Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility

Additional keywords: antimicrobial resistance, antimicrobial stewardship.

Neisseria (N.) gonorrhoeae, is the world’s second most prevalent sexually transmissible bacterial infection.¹ However, N. gonorrhoeae is a naturally competent organism that can acquire new genes from the microbial environment.² Those newly acquired genetic variations can render the organism less susceptible, or even resistant, to antimicrobial therapy.²

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.³ Cefixime is a highly useful antibiotic used for the treatment of gonorrhoea. N. gonorrhoeae remains susceptible to cefixime in most but not all countries.⁴ Currently, the World Health Organization (WHO) recommends cefixime, in combination with azithromycin, as dual therapy for oropharyngeal, genital and anorectal gonococcal infection.⁵ 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 USA, the Centers for Disease Control and Prevention also recommends cefixime, in combination with azithromycin, as an alternative regimen where ceftriaxone is not available.⁶ In the UK, oral cefixime in combination with azithromycin is also recommended in penicillin-allergic patients for whom intramuscular injection is contraindicated or refused.⁷ Cefixime has a serum half-life of 3–4 h in patients with normal renal function, high bioavailability after a single oral dose and is very well tolerated even in penicillin-allergic patients.⁶ However, in the last two decades, various investigators have reported cases of N. gonorrhoeae infection with strains that have decreased susceptibility to cefixime.^8–30 Furthermore, in the past 10 years, cases of N. gonorrhoeae treatment failure in patients treated with cefixime have also been reported in Japan,³¹ Norway,³² UK,^33,34 South Africa,²⁸ France,⁹ Australia³⁵ and Canada.²⁴ 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.^36–38

One target protein that is critical for the antimicrobial activity of many β-lactam antibiotics, including cefixime, is the penicillin-binding protein 2 (PBP2), which is a protein essential for the development of bacterial cell walls.³⁹ PBP2 is a transpeptidase encoded by the penA gene. Mosaicism of the penA gene in N. gonorrhoeae was first described by Ameyama et al.⁴⁰ 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. penA mosaicism, along with other point mutations in penA, helps N. gonorrhoeae to develop resistance against extended-spectrum cephalosporins like cefixime.^{14–27,41–43}

In this review, we describe molecular mechanisms of cefixime-decreased susceptibility in N. gonorrhoeae, summarise findings from published reports of various gene mutations contributing to that decreased susceptibility and make suggestions on how to develop a molecular-based cefixime susceptibility assay.

Historically, defining decreased susceptibility or resistance of N. gonorrhoeae to cefixime has been challenging due to the scarcity of treatment failure cases.³⁷ The most up-to-date recommendations of antimicrobial minimum inhibitory concentration (MIC) breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) in the USA are as follows:

EUCAST: susceptible ≤0.125 µg mL⁻¹; resistant >0.125 µg mL⁻¹.⁴⁴

CLSI: susceptible ≤ 0.25 µg mL⁻¹; intermediate: not applicable; resistant: not applicable.⁴⁵

However, those recommendations might be too liberal when we compare those breakpoints to reported cases of clinical treatment failure. Allen et al. reported that the frequency of treatment failure with cefixime for gonococcal infections with an MIC ≥0.12 µg mL⁻¹ was 25%, compared with 1.9% among those with an MIC <0.12 µg mL⁻¹.²⁴ For this review, we have described the molecular characteristics of all N. gonorrhoeae strains reported, to date, with a MIC ≥0.12 µg mL⁻¹ as having a ‘decreased susceptibility’ to cefixime.

One author (X. Deng) searched all articles published on PubMed from 1 January 1995 to 1 November 2018 under the search terms ‘Neisseria gonorrhoeae’, ‘cefixime’ and ‘molecular’ and reviewed relevant articles cited as references. We identified a total of 74 articles from that search. We included articles that presented epidemiological or experimental evidence of certain molecular alterations contributing to cefixime-decreased susceptibility in N. gonorrhoeae; there was a total of 25 reports. All N. gonorrhoeae strains with a reported MIC ≥0.12 µg mL⁻¹ and specific penA alterations associated with cefixime decreased susceptibility were included in Table 1, along with their specific MIC plus the time and location of collection.

Table 1.  Summary of penA alterations in reported gonococcal strains with a cefixime minimum inhibitory concentration of ≥0.12 µg mL^−1A
CFX, cefixime; WHO, World Health Organization

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 only²⁷), non-mosaic (point mutations only) and wild-type (penA peptide sequence identical to that of the N. gonorrhoeae reference strain, M32091^49,50). Each different DNA sequence of an existing amino acid sequence gets a decimal number;^27,28 for example, the eighth DNA sequence reported for penA allele type 2 is assigned allele number penA2.008. Our review has exposed conflicting nomenclature for the same penA peptide sequence (Table 2); likely due to the lack of a single, centralised database requiring new submissions of sequences to be compared with existing entries and subsequently given appropriate designations. We also noticed a general lack of consensus in the standard style of nucleotide or amino acid sequence reporting. Many sequences reported were truncated, leading to incomplete information that hindered data interpretation. We highly recommend future researchers to submit complete gene sequencing data to GenBank (https://www.ncbi.nlm.nih.gov/genbank/), which is supported by the National Center for Biotechnology Information (NCBI) and N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR, https://ngstar.canada.ca/), which is supported by the Government of Canada, for easy reference. To prevent misclassification in this manuscript, we further verified penA sequences from each research article against the penA profiles in NG-STAR, a centralised, comprehensive and publicly accessible database for standardised characterisation of molecular alterations in N. gonorrhoeae worldwide.²⁷ One author (X. Deng) conducted all the multiple peptide sequence alignment using the Multiple Sequence Alignment tool by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Table 2.  penA gene types with conflicting nomenclature
NG- STAR, Neisseria gonorrhoeae sequence typing for antimicrobial resistance

We proposed a parsimonious group of penA amino acid locations to predict decreased susceptibility to cefixime in N. gonorrhoeae strains. We estimated the sensitivity of a hypothetical assay for predicting decreased susceptibility using those locations by calculating the number of isolates with genotypic mutations in those locations divided by the number of phenotypically decreased susceptible isolates using the data summarised in Table 1.

Table 3 shows a list of penA types from the NG-STAR database and the presence or absence of amino acid alterations associated with 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^14,40 and other point mutations^19,47,48,51 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 an MIC <0.125 µg mL⁻¹ vs highly resistant with an MIC ≥ 0.5 µg mL⁻¹), indicating the involvement of other genes in decreasing susceptibility to cefixime.²⁷ Several candidate genes contributing to cefixime decreased susceptibility include mtrR, the transcriptional regulator for the mtrCDE efflux pump system;⁵² ponA, encoding for penicillin-binding protein 1 (PBP1);⁵³ pilQ, encoding for the type IV pilus secretin;⁵⁴ and penB (alias: porB1b), encoding for a major outer membrane protein, porB, a porin.⁵⁵

Table 3.  penA types: mosaicism and amino acid alterations associated with cefixime-decreased susceptible Neisseria gonorrhoeae infections
The one-letter abbreviation of amino acid indicates the substitution of the amino acid described in the header row. The lack of a one-letter abbreviation indicates that the position contains the wild-type amino acid. The x indicates the insertion of an aspartate at amino acid 345 position.

Mosaicism in penA was found to be the primary determinant of cefixime-decreased susceptibility by many studies.^{14–27,41–43} From an aggregate of 415 N. gonorrhoeae isolates with a MIC ≥0.12 µg mL⁻¹ from 25 reports representing 22 countries, 83.1% (345 out of 415) were found to have mosaic penA genes (Table 1). Among all the different mosaic patterns, penA10^10,56,57 and penA34^{15,17,21–23,27,42,56,58} 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 39.1% (135 out of 345) and 49.9% (172 out of 345) 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.⁵⁹

The I312M, V316T, N512Y and G545S amino acid substitutions are frequently seen in mosaic penA patterns. Researchers have found that amino acid substitutions, I312M,^19,25 V316T/P^19,25,48 and G545^19,25,60,61 are associated with reduced cefixime susceptibility. However, a gene transformation study by Tomberg et al.⁵¹ in 2010 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 35/02 (with penA10) back into wild-type returned its MIC to that similar to the wild-type penA MIC, indicating that those three mutations are important to cefixime resistance only in the context of other mutations found in mosaic penA10 alleles.

In the same study by Tomberg et al.,⁵¹ chimeric penA genes were created by replacing sequential portions of penA10 with the corresponding regions of a wild-type penA gene. Reversion of amino acid regions 309–353 (containing I312M and V316T and 13 other substitutions), 489–528 (containing N512Y and two other mutations) and 528–581 (containing G545S and nine other substitutions), showed significant decrease in MIC. Among the three, the reversion of region 528–581 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 528–581-wild-type penA10, the resulting strain’s cefixime MIC only rose to 0.05 µg mL⁻¹, suggesting that other mutations in the 528–581 region were necessary to significantly elevate the cefixime MIC to >0.125 µg mL⁻¹.

The reversion of region 489–528 (containing N512Y and two other mutations) decreased the MIC from ~0.125 to ~0.05 µg mL⁻¹. Y512N reversion alone presented a decreased MIC from ~0.125 to ~0.06 µg mL⁻¹. Accounting for most of the effect on cefixime resistance by mutations in the 489–528 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 98% of all isolates with a MIC ≥0.25 µg mL⁻¹ in the study by Grad et al. involving more than 1100 N. gonorrhoeae isolates collected in the USA, the percentage lowered to 91% when a lower and more clinically relevant MIC breakpoint (≥0.125 µg mL⁻¹) was used.²⁷ 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^8,10,14,18 and Europe¹⁷ were found to have non-mosaic penA alleles (Table 1). Among reports from Asia, over 20% 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.⁴⁷

Three amino acid substitutions, A501S/V/T/P, G542S and P551S/L/P, have been associated with cefixime-decreased susceptibility independent from penA mosaicism.^16,21 The study by Tomberg et al. demonstrated that an independent A501 substitution potentially decreased cefixime susceptibility by increasing the rigidity of the PBP2 active site.⁴⁷

In Table 1, we have summarised the cefixime-decreased susceptible related penA amino acid alterations in all N. gonorrhoeae strains with a cefixime MIC ≥0.12 µg mL⁻¹. One important finding is that 68 out of 70 (97.1%) non-mosaic penA strains with reduced susceptibility to cefixime have a 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.

In Figure 1, we have summarised the MIC levels of all N. gonorrhoeae strains with cefixime MIC ≥ 0.12 µg mL⁻¹ by different combinations of decreased susceptible-related penA amino acid alterations (n = 240). Strains lacking a specific cefixime MIC value or penA alteration records (n = 175) were excluded.

Fig. 1.  Summary of cefixime minimum inhibitory concentration (MIC) of Neisseria gonorrhoeae strains by different combinations of cefixime-decreased susceptibility-related penA amino acid alterations. 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 colours indicate different mutation combinations.

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.⁵² Changes in the promoter or coding sequence of the mtrR gene can potentially decrease antimicrobial susceptibility by increased efflux.⁶² 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,^13,22 plus A39T and G45D²² in the coding region. We did not find gene transformation studies that looked into mtrR alterations’ contribution to cefixime resistance independent from penA changes. Nonetheless, other studies report that mutations in the mtrR gene have little or no association with cefixime susceptibility.^58,62

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.^58,63 Alterations found in strains with cefixime-decreased susceptibility include G120K and A121N/D substitutions.⁴² While the study by Grad et al. in the USA suggested no correlation between cefixime-decreased susceptibility and G120K or G120D/A121D, the lack of G120K and A121N mutations strongly predicted susceptibility.²⁷

Two other mutations in the penB gene commonly found in cefixime-decreased susceptible strains are G101K/D and A102D/N/S. Combinations of mutations at those two sites were found in all 48 strains with a cefixime MIC >0.125 µg mL⁻¹ out of the total 329 N. gonorrhoeae strains in the Serra-Pladevall et al. study^17,22 and among all 127 strains with a cefixime MIC ≥0.125 µg mL⁻¹ out of the total 194 N. gonorrhoeae strains in the study by Jeverica et al.²²

We have found no evidence that alterations in the penB gene alone can confer decreased susceptibility to cefixime.

ponA encodes for penicillin-binding protein 1 (PBP1), an additional cell wall protein important in β-lactam antibiotic antimicrobial activity. Alterations in the ponA gene were associated with penicillin resistance by PBP1 target mutation, although its role in cefixime-decreased susceptibility is unclear.^19,37,58 One mutation, L421P,^17,42,64 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.⁶²

pilQ encodes for a type IV pili secretin.⁵⁴ 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.^58,63

While the study by Whiley et al.⁶³ concluded that changes in the pilQ gene are unlikely associated with cefixime-decreased susceptibility, the study by Grad et al.²⁷ found that a 176–183 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 23.7% of all isolates compared with ≤10% for any of the other three).

Antimicrobial stewardship is important in the control of antimicrobial resistance in infectious diseases.⁶⁵ Antimicrobial use results in selective pressure, favouring the development of resistant organisms.⁶⁵ Therefore, the use of molecular assays to predict antimicrobial susceptibility at the time of treatment allows for targeted therapy, enabling the use of antimicrobials shown to be highly effective and rapidly bactericidal, as well as the use of older medications previously deemed non-effective because of prevalent resistance.^66,67 Such molecular assays to predict susceptibility could be a valuable complement to antimicrobial stewardship and novel drug development in slowing the emergence of antimicrobial resistance.³⁸

Previously, we developed a polymerase chain reaction-based assay using high resolution melt analysis to predict N. gonorrhoeae susceptibility to ceftriaxone and cefixime by targeting the penA34 type.⁶⁸ While that assay showed greater than 98% sensitivity in predicting cefixime-decreased susceptibility in North America, where the penA34 is the most common penA type,^27,50 the assay is limited by its inability to identify cefixime-decreased susceptibility as a result of N. gonorrhoeae strains with non-mosaic penA or other non-34 mosaic penA patterns. As N. gonorrhoeae strains with various non-34 penA types account for many of the cefixime-resistant strains reported in Asia and Europe, it is likely only a matter of time for such strains to emerge in other parts of the world, including in the USA.

Additional amino acid alterations should be considered when developing a molecular assay to predict cefixime susceptibility, especially when a lower and more clinically relevant MIC breakpoint (<0.12 µg mL⁻¹) is used.

For the penA gene, either of the following patterns of alterations could lead to elevated cefixime MIC levels: (1) mosaic penA types with characteristic polymorphisms I312M, V316T, N512Y, G545S; or (2) non-mosaic penA types with mutations at any combinations of the three amino acid positions: A501, G542 or P551. A multiple peptide sequence alignment showed that a wild-type sequence of amino acid region 375–377 can reliably distinguish 32 out of 33 types of wild-type and non-mosaic penA types (with the only exception being the non-mosaic penA49 type possessing a A377V substitution) from all 21 reported mosaic penA types (data not shown). Other researchers have targeted other regions or used the characteristic amino acid polymorphisms in mosaic penA types such as I312M, V316T and G545S.^19,69

Non-mosaic penA types with critical point mutations can be identified by the absence of changes at amino acid region 375–377, and the presence of one of the three critical point mutations A501, G542 or P551. A molecular assay that detects any penA alterations at amino acid positions 375–377, 501, 542 or 551 would be the most parsimonious assay to predict decreased susceptibility to cefixime. That modified assay would be more effective in predicting cefixime susceptibility than the assay we previously developed,⁶⁸ with greater sensitivity for diverse international isolates. Based on the molecular characteristics of all reported strains with decreased susceptibility to cefixime as recorded in Table 1, the estimated sensitivity for the proposed assay would be 99.5% (413 out of 415).

Other genes, such as mtrR, ponA, penB and pilQ, have a less clear role in cefixime resistance and seem to further increase cefixime MIC values only when penA alterations are present. Although certain alterations in these genes were found to be highly associated with full susceptibility as mentioned above, detecting these changes appears to be unnecessary as N. gonorrhoeae will constantly undergo selection by antimicrobial agents and gradually gain mutations that eventually lead to cefixime resistance.

Because of the continued challenge in treating antimicrobial-resistant N. gonorrhoeae infections, there have been many reports published regarding the genetic alterations associated with decreased susceptibility to third-generation cephalosporins. Researchers have provided epidemiological and molecular evidence that alterations in the penA gene are the primary determinants of cefixime-decreased susceptibility, while other genes in the presence of an altered penA gene further contribute in reducing cefixime susceptibility but are neither necessary nor sufficient in independently conferring decreased susceptibility. Based on those data, we proposed the optimal targets for novel molecular assays aiming to predict N. gonorrhoeae susceptibility to cefixime.

The authors declare no conflicts of interest.