In vitro activity of ertapenem versus ceftriaxone against Neisseria gonorrhoeae isolates with highly diverse ceftriaxone MIC values and effects of ceftriaxone resistance determinants: ertapenem for treatment of gonorrhea?

Clinical resistance to the currently recommended extended-spectrum cephalosporins (ESCs), the last remaining treatment options for gonorrhea, is being reported. Gonorrhea may become untreatable, and new treatment options are crucial. We investigated the in vitro activity of ertapenem, relative to ceftriaxone, against N. gonorrhoeae isolates and the effects of ESC resistance determinants on ertapenem. MICs were determined using agar dilution technique or Etest for international reference strains (n = 17) and clinical N. gonorrhoeae isolates (n = 257), which included the two extensively drug-resistant (XDR) strains H041 and F89 and additional isolates with high ESC MICs, clinical ESC resistance, and other types of clinical high-level and multidrug resistance (MDR). Genetic resistance determinants for ESCs (penA, mtrR, and penB) were sequenced. In general, the MICs of ertapenem (MIC(50) = 0.032 µg/ml; MIC(90) = 0.064 µg/ml) paralleled those of ceftriaxone (MIC(50) = 0.032 µg/ml; MIC(90) = 0.125 µg/ml). The ESC resistance determinants mainly increased the ertapenem MIC and ceftriaxone MIC at similar levels. However, the MIC ranges for ertapenem (0.002 to 0.125 µg/ml) and ceftriaxone (<0.002 to 4 µg/ml) differed, and the four (1.5%) ceftriaxone-resistant isolates (MIC = 0.5 to 4 µg/ml) had ertapenem MICs of 0.016 to 0.064 µg/ml. Accordingly, ertapenem had in vitro advantages over ceftriaxone for isolates with ceftriaxone resistance. These in vitro results suggest that ertapenem might be an effective treatment option for gonorrhea, particularly for the currently identified ESC-resistant cases and possibly in a dual antimicrobial therapy regimen. However, further knowledge regarding the genetic determinants (and their evolution) conferring resistance to both antimicrobials, and clear correlates between genetic and phenotypic laboratory parameters and clinical treatment outcomes, is essential.

Enhancing gonococcal antimicrobial resistance surveillance: a real-time PCR assay for detection of penicillinase-producing Neisseria gonorrhoeae by use of noncultured clinical samples.

With increasing concerns regarding diminishing treatment options for gonorrhea, maintaining the efficacy of currently used treatments and ensuring optimal Neisseria gonorrhoeae antimicrobial resistance surveillance are of the utmost importance. Penicillin is still used to treat gonorrhea in some parts of the world. In this study, we developed and validated a real-time PCR assay for the detection of penicillinase-producing N. gonorrhoeae (PPNG) in noncultured clinical samples with the aim of enhancing penicillin resistance surveillance. The assay (PPNG-PCR2) was designed to be an indirect marker of penicillinase activity, by targeting a region of sequence predicted to be conserved across all N. gonorrhoeae plasmid types harboring the beta-lactamase gene while not specifically targeting the actual beta-lactamase-encoding sequence. The assay was evaluated by using a total of 118 N. gonorrhoeae clinical isolates and 1,194 clinical specimens, including 239 N. gonorrhoeae-positive clinical samples from which N. gonorrhoeae cells were isolated and for which phenotypic penicillinase results are available. Overall, the PPNG-PCR2 assay provided 100% sensitivity and 98.7% specificity compared to bacterial culture results for the detection of PPNG in clinical specimens. PPNG-PCR2 false-positive results, presumably due to cross-reactions with unrelated bacterial species, were observed for up to 1.3% of clinical samples but could be distinguished on the basis of high cycle threshold values. In tandem with phenotypic surveillance, the PPNG-PCR2 assay has the potential to provide enhanced epidemiological surveillance of N. gonorrhoeae penicillin resistance and is of particular relevance to regions where penicillin is still used to treat gonorrhea.

Alterations of the pilQ gene in Neisseria gonorrhoeae are unlikely contributors to decreased susceptibility to ceftriaxone and cefixime in clinical gonococcal strains.

OBJECTIVES: Gonorrhoea remains a global public health problem and the treatment options are diminishing through the emergence of gonococci resistant to most antimicrobials. Previous in vitro studies have indicated a role for Neisseria gonorrhoeae pilQ alterations in conferring resistance to antimicrobials, including penicillin. In this study, we investigated whether pilQ polymorphisms were associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs) in clinical gonococcal strains. METHODS: Full-length pilQ nucleotide and PilQ amino acid sequences from geographically and temporally diverse gonococcal clinical isolates (n = 63), including the 2008 WHO reference strains, representing a range of ceftriaxone and cefixime MICs (≤0.008-0.25 and <0.016-0.5 mg/L, respectively) and 38 N. gonorrhoeae multiantigen sequence types, were examined. Previously described alterations associated with decreased ESC susceptibility (mosaic penA, mtrR and penB alterations) were also examined. RESULTS: Fifteen different pilQ nucleotide sequence types and nine different PilQ amino acid sequence types were observed, with two PilQ types accounting for 53 (84%) of the isolates. Independent of other genetic resistance determinants (penA mosaic, mtrR promoter deletion and penB), only one pilQ alteration, a D526N substitution, provided a statistically significant association with ceftriaxone (P < 0.01) and cefixime (P < 0.05) MICs. However, the two isolates exhibiting D526N lacked all three previously described alterations associated with decreased ESC susceptibility, thereby providing an alternative basis for the low MICs (≤0.008 mg/L) observed for these strains. The previously described E666K (pilQ2) and F595L (pilQ1) mutations were absent in all 63 isolates. CONCLUSIONS: pilQ polymorphisms are unlikely contributors to decreased susceptibility to ESCs in clinical gonococcal strains.

Reduced susceptibility to ceftriaxone in Neisseria gonorrhoeae is associated with mutations G542S, P551S and P551L in the gonococcal penicillin-binding protein 2.

OBJECTIVES: Reduced susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae has, to date, been associated with three alterations: a mosaic penA allele encoding the penicillin-binding protein 2 (PBP2); A-del-mtrR, an adenine deletion in the mtrR promoter; and penB, comprising mutated alleles of PorBIb. In this study, we examined an association between reduced susceptibility to ceftriaxone and additional mutations in gonococcal PBP2. METHODS: N. gonorrhoeae isolates (n = 76) exhibiting reduced susceptibility to ceftriaxone but lacking the mosaic penA sequence were investigated for A-del-mtrR and penB as well as substitutions at PBP2 501, 542 and 551 using a previously described real-time PCR approach. To further investigate PBP2 542 and 551 substitutions, we reanalysed penA sequence data from a previous study of 98 gonococci exhibiting a range of ceftriaxone MICs. RESULTS: Of 76 N. gonorrhoeae isolates exhibiting reduced susceptibility to ceftriaxone and lacking the mosaic penA sequence, a 501 (A501V or A501T) substitution was present in 9/76, a 542 substitution in 39/76 and a 551 substitution in 26/76 isolates. Reanalysis of 98 gonococcal isolates from a previous study showed that substitutions at PBP2 542 (G542S) and 551 (P551S or P551L) were significantly associated with raised MICs to ceftriaxone (P = 0.0186 and 0.001, respectively) and penicillin (P = 0.0231 and 0.0007, respectively). CONCLUSIONS: Our findings provide strong evidence for the involvement of PBP2 G542S and P551S/P551L in reduced susceptibility to ceftriaxone and to penicillin. Further studies are needed to investigate the precise and relative roles played by these mutations.

Characteristics and population dynamics of mosaic penA allele-containing Neisseria gonorrhoeae isolates collected in Sydney, Australia, in 2007-2008.

Eighteen hundred Neisseria gonorrhoeae isolates collected in Sydney, Australia, in 2007 and 2008 were examined for mosaic penA alleles that mediated cephalosporin resistance, and the genotypes of the isolates were evaluated. In 2008, there were substantial increases in numbers (from 15 to 85) and proportions (from 1.5 to 10.3%) of mosaic-containing gonococci and major shifts in genotypic patterns, with 10 new genotypes representing 74 of the 85 mosaic-containing isolates and genotypes detected between 2001 and 2005 having disappeared. Enhanced surveillance of gonococcal resistance to cephalosporins is necessary.

Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens.

OBJECTIVES: The Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal isolates but is not validated for non-cultured clinical samples. This study sought to examine the performance of the NG-MAST system on a range of non-cultured samples. METHODS: Nucleic acid extracts of 73 N gonorrhoeae-positive samples, comprising eight cervical swabs, nine urethral swabs, 35 urine samples, one vaginal swab, 13 rectal swabs and seven throat swabs, were analysed by NG-MAST. For 27 specimens, corresponding gonococcal isolates were also analysed and the results compared. A panel of 44 non-gonococcal Neisseria strains and 100 N gonorrhoeae-negative clinical samples were used to investigate further the specificity of the NG-MAST PCR reactions. RESULTS: PCR amplification and DNA sequencing of gonococcal porB and tbpB genes was successful for all N gonorrhoeae-positive urogenital specimens, 11 of 13 rectal swabs and four of seven throat swabs. For the 27 N gonorrhoeae-positive specimens with corresponding gonococcal isolates, the porB and tbpB sequences obtained from the non-cultured specimen were identical to those obtained from the isolate. Cross-reaction with non-gonococcal Neisseria species was observed for both the porB and tbpB PCR reactions, and proved to be problematical for NG-MAST typing of throat swab specimens. CONCLUSIONS: The NG-MAST system can successfully be applied directly to non-cultured urogenital samples, but is less suitable for extragenital specimens, particularly throat swabs, due to cross-reaction with commensal Neisseria species.

Meeting the public health challenge of multidrug- and extensively drug-resistant Neisseria gonorrhoeae.

Globally, antimicrobial resistance (AMR) in Neisseria gonorrhoeae is increasing in prevalence, both within and across antibiotic classes, including extended-spectrum cephalosporins, raising concerns that gonorrhea may become untreatable in certain circumstances. The AMR surveillance that is essential to optimize standard treatments is often lacking or of poor quality in countries with high disease rates. Recent initiatives by the WHO to enhance global AMR surveillance that focus on multidrug- and extensively drug-resistant N. gonorrhoeae through revision of surveillance standards and use of a new panel of N. gonorrhoeae control strains are described. Keys to meeting these new challenges posed by gonococcal AMR remain the reduction in global burden of gonorrhea combined with implementation of wider strategies for general AMR control, and better understanding of mechanisms of emergence and spread of AMR.

Neisseria gonorrhoeae and emerging resistance to extended spectrum cephalosporins.

PURPOSE OF REVIEW: Antibiotic resistance in Neisseria gonorrhoeae poses on-going problems for individual case management and disease control for gonorrhoea. Considerable reliance is now placed on third-generation cephalosporins for the treatment of gonorrhoea following the loss of efficacy of penicillins and quinolones. Current clinical and laboratory perspectives on N. gonorrhoeae with decreased susceptibility to third-generation cephalosporins are provided. RECENT FINDINGS: Treatment failures following therapy with the oral third-generation cephalosporins cefixime and ceftibuten have been reported, but not with the injectable ceftriaxone. The gonococci involved have raised minimal inhibitory concentrations to these agents, including to ceftriaxone. The presence of multiple chromosomal changes form the basis for this 'resistance', prominent among which is a mosaic penicillin-binding protein 2 found in association with additional known and unknown mutations in other genes. The imprecise nature of laboratory criteria for detecting these gonococci means that the distribution and prevalence of these strains is also uncertain. SUMMARY: Concerns regarding the appearance of gonococci associated with treatment failure with oral cephalosporins are increasing. The origins, causes and patterns of spread of these clinically resistant gonococci are reminiscent of the earlier experiences with quinolone-resistant gonococci. Preventive measures require simultaneous implementation of disease-control principles, coupled with those for antimicrobial resistance.

Simple, rapid, and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR green-based real-time PCR.

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by high-resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.

Quinolone resistance in Neisseria gonorrhoeae: rapid genotyping of quinolone resistance-determining regions in gyrA and parC genes by melting curve analysis predicts susceptibility.

We report a duplex real-time PCR assay for the simultaneous screening of mutations involved in fluoroquinolone resistance within gyrA and parC quninolone resistance-determining regions (QRDRs) in Neisseria gonorrhoeae. Our assay clearly detects all mutated QRDRs and allows the identification of common genotypes, whether the QRDRs contain single or double mutations, providing valuable epidemiological tools. When this method is used in conjunction with similar assays and in vitro analyses, essential antibiotic resistance surveillance can be performed for public health purposes.

Ceftibuten resistance and treatment failure of Neisseria gonorrhoeae infection.

Neisseria gonorrhoeae infections have been empirically treated in Hong Kong with a single oral 400-mg dose of ceftibuten since 1997. Following anecdotal reports of the treatment failure of gonorrhea with oral extended-spectrum cephalosporins, the current study was undertaken to determine the antimicrobial susceptibility pattern and molecular characteristics of isolates of N. gonorrhoeae among patients with putative treatment failure in a sexually transmitted disease clinic setting. Between October 2006 and August 2007, 44 isolates of N. gonorrhoeae were studied from patients identified clinically to have treatment failure with empirical ceftibuten. The ceftibuten MICs for three strains were found to have been 8 mg/liter. These strains were determined by N. gonorrhoeae multiantigen sequence typing to belong to sequence type 835 (ST835) or the closely related ST2469. The testing of an additional eight archived ST835 strains revealed similarly elevated ceftibuten MICs. The penA gene sequences of these 11 isolates all had the mosaic pattern previously described as pattern X. Of note is that the ceftriaxone susceptibility results of these strains all fell within the susceptible range. It is concluded that ceftibuten resistance may contribute to the empirical treatment failure of gonorrhea caused by strains harboring the mosaic penA gene, which confers reduced susceptibility to oral extended-spectrum cephalosporins. Screening for such resistance in the routine clinical laboratory may be undertaken by the disk diffusion test. The continued monitoring of antimicrobial resistance and molecular characteristics of N. gonorrhoeae isolates is important to ensure that control and prevention strategies remain effective.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae.

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to ceftriaxone.

Increasing numbers of Neisseria gonorrhoeae strains with decreased susceptibilities to ceftriaxone and other oral cephalosporins widely used for the treatment of gonorrhea have been isolated in Sydney, Australia, over several years. In this study, we examined the complete penicillin-binding protein 2 (PBP 2) amino acid sequences of 109 gonococci, selected on the basis of their diverse temporal and geographic origins and because they exhibited a range of ceftriaxone MICs: < OR =0.03 microg/ml (n = 59), 0.06 microg/ml (n = 43), and 0.125 microg/ml (n = 7). Auxotyping, serotyping, and genotyping by N. gonorrhoeae multiantigen sequence typing sequence-based analysis was also performed. In total, 20 different amino acid sequence patterns were identified, indicating considerable variation in the PBP 2 sequences in this study sample. Only some of the N. gonorrhoeae isolates with significantly higher ceftriaxone MICs contained a mosaic PBP 2 pattern, while more isolates exhibited a nonmosaic PBP 2 pattern containing an A501V substitution. Although particular N. gonorrhoeae genotypes in our sample were shown to be less susceptible to ceftriaxone, the reduced susceptibility to ceftriaxone was not specific to any particular genotype and was observed in a broad range of auxotypes, serotypes, and genotypes. Overall, the results of our study show that N. gonorrhoeae strains exhibiting reduced sensitivity to ceftriaxone are not of a particular subtype and that a number of different mutations in PBP 2 may contribute to this phenomenon.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target.

AIMS: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. METHODS: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. RESULTS: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. CONCLUSIONS: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

Dynamics of appearance and expansion of a prolyliminopeptidase- negative subtype among Neisseria gonorrhoeae isolates collected in Sydney, Australia, from 2002 to 2005.

Recent studies have demonstrated a wide geographic circulation of isolates of Neisseria gonorrhoeae that fail to produce prolyliminopeptidase (PIP). Tests based on the production of this enzyme are important elements of a number of identification systems for gonococci. We documented the appearance, expansion, and contraction of subtypes of 165 PIP-negative gonococci detected in an extended and systematic sample of the 3,926 N. gonorrhoeae isolates collected in Sydney, Australia, from July 2002 to September 2005. Their appearance, peak, and decline followed an "epidemic" curve. At the peak of their prevalence in 2003, PIP-negative gonococci comprised 22% of all isolates. Closely related phenotypes accounted for 162/165 of the PIP-negative gonococci. Algorithms for confirmation of N. gonorrhoeae should take account of the temporal and geographic variability of gonococci by utilizing two or more distinct confirmatory methods.

Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge.

Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review.