Penicillinase-producing Neisseria Gonorrhoeae Research Articles

Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their blaTEM-135 Gene Variant in Bangkok, Thailand, 2015-2017.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) possessing blaTEM-135 is a serious public health threat. With only a single change in the amino acid sequence, blaTEM-135 could evolve into a TEM-type extended-spectrum beta-lactamase (ESBL), which hydrolyzes extended-spectrum cephalosporins, including ceftriaxone and cefixime. We investigated the molecular epidemiological characteristics, types of plasmids in PPNG isolates, and prevalence of PPNG clinical isolates producing TEM-135 beta-lactamases. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was used to determine the molecular epidemiological characteristics of 99 PPNG isolates collected from 2015 to 2017. A mismatch amplification mutation assay was used to examine the blaTEM-135 gene prevalence. Of the 89 identified NG-MAST sequence types, 65 (73.0%) were novel. Only 17.7% (43/243) of PPNG isolates belonged to 16 genogroups. The most frequent plasmid was African, followed by Rio/Toronto, and Asian. The blaTEM-135 allele was found in Rio/Toronto plasmids. The blaTEM-135 allele was present in 23.2% (23/99) of the PPNG isolates. PPNG isolates expressing TEM-135 beta-lactamase exhibited significantly higher penicillin MIC (minimum inhibitory concentration) values than TEM-1 PPNG isolates. The PPNG isolates showed high genetic diversity and a high proportion of blaTEM-135 alleles. Mutation of the blaTEM-135 allele is worrisome as only one mutation could cause TEM-1 to evolve into an ESBL variant that degrades ceftriaxone. Ongoing surveillance of blaTEM-135 and new PPNG isolates is imperative.

Beta lactamase-producing Neisseria gonorrhoeae alleviates Amoxicillin-induced chlamydial persistence in a novel in vitro co-infection model

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cause most bacterial sexually transmitted infections (STIs) worldwide. Epidemiological studies have shown high percentages of co-infections with CT/NG and indicate that NG co-infection can reactivate CT shedding during persistent chlamydial infection. These data also suggest that biological interaction between the two bacteria may increase susceptibility or transmissibility. CT is an obligate intracellular bacterium with a developmental cycle that alternates between two forms: infectious elementary bodies (EBs) which invade the epithelium and non-infectious reticulate bodies (RBs) which divide and replicate inside the inclusion. Adverse environmental conditions can interrupt chlamydial development, with a consequent temporary halt in RB division, reduction in infectious EB production and formation of enlarged chlamydiae (aberrant bodies, ABs) – characterizing chlamydial persistence. When the stressor is removed, the chlamydial developmental cycle is restored, together with production of infectious EBs. The beta-lactam amoxicillin (AMX) induces chlamydial persistence, both in vitro and in mice. We investigated the impact of penicillinase-producing NG strain (PPNG) on AMX-persistent chlamydial infection utilizing our recently developed, contact-independent in vitro model of co-infection. We hypothesized that co-infection with PPNG could prevent and/or reverse AMX-induced chlamydial persistence. Our results showed that PPNG can ameliorate AMX-persistence in two chlamydial species, CT and C. muridarum (CM), providing novel evidence for a range of Chlamydia/NG interactions.

High prevalence of blaTEM-135 and genetic epidemiology of blaTEM-135-carrying Neisseria gonorrhoeae isolates in Shandong, China, 2017-19.

Only one additional specific SNP in blaTEM-135 in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates is required for TEM-135 to evolve into an ESBL that is capable of degrading all extended-spectrum cephalosporins (ESCs). Identification of the blaTEM-135 gene was achieved by performing a mismatch amplification mutation assay PCR. PPNG isolates were then genotyped using MLST and N. gonorrhoeae multiantigen sequence typing (NG-MAST) techniques. Finally, phylogenetic analyses based on MLST and NG-MAST were performed on all of the PPNG isolates, and genogroups were classified according to the phylogenetic relatedness on the basis of each MLST and NG-MAST gene typing and the blaTEM allele varieties. Among the 197 PPNG isolates, one (0.5%) isolate possessed the blaTEM-220 allele, 90 (45.7%) possessed the blaTEM-135 allele, and 106 (53.8%) possessed the blaTEM-1 allele. Among the blaTEM-135-carrying PPNG isolates, MLST ST8109 (n = 30) and NG-MAST ST12199 (n = 5), ST12754 (n = 5) and ST15073 (n = 5) were the most prevalent in the Shandong province of China. The phylogenetic analyses revealed that MLST ST8109, ST8140 and ST14417, mainly carrying the blaTEM-135 allele, belonged to the same genogroup, G1, while NG-MAST ST12199 and ST15073, mainly carrying the blaTEM-135 allele, belonged to the same genogroup, Ga. This study has shown that the molecular epidemiology of PPNG isolates carrying the blaTEM-135 allele in Shandong has a dynamically changing status. Therefore, it is very pressing to continuously monitor the prevalence and mutation of the blaTEM-135 allele and the genetic epidemiology of PPNG isolates carrying the blaTEM-135 allele in this district.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996–2018

The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 β-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum β-lactamase (ESBL).

A β-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly.

Seven structurally related β-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903. To compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 β-lactamases. N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. β-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays. N. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow β-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 β-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours. This unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 β-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

High prevalence of TEM-135 expression from the Asian plasmid in penicillinase-producing Neisseria gonorrhoeae from Hangzhou, China

Penicillinase-producing Neisseria gonorrhoeae (PPNG) expressing the TEM β-lactamase variant TEM-135 are a global public-health concern as this variant requires only a single amino acid substitution to develop into an extended-spectrum β-lactamase. The aim of this study was to investigate the epidemiology of blaTEM-135 in 505 N. gonorrhoeae isolates from Hangzhou, China, during the periods 2011-2012 and 2015-2017. Investigation by nitrocefin test and mismatch amplification PCR showed that 41.0% (207/505) of the isolates were PPNG, of which 37.2% (77/207) contained the blaTEM-135 gene. Further PCR-based plasmid typing showed that blaTEM-135 was predominantly expressed from the Asian plasmid (94%). PPNG isolates consisted of three major clusters, namely Asian plasmid/blaTEM-135 (34.8%), Asian plasmid/blaTEM-1 (32.4%) and African plasmid/blaTEM-1 (28.0%), which showed significant differences in penicillin minimum inhibitory concentrations (MICs) determined by the agar dilution method. Representative isolates were investigated by quantitative real-time PCR (plasmid copy number and blaTEM gene expression), western blot analysis (TEM levels and TEM stability) and in vivo β-lactamase activity assays to elucidate the cause of the observed differences in penicillin MIC. Overall, isolates of the Asian plasmid/blaTEM-135 cluster showed the highest β-lactamase activity, which was explained by higher blaTEM gene expression (Asian versus African plasmid) and higher TEM stability (TEM-135 versus TEM-1). In conclusion, the blaTEM-135 gene is commonly present on the Asian plasmid in PPNG isolates from Hangzhou. The PPNG isolate cluster containing the Asian plasmid and blaTEM-135 showed the highest penicillin MICs, which might explain its abundance in the Hangzhou population.

Molecular screening for Neisseria gonorrhoeae antimicrobial resistance markers in Nigerian men who have sex with men and transgender women.

Antimicrobial-resistant Neisseria gonorrhoeae (NG) is a global public health issue that threatens effectiveness of current treatments of NG. Increased use of nucleic acid amplification tests (NAATs) in lieu of cultures makes obtaining clinical isolates for susceptibility testing difficult and samples collected in commercial transport buffer for NAATs do not preserve viable organism, while molecular methods of assessing antibiotic susceptibility do not require viable organism. We evaluated 243 NG-positive samples in Aptima transport media including urine, oral, and rectal swabs from Nigerian men who have sex with men for markers to penicillinase-producing NG, ciprofloxacin ( GyrA and ParC mutations), and extended spectrum cephalosporins (ESCs, PenA mosaic [allele X], PonA, mtrR, PorB mutations) by real-time PCR. NG DNA was recovered in 75% (183/243) of samples. Of these, 93% (171/183) were positive for at least one resistance marker. We observed a prevalence of dual resistance markers to penicillin and ciprofloxacin at 46.2% (79/171). Six percent of samples (10/171) tested positive for the PenA mosaic (allele X) ESC marker. These data indicate that antibiotic-resistant NG is common in Nigeria. Laboratory and clinical capacity building in Nigeria should include development of methods to culture NG and determine antimicrobial susceptibility.

Molecular Characterization of Markers Associated With Antimicrobial Resistance in Neisseria gonorrhoeae Identified From Residual Clinical Samples.

The emergence and spread of antimicrobial-resistant (AMR) Neisseria gonorrhoeae (NG) is a major public health concern. In the era of nucleic acid amplifications tests, rapid and accurate molecular approaches are needed to help increase surveillance, guide antimicrobial stewardship, and prevent outbreaks. Residual urethral swabs, collected prospectively in the Baltimore City Health Department during a 6-month period, were analyzed by real-time polymerase chain reaction assays for NG DNA and AMR determinants to fluoroquinolones, penicillin, and extended-spectrum cephalosporins. N. gonorrhoeae DNA was detected in 34.8% (73/210) of samples, including 67.3% (68/101) of the swabs that had been previously identified as NG positive by culture. Markers associated with decreased susceptibility to fluoroquinolones were detected in 22.4% of the polymerase chain reaction NG-positive samples. The rate of penicillinase-producing NG was very low (1.6%), and no markers associated with decreased susceptibility to extended-spectrum cephalosporins were detected in this cohort of men using the AMR assays herein described. Detection of molecular markers associated with AMR in NG can be performed directly from residual clinical samples, although the recovery rate of adequate DNA for molecular testing from these samples can be suboptimal. A high number of samples with mutations associated with decreased susceptibility to fluoroquinolones were identified.

Recent Increase in the Incidence of TEM-135 β-Lactamase-harboring Neisseria gonorrhoeae in Korea.

BackgroundWe investigated the molecular epidemiological characteristics and antimicrobial susceptibility pattern of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates to monitor the change in distribution of blaTEM in Korea.MethodsWe collected 804 PPNG isolates from diverse hospitals and clinics mainly located in Seoul, Korea, over a period of 11 years (2005–2015). Isolate susceptibility to seven antimicrobials was determined using the agar dilution test. The molecular epidemiological characteristics of the isolates were determined by Sanger sequencing of blaTEM, N. gonorrhoeae multiantigen sequence typing (NG-MAST) and plasmid typing.ResultsAmong 72 fully sequenced PPNG isolates, sixteen (22.2%) possessed TEM-135. All TEM-135 isolates had a common silent mutation (c.18C>T), which was previously unreported. We observed a pattern of continuous increase in the number of TEM-135 isolates since 2012. The median and 90% minimum inhibitory concentration of azithromycin were substantially lower in the TEM-135 group than in the non-PPNG and TEM-1 groups. All TEM-135 isolates showed different NG-MAST types and predominantly harbored Toronto/Rio (75%) plasmids. A comprehensive comparative analysis of PPNG with TEM-135 according to NG-MAST, plasmid type, and year of isolation revealed a wide distribution.ConclusionsThe proportion of TEM-135 PPNG has continuously increased since 2012, in association with clonal spread. The difference at position 18 of the TEM-135 sequence can be interpreted as the existence of multiple clonal complexes. The possibility that TEM-135 was acquired via foreign plasmids requires careful follow-up and continuous monitoring of TEM-135 to ascertain whether it constitutes a step towards evolutionary change.

Molecular Characterization of Penicillinase-Producing Neisseria gonorrhoeae Isolated in Two Time Periods, 2003-2004 and 2014-2015, in Italy.

The emergence of antibiotic resistant strains poses a great concern for gonorrhea treatment. The aim of this study was to characterize penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Italy in two time frames, 2003-2004 and 2014-2015. A total of 80 PPNG were characterized for the blaTEM gene variant and the plasmid type. Furthermore, gonococci were typed using Neisseria gonorrhoeae multiantigen sequence typing. Antibiotic susceptibility assay was performed for penicillin, ciprofloxacin, ceftriaxone, and spectinomycin by Etest and minimum inhibitory concentration (MIC) test strip methods. The β-lactamase production was detected using nitrocefin test. Among PPNG isolates, four blaTEM alleles were identified as follows: blaTEM-1, blaTEM-228, blaTEMP14S, and blaTEM-135. The African plasmid possessed the blaTEM-1, blaTEM-228, and blaTEMP14S, whereas blaTEM-135 was identified in Toronto/Rio and Asian plasmids. The percentage of isolates with the blaTEM-1-carrying African plasmid increased from 42.5% in 2003-2004 to 55% in 2014-2015; conversely, the isolates with blaTEM-135-carrying Toronto/Rio plasmid decreased from 57.5% to 35%. Among the isolates carrying the Toronto/Rio plasmids possessing blaTEM-135, sequence type (ST)661 and ST5624 were found to be the predominant STs in both periods 2003-2004 and 2014-2015, respectively. More than half of the PPNG isolates were resistant to ciprofloxacin. Increase in the isolates carrying the African plasmid possessing blaTEM-1 and a parallel decrease of the blaTEM-135-carrying Toronto/Rio plasmid was observed. Moreover, PPNG isolate harbored Toronto/Rio plasmid with blaTEM-135 belonged mainly to two major STs (ST661 and ST5624). Given the possible role of a mutated blaTEM gene as an additional mechanism to extended spectrum β-lactamase resistance, it is crucial to monitor gonococci carrying these resistance genes.

Antimicrobial Susceptibility Evaluation and Multiple-Locus Variable Number Tandem Repeat Analysis of Neisseria gonorrhoeae Isolates in China in 2012.

This study aimed to gain information on the antimicrobial susceptibility and molecular epidemiological typing of Neisseria gonorrhoeae (NG) isolates in China in 2012. A total of 244 NG isolates were consecutively recovered from patients with uncomplicated gonorrhea attending sexually transmitted disease clinics in 3 Chinese cities-Guangzhou, Nanjing, and Tianjin-in 2012. Neisseria gonorrhoeae susceptibilities to penicillin and tetracycline were examined by detecting penicillinase-producing NG (PPNG) and high-level tetracycline-resistant NG, and NG susceptibilities to ciprofloxacin, spectinomycin, ceftriaxone, and cefixime were determined using an agar dilution method. Neisseria gonorrhoeae isolates were typed by multiple-locus variable number tandem repeat analysis. We conducted a χ analysis to compare clusters with Bonferroni correction and Kruskal-Wallis test. Neisseria gonorrhoeae isolates gathered from the 3 cities differed significantly in the prevalence of tetracycline-resistant NG (P < 0.001) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.001). The analysis of the combination of the 7 variable number of tandem repeats loci for all of the 244 isolates yielded 110 multiple-locus variable number tandem repeat analysis types falling into 5 clusters. Cluster III was associated with PPNG, whereas cluster II was associated with non-PPNG (P < 0.05) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.05). Antimicrobials that can be used with confidence to treat NG infection currently in China include ceftriaxone and spectinomycin, but not penicillin, tetracycline, ciprofloxacin, and cefixime. Moreover, some of the resulting clusters were associated with PPNG and NG with decreased ceftriaxone susceptibility.

Mutation Pattern in the Genome of Neisseria gonorrhoeae and Its Association with Multidrug-resistant Isolates from Delhi, India

Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.

Genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae from England and Wales.

The objective of this study was to investigate the genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in 2012 from England and Wales. PPNG isolates were from the 2012 Gonococcal Resistance to Antimicrobial Surveillance Programme (GRASP). Their susceptibility to seven antimicrobials was determined using agar dilution methodology. β-Lactamase production was detected using a nitrocefin test. β-Lactamase plasmid types were determined and blaTEM genes were sequenced. Isolates were also typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Seventy-three PPNG isolates were identified in the 2012 GRASP collection (4.6%, 73/1603). Three different blaTEM alleles were identified, encoding three TEM amino acid sequences: TEM-1 (53%), TEM-1 with a P14S substitution (19%) and TEM-135 (27%). The blaTEM-135 allele was present in nine different NG-MAST types and was found mostly on Asian (60%) and Toronto/Rio (35%) plasmids. By contrast, most TEM-1-encoding plasmids were African (98%). All the TEM-135 isolates displayed high-level ciprofloxacin and tetracycline resistance. The high proportion of blaTEM-135 alleles (27%) demonstrates that this variant is circulating within several gonococcal lineages. Only a single specific mutation near the β-lactamase active site could result in TEM-135 evolving into an ESBL. This is concerning particularly because the TEM-135 isolates were associated with high-level ciprofloxacin and tetracycline resistance. It is encouraging that no further TEM alleles were detected in this gonococcal population; however, vigilance is vital as an ESBL in N. gonorrhoeae would render the last remaining option for monotherapy, ceftriaxone, useless.

Penicillinase-producing Neisseria gonorrhoeae and its blaTEM-135 gene variants at several gonococcal antimicrobial surveillance sites in China: an epidemiological study

Objective To determine the prevalence of penicillinase-producing Neisseria gonorrhoeae(PPNG) and the distribution of blaTEM-135 gene variants in PPNG at several gonococcal antimicrobial surveillance sites in China, to compare N. gonorrhoeae multi-antigen sequence typing (NG-MAST) types of PPNG and its blaTEM-135 gene variants, and to assess the difference and association in NG-MAST types of blaTEM-135 gene variants among different regions. Methods A total of 572 N. gonorrhoeae isolates were collected at 6 gonococcal antimicrobial surveillance sites from Jiangsu, Shanghai, Zhejiang, Tianjin, Guangdong and Guangxi in 2012. After isolation, purification, and identification, cefalotin paper discs were used for detection of PPNG. DNA was extracted by QIAxtractor DX kits after cultivation of the PPNG strains. Then, mismatch amplification mutation assay (MAMA) PCR was performed to identify blaTEM-135variants, and NG-MAST analysis to determine N. gonorrhoeae genotypes. Results Among the 572 N. gonorrhoeae strains, 38.1%(218/572) were identified as PPNG, and of the PPNG strains, 52.3%(114/218) were blaTEM-135 variants. The detection rate of PPNG at these surveillance sites from high to low was as follows: 51.7%(45/87, Zhejiang), 45.6%(36/79, Shanghai), 38.0%(78/205, Guangdong), 37.5%(12/32, Guangxi), 31.2%(24/77, Jiangsu) and 25.0%(23/92, Tianjin), and that of blaTEM-135 variants was as follows: 68.9%(31/45, Zhejiang), 58.3%(14/24, Jiangsu), 50.0%(39/78, Guangdong), 47.2%(17/36, Shanghai), 39.1%(9/23, Tianjin) and 33.3%(4/12, Guangxi). NG-MAST analysis showed that the ST2318, ST1768, ST1866, ST1053 and ST8726 types predominated among these blaTEM-135 variants, and a strong correlation was found between blaTEM-135 variants and some NG-MAST types, such as ST1768, ST1053 and ST8726 types. The distribution of NG-MAST types was significantly different between the surveillance site in Tianjin (in the Northern part of China) and the other sites (in the Southern part of China), but highly similar among the surveillance sites in Jiangsu, Zhejiang and Shanghai regions. Conclusions There is a high prevalence of PPNG and its blaTEM-135 variants at several gonococcal antimicrobial surveillance sites in China, with significant differences in NG-MAST genotype distribution of PPNG and its blaTEM-135 variants among different regions. Key words: Neisseria gonorrhoeae; beta-Lactamases; Epidemiology; blaTEM-135 Gene variant; Neisseria gonorrhoeae multi-antigen sequence typing

Infection with neisseria gonorrhoeae and association between rising ceftriaxone minumum inhibitory concentration and men who have sex with men

Objectives The aim of this study was to review patients presenting with Neisseria gonorrhoeae over a 5 year period. Methods We performed a retrospective cohort study, using a prospective database and retrospective review of medical records, of patients with gonorrhoea infection presenting between January 2008 and November 2013. Results 215 patients screened positive by NAAT and treatment failure occurred in 2 patients after both initially received treatment with ciprofloxacin. of the 106 culture positive Neisseria gonorrhoeae isolates, 37 had ceftriaxone MIC >0.008. In addition, there were 4 penicillinase producing Neisseria gonorrhoeae (PPNG) isolates, 30 quinolone resistant Neisseria gonorrhoeae (QRNG) isolates, 9 tetracycline resistant Neisseria gonorrhoeae (TRNG) isolates and 4 isolates resistant to azithromycin. On a multivariate model, an association was seen between ceftriaxone MIC >0.008 and a history of men who have sex with men (MSM, OR 11.81, 95% CI 2.51–55.59, p = 0.02). Univariate analysis was statistically significant for infection involving either rectal or pharyngeal sites (OR 3.02, 95 CI 1.24–7.34, p = 0.02). Conclusion Screening for Neisseria gonorrhoea in rectal and pharyngeal sites is important amongst patients presenting with a history of MSM. Rising ceftriaxone MIC is associated with MSM and infection in rectal or pharyngeal sites.

Surveillance of antimicrobial susceptibilities of Neisseria gonorrhoeae in Nanning, China, 2000 to 2012.

To monitor the frequency of antibiotic resistance of Neisseria gonorrhoeae (NG) in Nanning, China, between 2000 and 2012. The production of β-lactamase by NG isolates was determined using the paper acidometric testing method. Antimicrobial susceptibility testing was performed for tetracycline, ciprofloxacin, spectinomycin, and ceftriaxone using the agar dilution method. The χ(2) test, t test, and univariate and multivariate analyses were used to analyze the statistical difference of the results. A total of 923 NG isolates were collected in Nanning between 2000 and 2012. Among these, 131 (14.2%) were penicillinase-producing NG, 520 (56.3%) isolates were tetracycline-resistant NG, and 857 (92.9%) isolates were ciprofloxacin-resistant strains. One spectinomycin-resistant strain was identified in 2000. There were 304 (32.9%) isolates with decreased susceptibility to ceftriaxone; the proportion of such isolates increased from 22.8% in 2000 to 2002 to 48.9% in 2006 to 2008 (P < 0.001), followed by a fall to 32.2% in 2009 to 2012 (P = 0.001). Patients' age of 16 to 25 years and isolate collection period of 2008 to 2012 (except 2011) were demonstrated to be risk factors for infection with isolates with decreased susceptibility to ceftriaxone. Antimicrobial susceptibility of NG isolates obtained from patients in Nanning from 2000 to 2012 was characterized by high occurrence of penicillinase-producing NG, tetracycline-resistant NG, and ciprofloxacin-resistant strains. Spectinomycin and ceftriaxone can be considered drugs of choice for empirical treatment of NG infection in Nanning. Moreover, we recommend a combination of 500 mg or higher dose of intramuscular ceftriaxone and 1 g oral azithromycin be used for the treatment of NG infection in Nanning and possibly in China.

Prevalence and Molecular Epidemiological Typing of Penicillinase-Producing Neisseria gonorrhoeae and Their blaTEM-135 Gene Variants in Nanjing, China

This study aimed to investigate the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and their blaTEM-135 gene variant in 2007 and 2012 in Nanjing, China. In addition, molecular epidemiological typing of all isolates was performed to elucidate the genetic relationships of the PPNG strains. A total of 199 and 77 N. gonorrhoeae isolates were collected at the National Center for STD Control in 2007 and 2012, respectively. Nitrocefin tests were performed to identify PPNG. Mismatch amplification mutation assay was used to identify blaTEM-135. All isolates were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST), and additionally, porB-based phylogenetic analysis was performed for the PPNG isolates. The total prevalence of PPNG isolates was 41% (114/276) and 58% (66/114) of these PPNG isolates possessed bla(TEM-135). In 2007, 45% (90/199) produced β-lactamase, and of those PPNG, 58% (52/90) possessed bla(TEM-135). In 2012, 31% (24/77) were PPNG, and 58% (14/24) of those isolates contained bla(TEM-135). There were 162 NG-MAST STs among the 276 isolates, and 89 of those were novel STs. A strong association between specific NG-MAST STs and bla(TEM-135) was found, and the porB-based phylogenetic analysis showed a distant evolutionary relationship between isolates in 2007 and isolates in 2012. A high prevalence of PPNG and blaTEM-135 was found in Nanjing, China. bla(TEM-135) might be a precursor in the evolution into an extended-spectrum β-lactamase that can degrade ceftriaxone, which stresses the need to continuously monitor PPNG, blaTEM-135, and additional evolving blaTEM gene variants.

Molecular Epidemiology of β-Lactamase–Producing Neisseria gonorrhoeae Strains in Manaus, AM, Brazil

We report new sequence types of 14 penicillinase-producing Neisseria gonorrhoeae, isolated from sexually transmitted disease clinic attendees in Manaus, Brazil. They were characterized by WI/WII/WIII groups, susceptibility testing and Multi-Antigen Sequencing Typing/Mutilocus Sequence Typing protocols. Twelve were classified as WII/III and 2 as WI and were presented resistance to penicillin and tetracycline. New alleles for por and AroE genes and novel sequence types were identified, revealing molecular characteristics not described previously. ST1590 is the common ancestor after eBURST analysis, and these findings represent an important contribution of molecular epidemiology approach in gonococci's research in Amazonas.

1229

1229

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.