacterium is Neisseria gonorrhoeae (Ngo) or Neisseria meningitidis (Nme). In some embodiments, the composition is administered topically. In some embodiments, the composition does not kill and / or inhibit the growth of commensal 25 strains of Neisseria. In some embodiments, the composition sensitizes the target bacterium to an antibiotic. Additional embodiments are described herein. BRIEF DESCRIPTION OF THE DRAWINGS 30 Fig. 1. Low passage and antibiotic resistant Neisseria gonorrhoeae isolates are killed by N. elongata DNA dissolved in Tris buffer. ns, not significant compared to Nel DNA. 3

[0004] Abbreviations: *P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant; AR, antibiotic resistant. Fig. 2. Killing of Neisseria gonorrhoeae (Ngo) strain MS11 by DNA constructs. A, Names of each construct and diagram of the loci selected for testing. B, Killing efficiency of 5 DNA constructs in (A). Fig. 3. MB 14-1-15 efficiently kills all tested Neisseria gonorrhoeae isolates, whether dissolved in buffer (10 mM Tris) or personal lubricants. A, Laboratory adapted isolates; B, Low- passage clinical isolates from the STD clinic, Durham North Carolina; C, Antibiotic resistant isolates from the AR bank, CDC. MB 14-1-15 was incorporated to commercially available 10 personal lubricants KY Jelly and Astroglide. Fig. 4. MBA, MB-B and MB-C efficiently kills Neisseria gonorrhoeae isolates but not commensal Neisseria subflava. A, Laboratory adapted isolates; B, Low-passage clinical isolates from the STD clinic, Durham North Carolina; C, Antibiotic resistant isolates; D, N. subflava. . Fig. 5. Ciprofloxacin resistance of 4 surviving Ngo AR173 colonies after a killing assay. 15 Fig. 6. Exemplary sequences. Definitions To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. 20 Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention 25 may be readily combined, without departing from the scope or spirit of the invention. In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, 30 and “the” include plural references. The meaning of “in” includes “in” and “on.” 4

[0005] As used herein, the terms “subject” and “patient” refer to any animal, such as a mammal or other animal, for example, a dog, cat, bird, livestock, and preferably a human. As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for 5 therapeutic use. The terms “pharmaceutically acceptable” or “pharmacologically acceptable”, as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. As used herein, the term “administration” refers to the act of giving a drug, prodrug, 10 antibody, vaccine, or other agent, or therapeutic treatment to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like. 15 “Coadministration” refers to administration of more than one chemical agent or therapeutic treatment to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). As used herein, administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. “Coadministration” of therapeutic treatments may be concurrent, or in any temporal 20 order or physical combination. As used herein, “carriers” include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH-buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, 25 citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar 30 alcohols such as mannitol or sorbitol; personal lubricants, salt-forming counterions such as sodium; and / or nonionic surfactants. 5

[0006] As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The nucleic acid molecule may comprise one or more nucleotides. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6- 5 methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 10 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2- 15 thiocytosine, and 2,6-diaminopurine. The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand 20 binding, signal transduction, immunogenicity, etc.) of the full-length or fragments are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of 1 kb or more on either end, which function in regulating expression of said gene. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences 25 located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. As used herein, the term "oligonucleotide," refers to a short length of single-stranded polynucleotide chain. Oligonucleotides are typically less than 200 residues long (e.g., between 30 15 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24 6

[0007] residue oligonucleotide is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. The terms “protein” and “polypeptide” refer to compounds comprising amino acids 5 joined via peptide bonds and are used interchangeably. A “protein” or “polypeptide” encoded by a gene is not limited to the amino acid sequence encoded by the gene, but includes post- translational modifications of the protein. Where the term “amino acid sequence” is recited herein to refer to an amino acid sequence of a protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or 10 “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. 15 As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues (e.g., kidney tissue or cells), and gases. Biological samples include blood products, such as plasma, serum and the like. Such examples are not however to 20 be construed as limiting the sample types applicable to the present disclosure. DETAILED DESCRIPTION Nme and Ngo descended from a commensal Neisseria ancestor and are genetically related to present day commensal Neisseria spp (Quillin, S.J. and H.S. Seifert, Neisseria 25 gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol, 2018. 16(4): p. 226-240). This shared ancestry with commensals supports that their tendency to cause persistent asymptomatic infection may be governed by a shared repertoire of host interaction factors. Indeed, of the approximately 177 Ngo and Nme genes previously reported to encode host interaction factors, 69 are conserved in all 19 other Neisseria spp. evaluated (Marri, P.R., et al., 30 Genome sequencing reveals widespread gene exchange among human Neisseria species. PLoS One, 2010. 5(7): p. e11835). While genes unique to Ngo and Nme undoubtedly contribute to 7

[0008] pathogenesis, the importance of the shared host interaction factors to infection cannot be discounted. Moreover, these shared host interaction factors may also contribute to the ability of commensal Neisseria spp to sporadically cause disease. It was reported that DNA from commensal Neisseria kills Ngo and Nme (Kim, W.J., et 5 al., Cell Host & Microbe, 2019, 26, 1-12 PMCID: PMC6728082). The DNA kills when it is taken up by the pathogen and when these DNA fragments attempt to recombine with the many homologous sequences in the pathogen genome. Restriction endonucleases in the pathogen recognize that the incoming DNA is methylated differently than pathogen DNA and cleave the heteroduplexes, damaging chromosome integrity and causing cell death. Taking advantage of the 10 knowledge that pathogenic Neisseria have numerous genes in common with commensal Neisseria, that they readily take up DNA with the Neisseria DNA Uptake Sequence (DUS), and that pathogen DNA is methylated differently than commensal Neisseria DNA, experiments were performed that resulted in the development of a DNA-based microbicide that kills Ngo and Nme and leaves commensals unharmed. 15 US 10,286,016, Compositions and Methods for Treating Gonorrhea, describes the use of DNA from Neisseria elongata, a commensal, as a microbicidal agent for preventing Ngo infection. N. elongata DNA kills Ngo in vitro and in vivo. In fact, any DNA is able to kill the pathogen, providing it is methylated differently than Ngo DNA, has sequence homology to this pathogen's chromosome, and is taken up (internalized) by the pathogen (Kim, W.J., Higashi, D., 20 Goytia, M., Rendon, M.A., Pilligua-Lucas, M., Bronnimann, M., McLean, J.A., Duncan, J., Trees, D., Jerse, A.E. and So, M. (2019) Commensal Neisseria Kill Neisseria gonorrhoeae through a DNA-Dependent Mechanism. Cell Host & Microbe 26, 1-12. PMCID: PMC6728082). The present disclosure expands upon this observation by providing specific Neisseria gonorrhoeae sequences that are able to kill pathogenic strains of Neisseria. 25 Experiments described herein demonstrated that DNA molecules kill Ngo if they contain sequences with homology to the Ngo genome and a Neisseria-specific DNA Uptake Sequence (DUS), and if their nucleotides are methylated differently than Ngo DNA. For example, DNA molecules containing sequences present in Ngo and Nme but absent from the majority of commensal Neisseria spp, such as those in the tdfF, tdfH, and iga loci, and DNA molecules 30 containing sequences of genes that are essential for pathogen viability, such as DNA methyltransferase genes and DNA replication and maintenance genes, kill the pathogen, 8

[0009] provided they contain a DUS, have homology to Ngo chromosomal DNA, and is methylated differently than Ngo DNA. In addition, a concatemer of Ngo DNA methyltransferase genes, MB14-1-5, kills Ngo with the same high efficiency when dissolved in buffer and suspended in personal lubricants, making it possible to use a personal lubricant containing DNA molecules 5 such as MB14-1-5 as a topical microbicide. Further experiments demonstrated that DNA molecules containing Ngo DNA replication and maintenance genes kill Ngo with high efficiency. In addition, DNA molecules containing alleles of these genes that confer sensitivity to antibiotics sensitize DNA killing survivors to antibiotic sensitivity. Accordingly, in some embodiments, provided herein is a composition 10 comprising a nucleic acid comprising at least one DNA uptake sequence (DUS) and at least one nucleic acid encoding at least a portion (e.g., 10, 50, 100, 1000, 2000, or 5000 nucleotides up to the entire gene) of a protein essential for viability of a pathogenic Neisseria, wherein the protein is selected from, for example, GyrA, GyrB or ParC. In some embodiments, the nucleic acid further comprises a nucleic acid encoding one or 15 more additional proteins essential for viability of a pathogenic Neisseria (e.g., Neisseria gonorrhoeae (Ngo) or Neisseria meningitidis (Nme)). In some embodiments, the one or more additional proteins are DNA methyltransferases e.g., NgoAXIV, NgoAI, and NgoAXV) and / or DNA replication and maintenance proteins. In some embodiments, the one or more additional nucleic acids encode tdfF, tdfH, or iga. 20 Also provided is a method for the inhibition of bacterial growth and / or for the killing of a target bacterium, comprising the step of adding to the target bacterium a composition described herein, thereby inhibiting bacterial growth and / or killing the bacteria. These nucleic acid sequences can span the entire gene or a portion of the genes in question (e.g., 10, 50, 100, 1000, 2000, or 5000 nucleotides up to the entire gene) and include 25 both coding and non-coding regulatory portions of the genes. The present disclosure is not limited to the Ngo sequences described herein. Any Ngo sequences essential for Ngo viability (e.g., DNA replication and repair proteins and / or DNA methyltransferases (e.g., ngoAXIV, ngoAI, and ngoAXV)) are specifically contemplated. These nucleic acid sequences can span the entire gene or a portion of the genes in 30 question. These nucleic acid sequences can span the entire gene or a portion of the genes in 9

[0010] question (e.g., at least 10, 20, 50, 100, 200, 500, 1000, 5000 or more nucleotides up to the entire sequence) and include both coding and non-coding regulatory regions of the genes. The present disclosure is not limited to particular nucleic acids. Examples include but are not limited to a plasmid, linear DNA, a bacterial artificial chromosome, genomic DNA, or DNA 5 synthesized in vitro. In some embodiments, the one or more nucleic acids are ngoAXIV, ngoAI, and ngoAXV (e.g., present as a concatemer). In some embodiments, the one or more nucleic acids are tdfF, tdfH, and iga (e.g., present as a concatemer). In some embodiments, the DNA replication or maintenance protein is one, two, or all of a nucleic acid encoding at least a portion (e.g., 10, 50, 100, 1000, 2000, or 5000 nucleotides up to 10 the entire gene) of a DNA replication or maintenance protein selected from, for example GyrA, GyrB or ParC. In some embodiments, the DNA replication or maintenance protein is a gene that is essential for cell viability or alleles of a gene that confers sensitivity to an antibiotic. In some embodiments, the one or more nucleic acids are synthesized in vitro or produced by a different microorganism and the methylation signature is different than Ngo or Nme (e.g., 15 E. coli). The present invention is not limited to a particular Neisseria DUS (See e.g., Frye S.A., Nilsen, M., Tønjum, T., Ambu, O.H. Dialects of the DNA uptake sequence in Neisseria. PLoS Genet. Apr;9(4):e1003458. doi: 10.1371 / journal.pgen.1003458. Epub 2013 Apr 18 (2013).; herein incorporated by reference in its entirety).Examples include but are not limited to the 20 sequence N1N2N3N4N5N6N7CTGN8A (SEQ ID NO:4), wherein N1is A or T, N2is T, G, or A, N3is G or C, N4is C or T, N5is C, T, or A, N6is G or A, N7is T or C, and N8is C or A (e.g., A[T / G]GCCGTCTGAA (SEQ ID NO:5) or GCCGTCTGAA (SEQ ID NO:6)). In some embodiments, the composition is a pharmaceutical composition (e.g., a personal lubricant). In some embodiments, the target bacterium is Neisseria gonorrhoeae (Ngo) or 25 Neisseria meningitidis (Nme). In some embodiments, the composition is administered topically. In some embodiments, the composition does not kill and / or inhibit the growth of commensal strains of Neisseria. In some embodiments, the composition is an antiseptic. Antiseptics are antimicrobial substances that are applied to living tissue / skin to reduce the possibility of infection and / or 30 sepsis, and / or putrefaction. Antiseptics are generally distinguished from antibiotics in that it is applied to the surfaces of the body, while the latter is transported through the lymphatic system 10

[0011] to destroy bacteria within the body. Antiseptics are distinguished from disinfectants, which destroy microorganisms found on non-living objects. In some embodiments, antiseptic compositions comprising an effective amount of a commensal species of Neisseria (e.g., an effective amount of an extract of a commensal species of Neisseria) (e.g., N. elongata (Nel), N. 5 polysaccharea (Npo)) capable of inhibiting the growth of Ngo or Nme and / or is killing Ngo or Nme are provided. For example, in some embodiments, such an antiseptic composition can be applied to the tissue of a subject (e.g., a human subject) for purposes of preventing the growth or inducing the killing of Ngo or Nme. In some embodiments, the composition is a disinfectant. In some embodiments, the 10 compositions are used for anti-fouling. Anti-fouling is the process of removing or inhibiting the accumulation of biofouling. Biofouling or biological fouling is the undesirable accumulation of microorganisms, plants, algae, and animals on surfaces. The present disclosure further provides pharmaceutical compositions (e.g., comprising the compounds described above). The pharmaceutical compositions of the present disclosure 15 may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, 20 subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include personal lubricants, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily 25 bases, gels, thickeners and the like may be necessary or desirable. The personal lubricant composition is oil base or water based and can be a liquid, emulsion, frost or gel. The example of oil-based composition comprises polytrimethylene ether glycol, thickening agent, ethylene glycol and alpha-hydroxy acid (AHA). AHA products such as 30 glycolic or lactic acid helps to regulate the acidity of compositions. Examples include the homopolymer of 1,3-PD, the copolymer of 1,3-PD and ethylene glycol; hydroxypropyl cellulose 11

[0012] as thickening agent; biologically-derived (biologically-derived) 1,3-PD as additive; and lactic acid. Other personal lubricant compositions are hydrogel compositions. Hydrogel compositions become relative flowable liquid when contacting with the warm surfaces of skin 5 after using. These compositions are physiologically acceptable and wash off neatly from skin. Bio-derived material in the aqueous solution is particularly useful aspect of a vagina wetting agent and personal lubricant. The form of personal lubricant compositions of the present invention can be liquid, semisolid or solid. In some embodiments, the lubricant is an emulsion, frost or gel. The lubricant 10 can also be formulated as syrupy liquigel, dumpable gel or thick gel. Other components of personal lubricant are, for example, glycerol, 1; 2,3-glycerol, polyethylene glycol (PEG) are such as PEG200 or PEG 400, polypropylene glycol, polyisobutylene, polyoxyethylene, behenic acid, sugar-alcohol (such as sorbitol) and some organo-silicon compound (such as polydimethylsiloxane). 15 Suitable thickening agents for lubricants include, for example, chemically treated cellulose derivative (such as hydroxyethyl-cellulose or hydroxy methocel), radix acaciae senegalis, agar, alginate, carrageenin, Tragacanth, xanthan gum, collagen, carbomer, glyceryl monostearate, polyvinylpyrrolidone and polyacrylamide. Other component can be added in the lubricant compositions, for example, antiseptic 20 (DMDM Hydantoin for example, chlorhexidine gluconate), antigraining agent (for example gluconic acid-δ-lactate), spice, sweeting agent, flavour enhancer, coloring agent, EDTA, lanolin, aloe extract, hydrocortisone, antiviral agent (for example zinc salt), hormone (for example estrogen) or spermicide (for example Nonyl pheno (9) ether). In some embodiments, the personal lubricant is a commercially available lubricant (e.g., 25 ASTROGLIDE lubricant (Combe, White Plains, NY) or K-Y JELLY lubricant (Reckit, Slough, England). Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. 30 Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and 12

[0013] other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be 5 generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into 10 association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions of the present disclosure may be formulated into any of many possible 15 dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. 20 In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. Agents that enhance uptake of oligonucleotides at the cellular level may also be added to 25 the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97 / 30731), also enhance the cellular uptake of oligonucleotides. The compositions of the present disclosure may additionally contain other adjunct 30 components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for 13

[0014] example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly 5 interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. 10 In some embodiments, the composition is a gel (e.g., formulated for delivery to a mucosal surface). In some embodiments, the gel coats a product for use in treating or preventing infection by Ngo or Nme (e.g., a condom). In some embodiments, the composition stabilizes the nucleic acid from degradation by enzymes in the mucosa. In some embodiments, the composition is formulated for delivery to the oropharynx 15 (e.g., as a toothpaste or mouthwash). In some embodiments, the composition is added to a personal lubricant (e.g., water based, silicone based, or oil based). In some embodiments, any one of many commercially available personal lubricants are utilized (e.g., available from Johnson and Johnson, New Brunswick, NJ or Biofilm, Inc, Vista, CA). 20 In some embodiments, the composition is coated onto a condom (e.g., male or female condom). Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be 25 calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 30 0.01 µg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating clinician can estimate repetition rates for dosing based on measured 14

[0015] residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 µg to 100 g per kg of body weight, once or more daily, to once every 5 20 years. The composition according to the present invention can be co-administered to an individual in need thereof in combination with one or more drugs such as one or more drugs with antibacterial effect. The one or more antibiotics can be selected from the group consisting of Amikacin disulfate salt, Amikacin hydrate, Anisomycin from Streptomyces griseolus, 10 Apramycin sulfate salt, Azithromycin, Blasticidine S hydrochloride, Brefeldin A, Brefeldin A from Penicillium brefeldianum, Butirosin sulfate salt, Butirosin A from Bacillus vitellinus, Chloramphenicol, Chloramphenicol base, Chloramphenicol succinate sodium salt, Chlortetracycline hydrochloride, Chlortetracycline hydrochloride from Streptomyces aureofaciens, Clindamycin 2-phosphate, Clindamycin hydrochloride, Clotrimazole, 15 Cycloheximide from microbial, Demeclocycline hydrochloride, Dibekacin sulfate salt, Dihydrostreptomycin sesquisulfate, Dihydrostreptomycin solution, Doxycycline hyclate, Duramycin from Streptoverticillium cinnamoneus, Emetine dihydrochloride hydrate), Erythromycin, Erythromycin USP, Erythromycin powder, Erythromycin, Temephos, Erythromycin estolate, Erythromycin ethyl succinate, Erythromycin standard solution, 20 Erythromycin stearate, Fusidic acid sodium salt, G 418 disulfate salt, G 418 disulfate salt powder, G 418 disulfate salt solution liquid, Gentamicin solution liquid, Gentamicin solution, Gentamicin sulfate Micromonospora purpurea, Gentamicin sulfate salt, Gentamicin sulfate salt powder USP, Gentamicin-Glutamine solution liquid, Helvolic acid from Cephalosporium caerulens, Hygromycin B Streptomyces hygroscopicus, Hygromycin B Streptomyces 25 hygroscopicus powder, Hygromycin B solution Streptomyces hygroscopicus, Josamycin, Josamycin solution, Kanamycin B sulfate salt, Kanamycin disulfate salt from Streptomyces kanamyceticus, Kanamycin monosulfate from Streptomyces kanamyceticus, Kanamycin monosulfate from Streptomyces kanamyceticus powder USP, Kanamycin solution from Streptomyces kanamyceticus, Kirromycin from Streptomyces collinus, Lincomycin 30 hydrochloride, Lincomycin standard solution, Meclocycline sulfosalicylate salt, Mepartricin, Midecamycin from Streptomyces mycarofaciens, Minocycline hydrochloride crystalline, 15

[0016] Neomycin solution, Neomycin trisulfate salt hydrate, Neomycin trisulfate salt hydrate powder, Neomycin trisulfate salt hydrate USP powder, Netilmicin sulfate salt, Nitrofurantoin crystalline, Nourseothricin sulfate, Oleandomycin phosphate salt, Oleandomycin triacetate, Oxytetracycline dihydrate, Oxytetracycline hemicalcium salt, Oxytetracycline hydrochloride, Paromomycin 5 sulfate salt, Puromycin dihydrochloride from Streptomyces alboniger, Rapamycin from Streptomyces hygroscopicus, Ribostamycin sulfate salt, Rifampicin, Rifamycin SV sodium salt, Rosamicin Micromonospora rosaria, Sisomicin sulfate salt, Spectinomycin dihydrochloride hydrate, Spectinomycin dihydrochloride hydrate powder, Spectinomycin dihydrochloride pentahydrate, Spiramycin, Spiramycin from Streptomyces sp., Spiramycin solution, 10 Streptomycin solution, Streptomycin sulfate salt, Streptomycin sulfate salt powder, Tetracycline, Tetracycline hydrochloride, Tetracycline hydrochloride USP, Tetracycline hydrochloride powder, Thiamphenicol, Thiostrepton from Streptomyces azureus, Tobramycin, Tobramycin sulfate salt, Tunicamycin A1 homolog, Tunicamycin C2 homolog, Tunicamycin Streptomyces sp., Tylosin solution, Tylosin tartrate, Viomycin sulfate salt, Virginiamycin M1, (S)-(+)-15 Camptothecin, 10-Deacetylbaccatin III from Taxus baccata, 5-Azacytidine, 7- Aminoactinomycin D, 8-Quinolinol crystalline, 8-Quinolinol hemisulfate salt crystalline, 9- Dihydro-13-acetylbaccatin III from Taxus canadensis, Aclarubicin, Aclarubicin hydrochloride, Actinomycin D from Streptomyces sp., Actinomycin I from Streptomyces antibioticus, Actinomycin V from Streptomyces antibioticus, Aphidicolin Nigrospora sphaerica, Bafilomycin 20 A1 from Streptomyces griseus, Bleomycin sulfate from Streptomyces verticillus, Capreomycin sulfate from Streptomyces capreolus, Chromomycin A3Streptomyces griseus, Cinoxacin, Ciprofloxacin BioChemika, cis-Diammineplatinum(II) dichloride, Coumermycin A1, Cytochalasin B Helminthosporium dematioideum, Cytochalasin D Zygosporium mansonii, Dacarbazine, Daunorubicin hydrochloride, Daunorubicin hydrochloride USP, Distamycin A 25 hydrochloride from Streptomyces distallicus, Doxorubicin hydrochloride, Echinomycin, Echinomycin BioChemika, Enrofloxacin BioChemika, Etoposide, Etoposide solid, Flumequine, Formycin, Fumagillin from Aspergillus fumigatus, Ganciclovir, Gliotoxin from Gliocladium fimbriatum, Lomefloxacin hydrochloride, Metronidazole purum, Mithramycin A from Streptomyces plicatus, Mitomycin C Streptomyces caespitosus, Nalidixic acid, Nalidixic acid 30 sodium salt, Nalidixic acid sodium salt powder, Netropsin dihydrochloride hydrate, Nitrofurantoin, Nogalamycin from Streptomyces nogalater, Nonactin from Streptomyces 16

[0017] tsusimaensis, Novobiocin sodium salt, Ofloxacin, Oxolinic acid, Paclitaxel from Taxus yannanensis, Paclitaxel from Taxus brevifolia, Phenazine methosulfate, Phleomycin Streptomyces verticillus, Pipemidic acid, Rebeccamycin from Saccharothrix aerocolonigenes, Sinefungin, Streptonigrin from Streptomyces flocculus, Streptozocin, Succinylsulfathiazole, 5 Sulfadiazine, Sulfadimethoxine, Sulfaguanidine purum, Sulfamethazine, Sulfamonomethoxine, Sulfanilamide, Sulfaquinoxaline sodium salt, Sulfasalazine, Sulfathiazole sodium salt, Trimethoprim, Trimethoprim lactate salt, Tubercidin from Streptomyces tubercidicus, 5- Azacytidine, Cordycepin, Formycin A, (+)-6-Aminopenicillanic acid, 7- Aminodesacetoxycephalosporanic acid, Amoxicillin, Ampicillin, Ampicillin sodium salt, 10 Ampicillin trihydrate, Ampicillin trihydrate USP, Azlocillin sodium salt, Bacitracin Bacillus licheniformis, Bacitracin zinc salt Bacillus licheniformis, Carbenicillin disodium salt, Cefaclor, Cefamandole lithium salt, Cefamandole nafate, Cefamandole sodium salt, Cefazolin sodium salt, Cefinetazole sodium salt, Cefoperazone sodium salt, Cefotaxime sodium salt, Cefsulodin sodium salt, Cefsulodin sodium salt hydrate, Ceftriaxone sodium salt, Cephalexin hydrate, 15 Cephalosporin C zinc salt, Cephalothin sodium salt, Cephapirin sodium salt, Cephradine, Cloxacillin sodium salt, Cloxacillin sodium salt monohydrate, D-{tilde over ( )}( )-Penicillamine hydrochloride, D-Cycloserine microbial, D-Cycloserine powder, Dicloxacillin sodium salt monohydrate, D-Penicillamine, Econazole nitrate salt, Ethambutol dihydrochloride, Lysostaphin from Staphylococcus staphylolyticus, Moxalactam sodium salt, Nafcillin sodium salt 20 monohydrate, Nikkomycin, Nikkomycin Z Streptomyces tendae, Nitrofurantoin crystalline, Oxacillin sodium salt, Penicillic acid powder, Penicillin G potassium salt, Penicillin G potassium salt powder, Penicillin G potassium salt, Penicillin G sodium salt hydrate powder, Penicillin G sodium salt powder, Penicillin G sodium salt, Phenethicillin potassium salt, Phenoxymethylpenicillinic acid potassium salt, Phosphomycin disodium salt, Pipemidic acid, 25 Piperacillin sodium salt, Ristomycin monosulfate, Vancomycin hydrochloride from Streptomyces orientalis, 2-Mercaptopyridine N-oxide sodium salt, 4-Bromocalcimycin A23187 BioChemika, Alamethicin Trichoderma viride, Amphotericin B Streptomyces sp., Amphotericin B preparation, Calcimycin A23187, Calcimycin A23187 hemi(calcium-magnesium) salt, Calcimycin A23187 hemicalcium salt, Calcimycin A23187 hemimagnesium salt, Chlorhexidine diacetate salt 30 monohydrate, Chlorhexidine diacetate salt hydrate, Chlorhexidine digluconate, Clotrimazole, Colistin sodium methanesulfonate, Colistin sodium methanesulfonate from Bacillus colistinus, 17

[0018] Colistin sulfate salt, Econazole nitrate salt, Hydrocortisone 21-acetate, Filipin complex Streptomyces filipinensis, Gliotoxin from Gliocladium fimbriatum, Gramicidin A from Bacillus brevis, Gramicidin C from Bacillus brevis, Gramicidin from Bacillus aneurinolyticus (Bacillus brevis), lonomycin calcium salt Streptomyces conglobatus, Lasalocid A sodium salt, Lonomycin 5 A sodium salt from Streptomyces ribosidificus, Monensin sodium salt, N-(6-Aminohexyl)-5- chloro-1-naphthalenesulfonamide hydrochloride, Narasin from Streptomyces auriofaciens, Nigericin sodium salt from Streptomyces hygroscopicus, Nisin from Streptococcus lactis, Nonactin from Streptomyces sp., Nystatin, Nystatin powder, Phenazine methosulfate, Pimaricin, Pimaricin from Streptomyces chattanoogensis, Polymyxin B solution, Polymyxin B sulfate salt, 10 DL-Penicillamine acetone adduct hydrochloride monohydrate, Polymyxin B sulfate salt powder USP, Praziquantel, Salinomycin from Streptomyces albus, Salinomycin from Streptomyces albus, Surfactin from Bacillus subtilis, Valinomycin, (+)-Usnic acid from Usnea dasypoga, (±)- Miconazole nitrate salt, (S)-(+)-Camptothecin, 1-Deoxymannojirimycin hydrochloride, 1- Deoxynojirimycin hydrochloride, 2-Heptyl-4-hydroxyquinoline N-oxide, Cordycepin, 1,10- 15 Phenanthroline hydrochloride monohydrate puriss., 6-Diazo-5-oxo-L-norleucine, 8-Quinolinol crystalline, 8-Quinolinol hemisulfate salt, Antimycin A from Streptomyces sp., Antimycin A1, Antimycin A2, Antimycin A3, Antipain, Ascomycin, Azaserine, Bafilomycin A1 from Streptomyces griseus, Bafilomycin B1 from Streptomyces species, Cerulenin BioChemika, Chloroquine diphosphate salt, Cinoxacin, Ciprofloxacin, Mevastatin BioChemika, 20 Concanamycin A, Concanamycin A Streptomyces sp, Concanamycin C from Streptomyces species, Coumermycin A1, Cyclosporin A from Tolypocladium inflatum, Cyclosporin A, Econazole nitrate salt, Enrofloxacin, Etoposide, Flumequine, Formycin A, Furazolidone, Fusaric acid from Gibberella fujikuroi, Geldanamycin from Streptomyces hygroscopicus, Gliotoxin from Gliocladium fimbriatum, Gramicidin A from Bacillus brevis, Gramicidin C from Bacillus brevis, 25 Gramicidin from Bacillus aneurinolyticus (Bacillus brevis), Gramicidin from Bacillus brevis, Herbimycin A from Streptomyces hygroscopicus, Indomethacin, Irgasan, Lomefloxacin hydrochloride, Mycophenolic acid powder, Myxothiazol BioChemika, N-(6-Aminohexyl)-5- chloro-1-naphthalenesulfonamide hydrochloride, Nalidixic acid, Netropsin dihydrochloride hydrate, Niclosamide, Nikkomycin BioChemika, Nikkomycin Z Streptomyces tendae, N- 30 Methyl-1-deoxynojirimycin, Nogalamycin from Streptomyces nogalater, Nonactin □80% from Streptomyces tsusimaensis, Nonactin from Streptomyces sp., Novobiocin sodium salt, Ofloxacin, 18

[0019] Oleandomycin triacetate, Oligomycin Streptomyces diastatochromogenes, Oligomycin A, Oligomycin B, Oligomycin C, Oligomycin Streptomyces diastatochromogenes, Oxolinic acid, Piericidin A from Streptomyces mobaraensis, Pipemidic acid, Radicicol from Diheterospora chlamydosporia solid, Rapamycin from Streptomyces hygroscopicus, Rebeccamycin from 5 Saccharothrix aerocolonigenes, Sinefungin, Staurosporine Streptomyces sp., Stigmatellin, Succinylsulfathiazole, Sulfadiazine, Sulfadimethoxine, Sulfaguanidine purum, Sulfamethazine, Sulfamonomethoxine, Sulfanilamide, Sulfaquinoxaline sodium salt, Sulfasalazine, Sulfathiazole sodium salt, Triacsin C from Streptomyces sp., Trimethoprim, Trimethoprim lactate salt, Vineomycin A1 from Streptomyces albogriseolus subsp., Tectorigenin, and Paracelsin 10 Trichoderma reesei. In a further embodiment the present invention relates to a kit of parts comprising the composition according to the present invention. The kit of parts comprises at least one additional component, such as instructions for use, and / or one or more drugs for co-administration. Such compositions are not limited to particular uses. In some embodiments, the 15 compositions are capable of a static action wherein Ngo or Nme growth is inhibited. In some embodiments, the compositions are capable of a cidal action wherein Ngo or Nme organisms are killed. In some embodiments, the compositions are capable of a lytic action wherein Ngo or Nme organisms are lysed and killed. 20 EXPERIMENTAL Example 1 Methodology Bacterial strains, tissue culture cells and growth conditions 25 Bacteria used in this study are listed in Table 1. Neisseria strains were routinely grown in GCB agar or GC broth with Kellogg’s supplements at 37°C with 5% CO2. E. coli strains were grown in LB agar or broth and incubated at 37°C. Lactobacillus iners was grown in MRS agar. Gardnerella vaginalis was grown on BHI with 5% defibrinated sheep blood. Lactobacillus iners and Gardnerella vaginalis were incubated at 37°C under anaerobiosis using GasPak (BD). When 30 needed kanamycin was added to a final concentration of 50 mg / L, and erythromycin at 10mg / L. 19

[0020] Chromosomal DNA extraction Neisseria elongata (Nel) chromosomal DNA was extracted in a modified according to the protocol described by Kim, 2019(45). Briefly, Nel was grown on GCB agar for 16 h at 37°C / 5% CO2. Bacteria was harvested and lysed in GC lysis buffer, followed by sequential phenol, 5 phenol-chloroform, and chloroform extractions. The DNA was precipitated using cold ethanol and 7.5 M ammonium acetate, then washed 2 times with ice-cold 70% ethanol. DNA pellet was resuspended in 10 mM Tris pH 8. RNA was removed by treatment with RNAse (Thermo Scientific), followed by a second round of phenol-chloroform extraction. The purity of the DNA was assessed by determination of the 260 / 280 and 260 / 230 ratios using a Nanodrop instrument. 10 DNA was considered pure and suitable for killing assay when the ration 260 / 80 was between 1.8 and 1.9 and the ratio 260 / 230 was > 2. Killing assay Neisseria killing assay was done as described by Kim et al, 2019 (45). Briefly, piliated 15 colonies were lawned onto GCB agar and incubated at 37°C / 5% CO2 for 14 h. 5 x 105CFUs were suspended in 250 µl of GCB containing Kellogg’s supplements and added to one well of a 12-well plate. DNA was added to a final concentration of 10 µg / ml. As positive control Nel DNA was used, as negative control a reaction containing 10 mM Tris was performed. Bacteria was incubated for 5 h at 37°C / 5% CO2. Bacteria were recovered, and serial dilutions were 20 performed and plated onto GCB agar to enumerate the surviving bacteria. Bacteria growing in the presence 10 mM Tris were considered to have a 100% survival rate. PCR and Cloning Internal tdfF, tdfH, iga, ngoAXIV, ngoAI, ngoAXV, parC, gyrA, and gyrB fragments were 25 PCR amplified with Phusion-HF (New England Biolabs), primers are listed in Table 2. Each amplicon was purified and cloned into pCR®-Blunt (Life Technologies), ligated products were transformed into E. coli DH5-alpha. The insertion of the fragment was confirmed by digestion with EcoRI, followed by sequencing (Eurofin genomics). Primers used to concatenate tdfF, tdfH and iga, into a single molecule, and ngoAXIV, ngoAI, and ngoAXV, into single molecule, were 30 designed with NEB builder assembly tools (New England Biolabs). Individual tdfF, tdfH and iga, or ngoAXIV, ngoAI, and ngoAXIV were obtained by PCR with Phusion-HF using primers 20

[0021] listed in Table 2. Sequences were concatenated by Gibson assembly (New England Biolabs) following the manufacturer’s instructions. Concatenated products were cloned pCR®-Blunt (Life Technologies) and transformed into E. coli DH5-alpha. Transformants were selected on LB with kanamycin 50 mg / L. The fragment insertion was confirmed by digestion with EcoRI, followed 5 by sequencing (Eurofin genomics). Plasmids created are listed in Table 3. Plasmid extraction Small scale (5 ml) plasmids were extracted using Gene Jet Plasmid kit (Thermo Scientific) following the manufacturer’s instructions. Large quantities of plasmid were extracted 10 by alkaline lysis (79). Briefly, E. coli was harvested from a 100 ml LB culture grown at 37ºC with 250 rpm shaking for ~18 h. Bacterial pellet was resuspended in 10 ml of buffer 1 (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 mg / mL RNase A), then lysed by adding 20 ml of buffer 2 (0.2 M NaOH; 1 % (w / v) SDS), the solution was neutralized with 15 ml of buffer 3 (3 M potassium acetate, pH 5.5). Cell debris were removed by centrifugation. The plasmid was 15 precipitated with ethanol. Pellet was washed 3 times with 70 % ethanol, then dried at room temperature. Plasmid pellet was suspended in 10 mM tris pH 8. RNA was removed by treatment with RNAse (Thermo Scientific), followed by a phenol-chloroform extraction. The purity of the plasmid was assessed by determination of the 260 / 280 and 260 / 230 ratios using a Nanodrop. Plasmid was considered pure and suitable for killing assay when the ration 260 / 80 was between 20 1.8 and 1.88 and the ratio 260 / 230 was > 2. Antibiotic resistance determination Strip test was used to verify antibiotic resistance of the strains. Bacteria were prepared by suspending half of a plate from a 16 h lawn of Ngo grown on GCB agar. Bacteria were adjusted 25 to an OD600 equivalent of a 0.5 McFarland standard. Using a sterile swab, bacteria were spread on the surface of a GCB agar plate. After the plate dried for about 10 min a ciprofloxacin sensidisc (BD, catalog number 231657) was placed on the plate. Plates were incubated at 37°C in 5% CO2 for 20 h. A sensi discs assay was used to determine ciprofloxacin resistance of survivors after the 30 killing assay. A survivor colony was picked with a sterile calcium alginate swab and spread on one quarter GCB agar plate. A sensidisc (BD, catalog number 231657) was placed in the center. 21

[0022] Plates were incubated at 37°C in 5% CO2for 20 h. The diameter of zones of clearance (no- growth zones) was measured in mm. Statistical analysis 5 Statistical differences in killing assay were evaluated using one-way ANOVA with Turkey’s Post Test correction, DNA transformation assay was evaluated using a Student’s T-test using GraphPad Prism 9 software. Example 2 10 Nel DNA kills a variety of Ngo isolates As a first step towards testing the hypothesis that differentially methylated DNA can kill Ngo, it was determined whether commensal Neisseria elongata (Nel) DNA can kill a diverse group of Ngo isolates (Fig. 1) from an STD clinic in Durham, North Carolina, one of which, UNC020, has been shown to lose viability when cocultured with Nel (45), and from the 15 Antibiotic Resistance Isolate Bank (CDC-FDA Antibiotic Resistant Isolate Bank. Atlanta (GA):CDC) (49). The killing assay was performed as described by Kim et al (2019)(45), using 10 µg / ml of purified Nel DNA (see methods). Nel DNA killed 4 of the 5 tested isolates as efficiently as it killed lab strain MS11, the positive control (90-95% cells killed). It did not kill low passage isolate UNC003 (Fig. 1). 20 Example 3 MB 14-1-15 Activity DNA molecules that specifically kill Ngo and not commensals were designed. To this end, DNA molecules were constructed whose sequences are present in Ngo but rare in, or absent 25 from, human-adapted commensal Neisseria (50). Sequences in this group include tdfF (NGFG_RS00105), tdfH (NGFG_RS05315) and iga (NGFG_RS01485). tdfF was found in some commensals; tdfH was present in all closed Neisseria genomes except Nel, but not in draft Neisseria genomes (Table 4); iga was not found in any human commensal species. A second set of DNA molecules whose sequences have homology to Ngo genes essential for DNA replication 30 and maintenance was constructed , reasoning that these DNAs, when attempting to recombine with the essential Ngo genes, will lead to cell death. DNA sequences in this category are cytosine 22

[0023] DNA methyltransferase genes known to be active (46, 51), namely, ngoAXIV (NGFG_RS02014), ngoAI (NGFG_RS11080) and ngoAXV (NGFG_RS03570) (Data set1). Finally, a third group of DNAs was constructed to test the idea that any sequence encoding an essential Neisseria gene can act as a microbicide. These sequences, from genes involved in DNA 5 replication and maintenance, correspond to parC (NGFG_02066), gyrA (NGFG_01171), and gyrB (NGFG_01684). Promoter-less sequences within the open reading frame of each locus, designed to omit start codon and termination signals, and containing DNA Uptake Sequence (DUS) preferred by Ngo, DUS-12 (51) flanking both ends, were PCR-amplified using primers listed in Table 2. The 10 amplicons were cloned into pCR®-Blunt and transformed into E. coli DH5 alpha. The resulting plasmids were used for killing assays. The names of the constructs begin with the letters MB, for MicroBicide, followed by a letter or number that identifies the cloned sequence; for instance, MB-I indicates the DNA molecule contains iga sequences (Table 3, Fig. 2). Individual DNA molecules were also concatenated: constructs MB-FHI (tdfF, tdfH and iga); MB-14-1 (ngoAXVI 15 and ngoAI), and MB-14-1-15 (ngoAXIV, ngoAI and ngoAXV) (see methods). The DNA molecules were tested for their ability to kill Ngo lab strain MS11. The killing efficiency of each DNA was calculated as the number of viable bacteria in the DNA-treated sample divided by the number of viable bacteria in the buffer-treated sample (10 mM Tris pH 8), expressed as a percentage. Nel DNA served as the positive control and Tris buffer alone as the 20 negative control. The killing efficiency of the DNAs varied (Fig. 2). MB-F (tdfF), MB-H (tdfH), and MB-I (iga) killed MS11 at moderate efficiency (22-38%), while the concatenate MB-FHI killed at a higher efficiency (56%). Notably, MB-1, MB-14, MB-15, and the concatenates MB- 14-1 and MB-14-1-15, were as effective in killing MS11 as Nel DNA (>90% efficiency). High killing efficiency was also observed for MB-A (gyrA), MB-B (gyrB) and MB-C (parC). The two 25 concatenates together, MB-FHI and MB-14-1-15 killed MS11 less efficiently than when they were tested alone. MB14-1-15 was used for subsequent studies. The effect of MB14-1-15 was tested on human-adapted commensal Neisseria spp. and two common bacterial inhabitants of the female lower genital tract, Lactobacillus crispatus and Gardnerella vaginalis (Table 5). MB 14-1-15 did not kill any of the commensal Neisseria 30 isolates. By contrast, Nel DNA exhibited a modest killing effect on N. lactamica and N. subflava. The reason for this is not clear. This may be because the methylation patterns of N. lactamica 23

[0024] and N. subflava DNA are more similar to that of Ngo than to Nel DNA. N. lactamica encode between 14 and 17 restriction modification systems (Table 6); at least 6 of those are predicted to methylate cytosines (http: / / rebase.neb.com / rebase / rebase.html). This may explain why Nel DNA has a moderate to high killing effect on N. lactamica. N. subflava encode between 3 and 9 5 putative RM systems. It is contemplated that some of the RM systems present in N. subflava methylate cytosines. MB 14-1-15 DNA did not kill Lactobacillus or Gardnerella. These bacteria do not appear to have a Neisseria like DNA uptake system, and their DNA recombination systems are little understood. They would therefore not take up MB14-1-15. 10 One method for introducing DNA to the potential site of infection, the genital tract, is via a commonly used gel. To explore this, MB-14-1-15 was suspended in two commercially available personal lubricants, the water-based KY Jelly© lubricant and the silicone based Astroglide© lubricant, and tested the formulations for their ability to kill Ngo lab strain MS11. The negative and positive controls are lubricants containing Tris buffer and Nel DNA, 15 respectively (Fig. 3A). MB 14-1-15 in both water- and silicone-based personal lubricants killed MS11 as efficiently as Nel DNA incorporated into the same lubricants. KY jelly© and Astroglide© alone killed a small percentage of MS11; this may be due to the sensitivity of MS11 to the antiseptics in their formulation. These findings indicate that personal lubricants are suitable for use as delivery vehicles for the DNA molecules. 20 MB 14-1-15 incorporated into KY Jelly© , Astroglide© , or Tris buffer were compared for their ability to kill 2 additional lab-adapted Ngo isolates commonly used in the field (FA1090 and FA19), 4 low-passage clinical isolates (UNC012, UNC014, UNC016 and UNC020) from the Durham County, NC, STD clinic, and 4 antibiotic resistant isolates (AR173, AR174, AR200 and AR202) from the CDC and FDA Antibiotic Resistance Isolate Bank (Fig. 3, and Table 1). The 25 positive control was Nel DNA in KY 189 Jelly© , Astroglide© , or Tris buffer, and the negative controls were KY Jell© , Astroglide© , and Tris buffer without DNA. MB 14-1-15 DNA suspended in KY Jelly© and Astroglide© killed all tested isolates as well as, or better than, Nel DNA in the same medium, with an overall efficiency above 95%. KY Jelly© alone did not kill UNC014, and had only a mild negative effect on lab-adapted FA1090 30 and FA19 and low passage isolates UNC012, UNC016, and UNC020 (killing efficiencies ranging from 25% to 35%). It had a greater negative effect on AR174 (~80% killing efficiency). 24

[0025] Astroglide© alone had a similarly mild negative effect on UNC014, UNC020 and AR202; however, it had a much lower negative effect on the other strains (killing efficiencies ranging from 0-15%); it did not kill AR174. Thus, the ability if MB 14-1-15 to kill Ngo is retained in the presence of commercially available lubricants. 5 MB-A, MB-B and MB-C were also tested on a small number of Ngo isolates (Fig. 4), in all cases the killing ability of these microbicides was high (>95%). These microbicides did not kill commensal N. subflava. Neisseria meningitidis (Nme), a close relative of Ngo, occasionally causes genital infections that can lead to septic shock in women, and anogenital infections in men (58, 59). As 10 Nel DNA also kills Nme (45), the ability of MB14-1-15 to kill Nme serogroup C strain 8013 was tested. Nel DNA and Tris buffer without DNA served as the positive and negative controls, respectively. As reported, Nel DNA killed Nme efficiently (82% ± 4.5, P=0.009) (45); MB-14- 1-15 killed Nme even more efficiently (92.9 % 2.6, P<0.0001). This result indicate that MB14- 1-15 can potentially prevent genital infections caused by Nme. 15 Example 4 Recovery of antimicrobial sensitivity To determine whether survivors of DNA killing may have incorporated the incoming DNA into its genome, a killing assay was performed on two isolates, AR173 and AR174 (Fig. 4), 20 that are resistant to the fluoroquinolone ciprofloxacin (Table 7), using MB-A and MB-C, which carry gyrA and parC sequences respectively, that confer ciprofloxacin sensitivity (28, 29). A disk diffusion antimicrobial susceptibility test then performed on random survivor cells. All survivors from the MB-A killing assay were resistant to ciprofloxacin as no zone of clearance was visible around the disc. A small number of survivors from the MB-C killing assay had recovered some 25 sensitivity to ciprofloxacin, a zone of clearance for 3 of 10 AR173 and 3 of 12 AR174 survivor colonies was visible and ranged from 22 to 24 mm (Fig. 5). Sequencing of parC showed that survivor cells had acquired the sensitivity allele from the input DNA molecule, indicating that these bacteria are able to take up MB-C without being harmed. 30 25

[0026] Table 1 Strain Strain Features Reference Lab adapted isolates 26

[0027] * Detailed information on antibiotic resistance can be found at https: / / www.cdc.gov / ARIsolateBank / Panel / PanelDetail?ID=11 Table 1 references 5 1. Meyer T, Mlawer N, So M. 1982. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell 30:42-52. 2. Nachamkin I, Cannon JG, Mittler RS. 1981. Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen. Infect Immun 32:641-8. 10 3. Maness MJ, Sparling PF. 1973. Multiple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae. J Infect Dis 128:321-30. 4. Kim WJ, Higashi D, Goytia M, Rendon MA, Pilligua-Lucas M, Bronnimann M, McLean JA, Duncan J, Trees D, Jerse AE, So M. 2019. Commensal Neisseria Kill Neisseria gonorrhoeae through a DNA-Dependent Mechanism. Cell Host Microbe 26:228-239 e8. 15 5. Liu H, Vidyaprakash E, Schmerer MW, Pham DC, St Cyr S, Kersh EN. 2020. A Culture Collection of 50 Neisseria gonorrhoeae Isolates. Microbiol Resour Announc 9. 6. Nassif X, Lowy J, Stenberg P, O'Gaora P, Ganji A, So M. 1993. Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells. Mol Microbiol 8:719-25. 20 7. Marri PR, Paniscus M, Weyand NJ, Rendón MA, Calton CM, Hernandez DR, Higashi DL, Sodergren E, Weinstock GM, Rounsley SD, So M. 2010. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PLoS One 5:e11835. 25 30 27

[0028] Table 2 5 28

[0029] Table 3 Ta 5 IJ9

[0030] Table 5. MB 14-1-15 DNA does not harm commensals. A 479 negative value indicates bacteria in the test sample grew more than bacteria in the negative 5 480 control sample. Table 6 30

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Claims

CLAIMS We claim: 5 1. A composition comprising a nucleic acid comprising at least one DNA uptake sequence (DUS) and at least one nucleic acid encoding at least a portion of a protein essential for viability of a pathogenic Neisseria, wherein said protein is selected from the group consisting of GyrA, GyrB and ParC. 10 2. The composition of claim 1, wherein said nucleic acid further comprises a nucleic acid encoding one or more additional proteins essential for viability of a pathogenic Neisseria.

3. The composition of claim 1 or 2, wherein said pathogenic Neisseria is Neisseria 15 gonorrhoeae (Ngo) or Neisseria meningitidis (Nme).

4. The composition of claim 2, wherein said one or more additional nucleic acids encode DNA methyltransferases and / or DNA replication and maintenance proteins. 20 5. The composition of claim 4, wherein said DNA methyltransferases are selected from the group consisting of NgoAXIV, NgoAI, and NgoAXV.

6. The composition of claim 5, wherein said DNA methyltransferases are NgoAXIV, NgoAI, and NgoAXV. 25 7. The composition of claim 6, wherein said NgoAXIV, NgoAI, and NgoAXV are present as a concatemer.

8. The composition of claim 2, wherein said one or more additional nucleic acids 30 encode a protein selected from the group consisting of TdfF, TdfH, and Iga.

399. The composition of any one of the preceding claims, wherein said nucleic acid is selected from the group consisting of a plasmid, a linear DNA molecule, a bacterial artificial chromosome, and genomic DNA. 5 10. The composition of any one of the preceding claims, wherein said gyrA, gyrB and parC nucleic acids have a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and sequences at least 90% homologous or complementary to SEQ ID NOs: 1-3.

11. The composition of any one of the preceding claims, wherein said gyrA, gyrB and 10 parC nucleic acids have a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

12. The composition of any one of the preceding claims, wherein said gyrA, gyrB and parC nucleic acids are coding and / or non-coding portions of gyrA, gyrB and parC genes. 15 13. The composition of any one of the preceding claims, wherein said DUS has the sequence N1N2N3N4N5N6N7CTGN8A (SEQ ID NO:4), wherein N1is A or T, N2is T, G, or A, N3is G or C, N4 is C or T, N5 is C, T, or A, N6 is G or A, N7 is T or C, and N8 is C or A. 20 14. The composition of claim 13, wherein said DUS has the sequence A[T / G]GCCGTCTGAA (SEQ ID NO:5) or GCCGTCTGAA (SEQ ID NO:6).

15. The composition of any one of the preceding claims, wherein said nucleic acid has a different methylation pattern than said pathogenic Neisseria. 25 16. The composition of any one of the preceding claims, wherein said composition is a pharmaceutical composition.

17. The composition of claim 16, wherein said composition is a personal lubricant. 30 4018. A method for the inhibition of bacterial growth and / or for the killing of a target bacterium, comprising the step of adding to the target bacterium the composition of any one of claims 1 to 17, thereby inhibiting bacterial growth and / or killing said bacteria. 5 19. The method of claim 18, wherein said composition is administered topically.

20. The method of claim 18 or 19, wherein said composition does not kill and / or inhibit the growth of commensal species of Neisseria. 10 21. The method of any of claims 18 to 20, wherein said composition sensitizes a target bacterium to an antibiotic.

22. The use of the composition of any one of claims 1 to 17 to inhibit bacterial growth and / or kill a target bacterium. 15 23. The composition of any one of claims 1 to 17 for use in inhibiting bacterial growth and / or to kill a target bacterium. 20 41

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