Patent Description:
The content of the ASCII text file of the sequence listing named "<NPL> and electronically submitted via EFS-Web.

This invention was made with Government support under Grant Number AI117256, awarded by the National Institutes of Health. The Government has certain rights in the invention.

The field generally relates to predicting whether a Neisseria species is susceptible to cefixime compounds and treating infections by Neisseria species with cefixime compounds.

Neisseria (N. ) gonorrhoeae has become resistant to most if not all available antibiotics. Cefixime, a third-generation oral cephalosporin, is a highly effective and safe single dose treatment for susceptible cases of gonorrhea. However, in the last two decades, gonococcal strains with decreased susceptibility to cefixime and cases of clinical treatment failure with cefixime have been reported worldwide.

As the organism has developed resistance to multiple classes of antibiotics such as sulfas, penicillins, tetracyclines, fluoroquinolones, and macrolides, the third-generation extended-spectrum cephalosporins, like cefixime, are among the few reliable efficacious treatment options left. Cefixime is a highly useful antibiotic used for the treatment of gonorrhea. gonorrhoeae remains susceptible to cefixime in most but not all countries. Currently, the World Health Organization (WHO) recommends cefixime, in combination with azithromycin, as dual therapy for oropharyngeal, genital, and anorectal gonococcal infection. In settings where local resistance data confirm cefixime susceptibility, the WHO recommends cefixime in a single dose for genital and anorectal gonococcal infection. In the United States, the Centers for Disease Control and Prevention also recommends cefixime, in combination with azithromycin, as an alternative regimen where ceftriaxone is not available. In the United Kingdom, oral cefixime in combination with azithromycin is also recommended in penicillin-allergic patients for whom intramuscular injection is contraindicated or refused. Cefixime has a serum half-life of <NUM> to <NUM> hours in patients with normal renal function, high bioavailability after a single oral dose and is very well tolerated even in penicillin allergic patients. However, in the last two decades, various investigators have reported cases of N. gonorrhoeae infection with strains that have decreased susceptibility to cefixime. Furthermore, in the past <NUM> years, cases of N. gonorrhoeae treatment failure in patients treated with cefixime have also been reported in Japan, Norway, UK, South Africa, France, Australia, and Canada. Various research teams and governmental institutions have expressed the need for more research on the mechanisms of cefixime resistance and the development of new tools to predict cefixime susceptibility.

In some embodiments, the present invention provides an in vitro method for inhibiting the growth of a given Neisseria species, which comprises.

In some embodiments, the present invention provides an in vitro method for characterizing whether a given Neisseria species is susceptible to a cefixime compound, which comprises.

In some embodiments, the given Neisseria species may be a Neisseria gonorrhoeae strain.

In some embodiments, the cefixime compound may be cefixime.

In some embodiments, the antibacterial other than cefixime compounds may be selected from the following groups of antibiotics: Sulfonamides, Tetracyclines, Aminoglycosides, Macrolides, Ketolides, Quinolones, Lincomycins, and Glycopeptides.

Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

This invention is further understood by reference to the drawings wherein:
<FIG> graphically summarizes cefixime MIC of N. gonorrhoeae strains by different combinations of cefixime decreased susceptibility-related penA amino acid alterations (amino acid alterations encoded by the penA gene of the given strain). Minimum inhibitory concentrations are on the y-axis. Combinations of penA amino acid alterations associated with cefixime decreased susceptibility are on the x-axis; different colors indicate different mutation combinations.

The penicillin-binding protein <NUM> (PBP2) encoded by penA gene is the primary target of cefixime antimicrobial activity. Neisseria gonorrhoeae strains having a cefixime minimum inhibitory concentration (MIC) ≥ <NUM>µg/mL are significantly more likely to fail treatment with cefixime than strains with an MIC < <NUM>µg/ml. Therefore, as provided herein, Neisseria gonorrhoeae strains having a cefixime MIC ≥ <NUM>µg/mL are characterized as having a decreased susceptibility (i.e., resistance) to cefixime and strains having a cefixime MIC < <NUM>µg/mL are characterized as being susceptible to cefixime.

Disclosed herein are genetic profiles of Neisseria gonorrhoeae strains that exhibit resistance to cefixime as determined from a literature review and analysis of PBP2 and penA contributing to cefixime resistance in N. gonorrhoeae. As disclosed herein, either of the following patterns of alterations lead to cefixime resistance: <NUM>) Mosaic penA types (e.g., alterations in the amino acid region <NUM>-<NUM> of wildtype PBP2 (Accession No. M32091. <NUM>)) having characteristic polymorphisms I312M, V316T, N512Y, G545S, and <NUM>) Non-mosaic penA types having any combination of amino acid alterations at amino acid positions A501, G542, and P551 of wildtype PBP2.

In some embodiments, the present invention is directed to in vitro methods for characterizing whether a given Neisseria species, such as a given Neisseria gonorrhoeae strain, is susceptible to a cefixime compound, e.g., cefixime, which comprises (A) determining the presence or absence of one or more amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> of its PBP2, and (B1) characterizing the given Neisseria species as being resistant to the cefixime compound where one or more alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> are present, or (B2) characterizing the given Neisseria species as being susceptible to the cefixime compound where amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> are absent.

In some embodiments, the present invention is directed to in vitro methods for characterizing whether a given Neisseria species, such as a given Neisseria gonorrhoeae strain, is susceptible to cefixime, which comprises (A) determining the presence or absence of one or more amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> of its PBP2, and (B <NUM>) characterizing the given Neisseria species as being resistant to cefixime where one or more alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> are present, or (B2) characterizing the given Neisseria species as being susceptible to cefixime where amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> are absent.

In some embodiments, the present invention is directed to in vitro methods for characterizing whether a given Neisseria species, such as a given Neisseria gonorrhoeae strain, is susceptible to cefixime, which comprises (A) determining the presence or absence of one or more amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> of its PBP2, and (B <NUM>) characterizing the given Neisseria species as being resistant to cefixime where one or more amino acid alterations at amino acid positions <NUM>, <NUM>, and <NUM> are present, (B2) characterizing the given Neisseria species as being resistant to cefixime where one or more amino acid alterations at amino acid positions <NUM>-<NUM> and one or more amino acid alterations at amino acid positions <NUM>, <NUM>, <NUM>, and <NUM> are present, or (B3) characterizing the given Neisseria species as being susceptible to cefixime where one or more amino acid alterations at amino acid positions <NUM>, <NUM>, and <NUM> are absent, and a combination of (a) one or more amino acid alterations at amino acid positions <NUM>-<NUM> and (b) one or more amino acid alterations at amino acid positions <NUM>, <NUM>, <NUM>, and <NUM> is also absent.

In some embodiments, at least one of the amino acid alterations is an amino acid substitution. In some embodiments, the amino acid substitution is A311V, A376X, A501P, A501R, A501S, A501T, A501V, E377X, G375X, G542S, G545S, I312M, N512Y, P551A, P551G, P551L, P551S, T483S, V316P, or V316T, wherein each X are independently any amino acid residue. In some embodiments, the amino acid insertion is an insertion after or before the residue at amino acid position <NUM>. In some embodiments, the amino acid insertion is Asp inserted after or before amino acid position <NUM> (herein denoted as "345insD") based on the wildtype PBP2 sequence. In some embodiments, the one or more amino acid alterations is 345insD; N512Y; 345insD and A501T; 345insD and A501V; 345insD and G542S; 345insD and P551A; 345insD and P551L; 345insD and P551S; I312M and V316T; 345insD, A501S, and G542S; 345insD, A501T, and G542S; 345insD, A501T, and P551L; 345insD, A501V, and G542S; 345insD, A501V, and P551A; 345insD, A501V, and P551G; 345insD, A501V, and P551L; 345insD, A501V, and P551S; N512Y, G545S, and P551A; T483S, N512Y, and G545S; 345insD, A501S, G542S, and P551S; 345insD, A501V, N512Y, and P551S; I312M, V316P, N512Y, and G545S; I312M, V316T, N512Y, and G545S; V316T, N512Y, G545S, and P551A; A311V, I312M, V316P, N512Y, and G545S; A311V, I312M, V316T, N512Y, and G545S; I312M, V316T, A501P, N512Y, and G545S; I312M, V316T, A501R, N512Y, and G545S; I312M, V316T, A501S, N512Y, and G545S; I312M, V316T, A501T, N512Y, and G545S; I312M, V316T, A501V, N512Y, and G545S; I312M, V316T, N512Y, G545S, and P551S; I312M, V316T, T483V, N512Y, and G545S; A311V, I312M, V316P, T483S, N512Y, and G545S; or A311V, I312M, V316T, T483S, N512Y, and G545S. In some embodiments, the one or more amino acid alterations comprise at least one of G375X, A376X, and E377X, wherein each X are independently any amino acid, and at least one of A311V, A501P, A501R, A501S, A501T, A501V, G542S, G545S, I312M, N512Y, P551A, P551G, P551L, P551S, T483S, V316P, V316T, and 345insD.

As provided herein, amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> of the PBP2 of a given Neisseria species are based on an optimal alignment of the PBP2 sequence of the given Neisseria species with the wildtype PBP2 sequence (Accession No. AAA25463. <NUM> (SEQ ID NO: <NUM>) (which is encoded by Accession No. M32091. <NUM>)) and using the amino acid numbering of the wildtype PBP2 sequence. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of <NPL>), by the homology alignment algorithm of <NPL>), by the search for similarity method of <NPL>), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, <NUM> Science Dr. , Madison, WI), or by visual inspection.

In some embodiments, the presence or absence of any one or more of the above amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> of the PBP2 protein of the given Neisseria species (as compared to the wildtype PBP2) are determined by assaying the penA gene of the given Neisseria species for mutations that result in the amino acid alterations.

The sensitivity of assay methods for characterizing whether a given Neisseria gonorrhoeae strain is susceptible to cefixime based on the absence of an amino acid alteration in amino acid positions <NUM>-<NUM> combined with an amino acid alteration at amino acid positions <NUM>, <NUM>, <NUM>, and <NUM> of its PBP2 as compared to the wildtype sequence is predicted to be at least about <NUM>%.

Articles published on PubMed from January <NUM>, <NUM> to November <NUM>, <NUM> were searched using the search terms, "Neisseria gonorrhoeae", "cefixime", and "molecular" and the relevant hits were reviewed. A total of <NUM> articles from that search were identified. Articles that presented epidemiological or experimental evidence of certain molecular alterations contributing to cefixime decreased susceptibility in N. gonorrhoeae totaled <NUM> reports. gonorrhoeae strains with reported MIC of ≥ <NUM>µg/mL and specific penA alterations are included in Table <NUM> along with their specific MIC plus the time and location of collection.

The nomenclature of penA reflects the differences in amino-acid sequence rather than nucleotide sequence. Historically, each new amino-acid sequence gets a sequential whole number, and is classified into mosaic, semi-mosaic (alterations of either the first or second half of the penA gene only), non-mosaic (point mutations only), and wild-type (penA peptide sequence identical to that of the N. gonorrhoeae reference strain M32091). Each different DNA sequence of an existing amino acid sequence gets a decimal number. For example, the 8th DNA sequence reported for penA allele type <NUM> is assigned allele number penA2. Based on an analysis of the literature in the art, there is conflicting nomenclature for the same penA peptide sequence (see Table <NUM>); likely due to the lack of a single, centralized database requiring new submissions of sequences to be compared with existing entries and subsequently given appropriate designations.

There was a general lack of consensus in the standard style of nucleotide/amino acid sequence reporting. Many sequences reported were truncated, leading to incomplete information that hindered data interpretation. To prevent misclassification, penA sequences from each research article were verified against the penA profiles from NG-STAR, a centralized, comprehensive, and publicly accessible database for standardized characterization of molecular alterations in N. gonorrhoeae worldwide. Multiple peptide sequence alignments of the penA sequences using the Multiple Sequence Alignment tool by Clustal Omega (https://www. uk/Tools/msa/clustalo/) were conducted.

Table <NUM> provides the penA profiles of N. gonorrhoeae strains that exhibit decreased susceptibility to cefixime:.

A parsimonious group of penA amino acid locations to predict decreased susceptibility to cefixime in N. gonorrhoeae strains was proposed. The sensitivity of a hypothetical assay for predicting decreased susceptibility using those locations was estimated by calculating the number of isolates with genotypic mutations in those locations divided by the number of phenotypically decreased susceptible isolates using the data summarized in Table <NUM>.

Table <NUM> shows a list of penA types from the NG-STAR database and the presence or absence of specific gene alterations and associated amino acid changes that predict cefixime decreased susceptibility. Decreased susceptibility to cefixime has been associated with many genetic alterations in the penA gene. There is strong laboratory and epidemiological research supporting mosaicism and other point mutations of the penA gene as mechanisms for cefixime decreased susceptibility by means of PBP2 target alteration. Evidence supporting the importance of alterations in other genes are not as compelling. However, there is evidence that isolates with identical penA alleles could have different MIC levels (susceptible with MIC < <NUM>µg/mL versus highly resistant with MIC ≥ <NUM>µg/mL), indicating the involvement of other genes in decreasing susceptibility to cefixime. Several candidate genes impacting decreased N. gonorrhoeae susceptibility include mtrR, the transcriptional regulator for the MtrCDE efflux pump system; ponA, encoding for penicillin binding protein <NUM> (PBP1); pilQ, encoding for the type IV pilus secretin; and penB (alias: porB1b), encoding for a major outer membrane protein porB, a porin.

Mosaicism of the penA gene in N. gonorrhoeae was first described by Ameyama et al. , who found that some penA nucleotide sequences of N. gonorrhoeae contained portions that highly resemble those of other non-pathogenic or commensal Neisseria species, such as N. perflava, N. cinerea, N. flavescens, and N. meningitidis. As used herein, penA mosaicism refers to a penA gene of a given Neisseria species that comprises a multitude of nucleotide and amino acid changes thought to be acquired from the penA gene of another by transformation or conjugation and the spontaneous generation of mutations. See, e.g., <CIT>.

Mosaicism in penA was found to be the primary determinant of cefixime decreased susceptibility by many studies. From an aggregate of <NUM> N. gonorrhoeae isolates with an MIC ≥ <NUM>µg/mL from <NUM> reports representing <NUM> countries, <NUM>% (<NUM> out of <NUM>) were found to have mosaic penA genes (Table <NUM>). Among all the different mosaic patterns, penA10 and penA34 were the most frequently reported mosaic penA patterns of all isolates with mosaic penA gene mutations among N. gonorrhoeae strains with reduced cefixime susceptibility (accounting for <NUM>% (<NUM> out of <NUM>) and <NUM>% (<NUM> out of <NUM>), respectively). While penA34 was found worldwide, penA10 was mostly found in Asia, and was also associated with resistance to, and treatment failure by, another third-generation cephalosporin, ceftibuten.

<NUM>, V316T, N512Y, and G545S amino acid substitutions are frequently seen in mosaic penA patterns. Researchers have found that amino acid substitutions I312M, V316T/P, and G545 are associated with reduced cefixime susceptibility. However, a gene transformation study showed that when introduced into a wild-type penA, I312M, V316T and G545S together only minimally elevated the cefixime MIC. The reversion of the triple mutation in a cefixime-resistant strain <NUM>/<NUM> (with penA10) back into wild-type returned its MIC to that similar to the wild-type penA MIC, indicating that these three mutations are important to cefixime resistance only in the context of other mutations found in mosaic penA10 alleles.

Chimeric penA genes were created by replacing sequential portions of pen A10 by the corresponding regions of a wild-type penA gene. Reversion of amino acid regions <NUM>-<NUM> (containing I312M and V316T and <NUM> other substitutions), <NUM>-<NUM> (containing N512Y and <NUM> other mutations) and <NUM>-<NUM> (containing G545S and <NUM> other substitutions), showed significant decrease in MIC. Among the three, the reversion of region <NUM>-<NUM> decreased the MIC to such a significant degree that the chimera could not be selected despite multiple attempts, indicating mutations in this region may be critical factors in influencing cefixime susceptibility. When the G545S substitution was re-introduced to the <NUM>-<NUM>-wild-type penA10, the resulting strain's cefixime MIC only rose to <NUM>µg/mL, suggesting that other mutations in the <NUM>-<NUM> region were necessary to significantly elevate the cefixime MIC to > <NUM>µg/mL.

The reversion of region <NUM>-<NUM> (containing N512Y and <NUM> other mutations) decreased the MIC from about <NUM>µg/mL to about <NUM>µg/mL. Y512N reversion alone presented a decreased MIC from about <NUM>µg/mL to about <NUM>µg/mL. Accounting for most of the effect on cefixime resistance by mutations in the <NUM>-<NUM> region, N512Y showed major importance in conferring cefixime resistance in the context of other mosaic changes.

In summary, I312M, V316T, N512Y, and G545S were found to be important to cefixime decreased susceptibility or resistance only in the context of other mutations found in mosaic penA alterations. All of them were associated with but none was necessary or sufficient for cefixime decreased susceptibility or resistance.

Although the penA34 mosaicism was present in <NUM>% of all isolates with an MIC ≥ <NUM>µg/mL in a study on more than <NUM> N. gonorrhoeae isolates collected in the United States, the percentage lowered to <NUM>% when a lower, and more clinically relevant MIC breakpoint (≥ <NUM>µg/mL), was used. That indicates the role of other non-penA34 mutations, most notably, other amino acid substitutions in the penA gene. In the last decade, more than a dozen cefixime-resistant N. gonorrhoeae strains reported in Asia and Europe were found to have non-mosaic penA alleles (Table <NUM>). Among reports from Asia, over <NUM>% of N. gonorrhoeae strains with decreased susceptibility to cefixime had non-mosaic penA mutations. Gene transformation experiments have identified multiple, important amino acid substitutions in non-mosaic penA that significantly decrease cefixime susceptibility.

Three amino acid substitutions, A501 S/V/T/P, G542S, and P551 S/L/P, have been associated with cefixime decreased susceptibility independent from penA mosaicism. An independent A501 substitution potentially decreased cefixime susceptibility by increasing the rigidity of PBP2 active site.

Table <NUM> summarizes the cefixime decreased susceptible related penA amino acid alterations in all N. gonorrhoeae strains with cefixime MIC ≥ <NUM>µg/mL. One important finding is that <NUM> out of <NUM> (<NUM>%) non-mosaic penA strains with reduced susceptibility to cefixime have point mutation in at least one of the three codons, A501, G542, and P551. Additionally, decreased susceptible N. gonorrhoeae strains with non-mosaic penA have mostly been reported in Asia and Europe.

<FIG> summarizes the MIC levels of all N. gonorrhoeae strains with cefixime MIC ≥ <NUM>µg/mL by different combinations of decreased susceptible related penA amino acid alterations (n = <NUM>). Strains lacking specific cefixime MIC value or penA alteration records (n = <NUM>) were excluded.

mtrR is a repressor gene that regulates the expression of the MtrCDE efflux pump system, an important mechanism in transporting antimicrobial agents out of the bacterial cell. Changes in the promoter or coding sequence of the mtrR gene can potentially decrease antimicrobial susceptibility by increased efflux. There are conflicting reports on the importance of the mtrR gene in cefixime decreased susceptibility. Mutations frequently found in strains with cefixime decreased susceptibility include a -35A deletion in the promoter region, plus A39T and G45D in the coding region. No reports about gene transformation studies looking at the contributions of mtrR alterations to cefixime resistance independent from penA changes were found in the art. Nonetheless, other studies report that mutations in mtrR gene have little or no association with cefixime susceptibility.

penB, also known as porB1b, encodes for an outer membrane porin and is thought to increase penicillin resistance by changing the bacterial membrane permeability to certain antibiotics when penA and mtrR mutations are also present, although its role in cefixime resistance is unclear. Alterations found in strains with cefixime decreased susceptibility include G120K and A121N/D substitutions. While a study in the United States suggested no correlation between cefixime decreased susceptibility and G120K or G120D/A121D, the lack of G120K and A121N mutations strongly predicted susceptibility.

Two other mutations in penB gene commonly found in cefixime decreased susceptible strains are G101K/D and A102D/N/S. Combination of mutations at those <NUM> sites were found in <NUM> N. gonorrhoeae strains having decreased susceptibility to cefixime.

No evidence that alterations in penB gene alone can confer decreased susceptibility to cefixime was found in the prior art.

ponA encodes for penicillin binding protein <NUM> (PBP1), an additional cell wall protein important in beta-lactam antibiotic antimicrobial activity. Alterations in ponA gene were associated with penicillin resistance by PBP1 target mutation, although its role in cefixime decreased susceptibility is unclear. One mutation, L421P, was frequently found in cefixime decreased susceptible strains, but a gene transformation study showed that a ponA L421P substitution does not contribute additional decreased susceptibility to cefixime without a mosaic penA gene.

pilQ encodes for a type IV pili secretin. Mutations in the pilQ gene are thought to increase penicillin resistance by changing the bacterial membrane permeability when penA, mtrR or penB mutations are also present, although its role in cefixime resistance is also unclear.

While one study concluded that changes in the pilQ gene are unlikely associated with cefixime decreased susceptibility, another study found that a <NUM>-<NUM> deletion (vs. full length), N341S, D526N/G, or N648S each strongly predicted N. gonorrhoeae susceptibility to cefixime. Notably, among those four mutations, N648S was the only mutation that was found to be relatively common (in <NUM>% of all isolates compared to ≤ <NUM>% for any of the other three).

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

As used herein, "cefixime compounds" refer to compounds having the following structural formula (I) as part of its backbone structure:
<CHM>
Exemplary cefixime compounds include Cefacetrile, Cefaclor, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloram, Cefaloridine, Cefalotin, Cefaparole, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefbuperazone, Cefcanel, Cefcapene, Cefclidine, Cefdaloxime, Cefdinir, Cefditoren, Cefedrolor, Cefempidone, Cefepime, Cefetamet, Cefetrizole, Cefivitril, Cefixime, Cefluprenam, Cefmatilen, Cefmenoxime, Cefmepidium, Cefmetazole, Cefmetazole, Cefminox, Cefodizime, Cefonicid, Cefoperazone, Cefoselis, Cefotaxime, Cefotetan, Cefotetan, Cefotiam, Cefovecin, Cefoxazole, Cefoxitin, Cefoxitin, Cefozopran, Cefpimizole, Cefpirome, Cefpodoxime, Cefprozil, Cefquinome, Cefradine, Cefrotil, Cefroxadine, Cefsumide, Ceftamere, Ceftaroline, Ceftazidime, Cefteram, Ceftezole, Ceftibuten, Ceftiofur, Ceftiolene, Ceftioxide, Ceftizoxime, Ceftobiprole, Ceftolozane, Cefuracetime, Cefuroxime, Cefuzonam, Cephamycin, Flomoxef, Latamoxef, Loracarbef, Nitrocefin, Oxacephems, and the like. In some embodiments, the cefixime compounds are third generation cephalosporins such as Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefoperazone, Cefotaxime, Cefovecin, Cefpimizole, Cefpodoxime, Ceftamere, Ceftazidime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, and Latamoxef. In some embodiments, the cefixime compound is Cefixime or Ceftriaxone, preferably Cefixime. Thus, antibacterials that are not cefixime compounds lack structural formula (I) as part of its backbone structure, such antibacterials included Sulfonamides, Tetracyclines, Aminoglycosides, Macrolides, Ketolides, Quinolones, Lincomycins, and Glycopeptides.

As used herein, the terms "subject", "patient", and "individual" are used interchangeably to refer to humans and non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

As used herein, the term "diagnosing" refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the diagnosis. Similarly, "providing a prognosis" refers to the physical and active step of informing, i.e., communicating verbally or by writing (on, e.g., paper or electronic media), another party, e.g., a patient, of the prognosis.

The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms "a", "an", and "the" can include plural referents unless the context clearly dictates otherwise.

As used herein, "and/or" means "and" or "or". For example, "A and/or B" means "A, B, or both A and B" and "A, B, C, and/or D" means "A, B, C, D, or a combination thereof" and said "A, B, C, D, or a combination thereof" means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

As used herein, the phrase "one or more of", e.g., "one or more of A, B, and/or C" means "one or more of A", "one or more of B", "one or more of C", "one or more of A and one or more of B", "one or more of B and one or more of C", "one or more of A and one or more of C" and "one or more of A, one or more of B, and one or more of C".

Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence "In some embodiments, the composition comprises A, B, or C" is to be interpreted as if written as the following three separate sentences: "In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C. " As another example, the sentence "In some embodiments, the composition comprises at least A, B, or C" is to be interpreted as if written as the following three separate sentences: "In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C.

Claim 1:
An in vitro method for inhibiting the growth of a given Neisseria species, which comprises
(A) determining the presence or absence of amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> in the penicillin-binding protein <NUM> (PBP2) of the given Neisseria species;
(B) characterizing the given Neisseria species as being susceptible to cefixime compounds where amino acid alterations at amino acid positions <NUM>, <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> are absent; and
(C) contacting the given Neisseria species with a cefixime compound where the given Neisseria species is characterized as being susceptible to cefixime compounds, and contacting the given Neisseria species with an antibacterial other than cefixime compounds where the given Neisseria species is characterized as being resistant to cefixime compounds.