Mosaic Penicillin-binding Protein Research Articles

1355. Cluster of Multi-drug Resistance Neisseria gonorrhoeae Isolates with Reduce Ceftriaxone Susceptibility, First Report in Thailand

Abstract Background Rapid emergence of global azithromycin resistance, with ceftriaxone reduce susceptibility/resistant strains, threatens the current recommends dual therapy with ceftriaxone and azithromycin for gonorrhea to ensure effective treatment. Thailand is a major risk area for gonorrhea because it as a key destination for sex tourism industry, where antibiotic resistance gonorrhea can spread easily and quickly across the region. Here, we identified a first cluster of Neisseria gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Methods A 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, during 2016–2018 were studied. Broth micro-dilution and plate dilution methods to define resistance or decreased susceptibilityaccording to CLSI guidelines. Select isolates were subjected to whole genome sequencing with an Illumina MiSeq V2 instrument and analysis using CLCBio Genomics Workbench v6.5. Molecular docking studies were carried out to understand binding mode analysis and orientation of ceftriaxone in the active site of PBP2. Biofilm formation measured by confocal laser scanning microscopy. Results Among the N. gonorrhoeae clinical isolates, two isolates had significant reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L), which were isolated from urethral swab in male heterosexual patients. Both were multidrug resistance and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). One isolate was resistant to azithromycin (MIC of 1 mg/L), and other one remained susceptible (MIC of 0.5 mg/L). Reduced susceptibility to ceftriaxone associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region with one belonged to ST7235 and second one had new allele number of tbpB with new sequence type (ST). Ceftriaxone weakly occupy the active site of mosaic XXXIV penicillin-binding protein 2 (PBP2) variant in both. Molecular epidemiology results reviled that both isolates display similarities with isolates from UK, USA, and Netherlands. Conclusion This first cluster of genetically related gonococcal isolates with decreased ceftriaxone susceptibility may bring threat of treatment failure in Thailand. Also highlighted the importance of maintaining surveillance for antimicrobial resistance. Disclosures All Authors: No reported disclosures

Mapping-free variant calling using haplotype reconstruction from k-mer frequencies.

MotivationThe standard protocol for detecting variation in DNA is to map millions of short sequence reads to a known reference and find loci that differ. While this approach works well, it cannot be applied where the sample contains dense variants or is too distant from known references. De novo assembly or hybrid methods can recover genomic variation, but the cost of computation is often much higher. We developed a novel k-mer algorithm and software implementation, Kestrel, capable of characterizing densely packed SNPs and large indels without mapping, assembly or de Bruijn graphs.ResultsWhen applied to mosaic penicillin binding protein (PBP) genes in Streptococcus pneumoniae, we found near perfect concordance with assembled contigs at a fraction of the CPU time. Multilocus sequence typing (MLST) with this approach was able to bypass de novo assemblies. Kestrel has a very low false-positive rate when applied to the whole genome, and while Kestrel identified many variants missed by other methods, limitations of a purely k-mer based approach affect overall sensitivity.Availability and implementationSource code and documentation for a Java implementation of Kestrel can be found at https://github.com/paudano/kestrel. All test code for this publication is located at https://github.com/paudano/kescases.Supplementary information Supplementary data are available at Bioinformatics online.

New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary.

ABSTRACTThe Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient.

Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S.pneumoniae strains except for pbp1a of a Romanian S.pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S.pneumoniae to S.mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S.pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCEStreptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S.pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S.mitis as donor species. Sequences identical to one such mosaic gene were identified in an S.mitis strain from the same patient, suggesting that in this case S.pneumoniae played the role of donor.

Emergence and Spread of Neisseria gonorrhoeae Isolates With Decreased Susceptibility to Extended-Spectrum Cephalosporins in Argentina, 2009 to 2013.

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins represents a major concern globally. The aim of this study was to examine the phenotypic and molecular characteristics of N. gonorrhoeae isolates with decreased susceptibility to ceftriaxone and cefixime in Argentina. A total of 1987 isolates were collected during 2009 and 2013. The susceptibility to penicillin G, tetracycline, ciprofloxacin, cefixime, ceftriaxone, and azithromycin was determined using the agar dilution method. The major extended-spectrum cephalosporin resistance determinants (penA, mtrR, and porB1b) were sequenced in 42 N. gonorrhoeae isolates that showed decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC], 0.06-0.125 mg/L) and cefixime (MIC, 0.125-0.25 mg/L). Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed. Between 2009 and 2013, there was a shift in the modal MICs for ceftriaxone. Among the 42 isolates exhibiting decreased susceptibility to ceftriaxone and cefixime, 95.2% were resistant to penicillin G, 95.2% to tetracycline, 97.6% to ciprofloxacin, and 33.3% to azithromycin. Thirty-five (83.3%) of the 42 isolates had a mosaic penA allele XXXIV, which has been previously associated with resistance to ceftriaxone and cefixime as well as treatment failures. The isolates that contained the mosaic penicillin-binding protein 2 (PBP2) XXXIV were associated with NG-MAST ST1407 or closely related genotypes. In Argentina, N. gonorrhoeae isolates with decreased susceptibility to cefixime and ceftriaxone have now emerged, mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407.

The Molecular Epidemiology and Antimicrobial Resistance of Neisseria gonorrhoeae in Australia: A Nationwide Cross-Sectional Study, 2012.

Antimicrobial resistance (AMR) by Neisseria gonorrhoeae is considered a serious global threat. In this nationwide study, we used MassARRAY iPLEX genotyping technology to examine the epidemiology of N. gonorrhoeae and associated AMR in the Australian population. All available N. gonorrhoeae isolates (n = 2452) received from Australian reference laboratories from January to June 2012 were included in the study. Genotypic data were combined with phenotypic AMR information to define strain types. A total of 270 distinct strain types were observed. The 40 most common strain types accounted for over 80% of isolates, and the 10 most common strain types accounted for almost half of all isolates. The high male to female ratios (>94% male) suggested that at least 22 of the top 40 strain types were primarily circulating within networks of men who have sex with men (MSM). Particular strain types were also concentrated among females: two strain types accounted for 37.5% of all isolates from females. Isolates harbouring the mosaic penicillin binding protein 2 (PBP2)-considered a key mechanism for cephalosporin resistance-comprised 8.9% of all N. gonorrhoeae isolates and were primarily observed in males (95%). This large scale epidemiological investigation demonstrated that N. gonorrhoeae infections are dominated by relatively few strain types. The commonest strain types were concentrated in MSM in urban areas and Indigenous heterosexuals in remote areas, and we were able to confirm a resurgent epidemic in heterosexual networks in urban areas. The prevalence of mosaic PBP2 harboring N. gonorrhoeae strains highlight the ability for new N. gonorrhoeae strains to spread and become established across populations.

New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan.

We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3'-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread.

Emergence and evolution of internationally disseminated cephalosporin-resistant Neisseria gonorrhoeae clones from 1995 to 2005 in Japan.

BackgroundNeisseria gonorrhoeae strains with resistance to extended-spectrum cephalosporins (ESCs), last options for first-line monotherapy of gonorrhoea, likely emerged and initially disseminated in Japan, followed by international transmission. In recent years, multi-locus sequence typing (MLST) ST1901 and N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolates with the mosaic penicillin-binding protein (PBP) 2 XXXIV have accounted for most ESC resistance globally. Our aim was to elucidate the initial emergence and transmission of ESC-resistant strains by detailed examination of N. gonorrhoeae isolates from 1995 to 2005 in Kanagawa, Japan.MethodsN. gonorrhoeae isolates were examined phenotypically (n = 690) and genetically (n = 372) by agar dilution method (cefixime, ceftriaxone and ciprofloxacin), penA gene sequencing, MLST and NG-MAST.ResultsAlready in 1995, one cefixime-resistant (CFM-R) isolate was found, which is the first CFM-R isolate described globally. After 1996, the prevalence of CFM-R and CFM-decreased susceptibility (CFM-DS) isolates significantly increased, with the peak resistance level in 2002 (57.1 % CFM-R). In 1997–2002, the CFM-R MLST ST7363 strain type with the mosaic PBP 2 X was predominant among CFM-R/DS isolates. The first CFM-R/DS MLST ST1901 clone(s), which became the predominant CFM-R/DS strain type(s) already in 2003–2005, possessed the mosaic PBP 2 X, which was possibly originally transferred from the MLST ST7363 strains, and subsequently acquired the mosaic PBP 2 XXXIV. The first MLST ST1901 and NG-MAST ST1407 isolate was identified in Kanagawa already in 2003.ConclusionsThe two main internationally spread cefixime-resistant gonococcal clones, MLST ST7363 and ST1901 (NG-MAST ST1407 most frequent internationally) that also have shown their capacity to develop high-level ceftriaxone resistance (superbugs H041 and F89), likely emerged, evolved and started to disseminate in the metropolitan area, including Kanagawa, in Japan, which was followed by global transmission.

Decreased susceptibility to ceftriaxone in Neisseria gonorrhoeae in the absence of a mosaic penicillin-binding protein 2 (PenA) allele

Decreased susceptibility to ceftriaxone in Neisseria gonorrhoeae in the absence of a mosaic penicillin-binding protein 2 (PenA) allele

Characteristics of Males Infected With Common Neisseria gonorrhoeae Sequence Types in the Gonococcal Isolate Surveillance Project, San Francisco, California, 2009

We analyzed 265 urethral Neisseria gonorrhoeae specimens collected from symptomatic males at San Francisco's municipal sexually transmitted disease clinic, a participant in the Gonococcal Isolate Surveillance Project, during 2009. We used N. gonorrhoeae multiantigen sequence typing to describe characteristics of patients infected with common sequence type families. Specimens were classified into 6 homology-based families and 1 additional family of all other identified strains. Strain family results were combined with results of culture-based antibiotic sensitivity minimum inhibitory concentration, sociodemographic and behavioral risk data collected at the clinic, and presence or absence of the mosaic penicillin-binding protein 2 (penA) allele. Characteristics of patients were compared across strain families through the use of χ(2) statistics. Among men who have sex with men, strain distribution differed by those reporting receptive oral sex as their only urethral exposure (P = 0.04), by number of sex partners (P = 0.03), and by race/ethnicity (P < 0.001); there were no differences by age or human immunodeficiency virus status. Also, among men who have sex with men, strain family distributions differed for culture specimens with reduced susceptibility to a range of antibiotics, as well as with presence of the mosaic penA allele (all P < 0.001). The combination of molecular, phenotypic, and epidemiologic data on N. gonorrhoeae infection could help develop a more complete epidemiology of gonorrhea in the United States.

Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain

Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

Multiplex Bead Suspension Array for Screening Neisseria gonorrhoeae Antibiotic Resistance Genetic Determinants in Noncultured Clinical Samples

The increasing threat of antibiotic-resistant Neisseria gonorrhoeae highlights the need for new diagnostic options. A high-throughput multiplex bead suspension array assay was developed for profiling 29 N. gonorrhoeae genomic mutations and 2 plasmid genes conferring resistance to 6 antimicrobial agents: penicillin, ciprofloxacin, cefixime, tetracycline, azithromycin, and spectinomycin. The three steps of this assay include amplification of 12 N. gonorrhoeae chromosomal and plasmid loci, multiplex allele-specific primer extension reaction, and multiplex bead suspension array detection. Antibiotic resistance genetic determinants were identified successfully in 239 cervicovaginal N. gonorrhoeae-positive noncultured swab samples. This molecular assay can be used for detection of gonococci in clinical specimens, molecular typing, mutation profiling, and predictive assessment of N. gonorrhoeae susceptibility to antibiotics without the need for culture.

Antimicrobial susceptibility and penicillin-binding protein 1 and 2 mutations in Neisseria gonorrhoeae isolated from male urethritis in Sapporo, Japan

The spread of antimicrobial-resistant Neisseria gonorrhoeae worldwide is a critical issue in the control of sexually transmitted infections. The purpose of this study was to clarify recent trends in the susceptibility of N. gonorrhoeae to various antimicrobial agents and to compare these data with our previous data. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined in N. gonorrhoeae strains clinically isolated from male gonococcal urethritis. In addition, amino acid sequencing of penicillin-binding protein (PBP) 2, encoded by the penA gene, was analyzed so that genetic analysis of mosaic PBP 2 could clarify the susceptibility of the strains to cefixime and other cephalosporins. The susceptibility rate for ceftriaxone, cefodizime, and spectinomycin, agents whose use is recommended by the guideline of the Japanese Society of Sexually Transmitted Infections (JSSTI), was 100 %. The susceptibility rates of the strains to penicillin G and ciprofloxacin were lower than those in previous reports. Mosaic PBP 2 structures were detected in 51.9 % of the strains and the MICs of the strains with the mosaic PBP 2 to cefixime were much higher than those of the strains without the mosaic PBP 2. In the clinical situation, the treatment regimen recommended by the JSSTI remains appropriate; however, the susceptibility to cephalosporins should be intensively surveyed because strains with mosaic PBP 2 were commonly detected.

The influence of target population on nonculture-based detection of markers of Neisseria gonorrhoeae antimicrobial resistance

With treatment options for gonorrhoea (Neisseria gonorrhoeae) diminishing, strengthening antimicrobial resistance (AMR) surveillance is paramount. In this study, we investigated polymerase chain reaction (PCR) based methods, in parallel with N. gonorrhoeae multi-antigen sequence typing (NG-MAST), for direct detection of four N. gonorrhoeae chromosomal mechanisms associated with emerging resistance to extended spectrum cephalosporins using noncultured samples: an adenine deletion in the mtrR promoter, a mosaic penicillin-binding protein (PBP) 2, an A501V PBP2 mutation, and alterations at positions 120 and 121 of the porB protein. The PCR assays were validated using a panel of characterised N. gonorrhoeae isolates (n=107) and commensal Neisseria (n=100) species. These PCR assays with NG-MAST were then applied to noncultured clinical specimens from distinct populations in Australia with differing levels of N. gonorrhoeae AMR: the Northern Territory (NT), where resistance has a low population prevalence, and Queensland (Qld), with higher AMR prevalence. The real-time PCR assays proved highly sensitive and specific. When applied to the noncultured samples, only 1 out of 50 (2%) samples from NT harboured a resistant mechanism, whereas the Qld samples (n=129) collected over different periods showed progressive acquisition of resistant mechanisms, and these were associated with specific NG-MAST types, including Type 225. The results suggest that our PCR-based methods could be used to rapidly pinpoint incursion of resistant strains into previously unaffected populations. Likewise, our results show that for molecular AMR surveillance, the population being investigated is as important as the genetic mechanisms being targeted.

Cefpodoxime 10 μg disc screening test for detection of Neisseria gonorrhoeae with mosaic PBP2 and decreased susceptibility to extended-spectrum cephalosporins for public health purposes

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae remains a global public health problem. Susceptibility to first-line treatment extended-spectrum cephalosporins (ESCs) is decreasing worldwide resulting in therapeutic failures with oral ESCs. This study describes a cefpodoxime 10 μg disc test for screening for gonococci containing a penA mosaic allele encoding a mosaic penicillin-binding protein 2 (PBP2) and decreased ESC susceptibility. Selected clinical gonococcal isolates (n = 315), containing a high proportion of gonococci with decreased ESC susceptibility and high geographical, temporal and genetic diversity, were examined using agar dilution (n = 149; cefpodoxime and ceftriaxone) and Etest (n = 315; cefixime), and disc diffusion using a commercially available cefpodoxime 10 μg disc (n = 315). penA sequencing was performed on all isolates. The 2008 WHO gonococcal reference strains (n = 8) were included as quality controls. Using a ≤11 mm annular radius of growth inhibition as the breakpoint for the cefpodoxime 10 μg disc, all 78, with exception of one isolate (13 mm), mosaic PBP2-containing isolates, which also displayed decreased susceptibility to oral ESCs, were identified. In addition, 85 non-mosaic PBP2-containing isolates (44% of which contained a PBP2 A501 alteration) had annular radii ≤11 mm and raised minimal inhibitory concentrations to the ESCs. Screening for detection of mosaic PBP2-containing gonococci and decreased ESC susceptibility, most pronounced to oral ESCs, using a commercially available cefpodoxime 10 μg disc was rapid, inexpensive and sensitive. This test can be used in AMR surveillance programmes for public health purposes especially in less-resourced settings. Further studies to refine this disc testing-based approach are in progress.

A duplex PCR method to identify mosaic penA gene and predict reduced susceptibility to oral cephalosporins in Neisseria gonorrhoeae

Neisseria gonorrhoeae with mosaic penicillin-binding protein 2 (PBP2) is associated with reduced susceptibility to third-generation oral cephalosporins. A simple and rapid PCR method using three primers was designed to identify mosaic PBP2 , which could help predict reduced susceptibility to expanded-spectrum cephalosporins in N. gonorrhoeae.

Characteristics and dissemination of mosaic penicillin-binding protein 2-harboring multidrug-resistant Neisseria gonorrhoeae isolates with reduced cephalosporin susceptibility in northern Taiwan.

Among 254 Neisseria gonorrhoeae isolates from a sexually transmitted infection (STI) clinic in northern Taiwan, 69 isolates were found to contain the mosaic penA (MA) gene and were associated with elevated cefixime and ceftriaxone MICs. Most of these MA gene-harboring isolates were also resistant to penicillin (71.4%) and ciprofloxacin (100%) and were from men who have sex with men (MSM) or from bisexual men (81.2%). Three major sequence types (ST835, ST2180, and ST2253) constituted 55.7% of these isolates. The major sequence types harboring the mosaic penA gene may represent major sexual networks responsible for the emergence/introduction and the spread of the multidrug-resistant clones in Taiwan.

Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone

To examine mutations within the penA, mtrR, porB, ponA and pilQ genes of Neisseria gonorrhoeae to determine their contribution to cephalosporin resistance. A total of 46 N. gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone (MICs > or = 0.12 mg/L) and two susceptible isolates were selected. The full sequence of penA and partial sequences previously reported as hot mutation sites of the other genes were analysed. Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. A mosaic penicillin-binding protein 2 (PBP 2) was found in a single isolate that exhibited the highest cefixime MIC (0.5 mg/L). The majority of the isolates with reduced susceptibility to cephalosporins contained non-mosaic PBP 2 sequences, of which PBP 2 pattern XIII was most common (28/46). All isolates with reduced susceptibility to cephalosporins also had mtrR and porB mutations. Two susceptible isolates had the PBP 2 pattern XIV and an incomplete MtrR protein, which was a new mutation. Isolates with identical PBP 2 patterns comprised multiple NG-MAST sequence types. Reduced susceptibility of N. gonorrhoeae to ceftriaxone and cefixime was associated with diverse penA mutations, particularly PBP 2 pattern XIII containing an Ala-501-->Val substitution, together with mtrR and porB mutations. The existence of only one strain having the mosaic penA sequence indicated that ceftriaxone and cefixime resistance in Korea is mostly not associated with a mosaic penA sequence. Highly heterogeneous NG-MAST sequence types excluded the clonal expansion of a particular subtype.

Mosaic Penicillin-Binding Protein 2 in Neisseria gonorrhoeae Isolates Collected in 2008 in San Francisco, California

Using a real-time PCR assay specific for a mosaic penA allele that has been associated with oral cephalosporin resistance in Asia, 54 available Neisseria gonorrhoeae isolates collected in San Francisco, CA, from January to October 2008 were analyzed. Five isolates tested positive for the mosaic penA gene by real-time PCR. DNA sequencing revealed two mosaic penA alleles (SF-A and SF-B). Isolates with SF-A and SF-B alleles possessed elevated MICs for the oral cephalosporins cefpodoxime and cefixime.

The use of cephalosporins for gonorrhea: the impending problem of resistance

Gonorrhea remains an important clinical and public health problem throughout the world. Gonococcal infections have historically been diagnosed by Gram stain and culture but are increasingly diagnosed through nucleic acid tests, thereby eliminating the opportunity for antimicrobial susceptibility testing. Gonococcal infections are typically treated with single-dose therapy with an agent found to cure > 95% of cases. Unfortunately, the gonococcus has repeatedly developed resistance to antimicrobials including sulfonamides, penicillin, tetracyclines and fluoroquinolones. This has now left third-generation cephalosporins as the lone class of antimicrobials recommended as first-line therapy for gonorrhea in some regions. However, resistance to oral third-generation cephalosporins has emerged and spread in Asia, Australia and elsewhere. The mechanism of this resistance seems to be associated with a mosaic penicillin binding protein (penA) in addition to other chromosomal mutations previously found to confer resistance to β-lactam antimicrobials (ponA, mtrR, penB, pilQ). Few good options exist or are in development for treating cephalosporin-resistant isolates, as most have had multidrug resistance. Preventing the spread of resistant isolates will depend on ambitious antimicrobial management programs, strengthening and expanding surveillance networks, and through effective sexually transmitted disease control and prevention.