Prevalence,phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007

Johansson E, Fredlund H, Unemo M. Prevalence, phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007. APMIS 2009; 117: 900–4. In Neisseria gonorrhoeae culture diagnostics, species confirmation is commonly performed using commercial biochemical tests relying on prolyliminopeptidase (PIP) activity. It was previously shown that one PIP‐negative strain was mainly globally transmitted during 2000–2004. The aims were to investigate the prevalence and phenotypic and genetic characteristics of PIP‐negative N. gonorrhoeae isolates in Sweden during 2000–2007. Gonococcal isolates (n = 1230) cultured in Sweden during 2000–2007 were characterized using PIP screening, antibiogram, serovar determination, pip and porB gene sequencing, and N. gonorrhoeae multiantigen sequence typing (NG‐MAST). Fifteen (1.2%) of the isolates were PIP‐negative. Of those, 13 (87%) were indistinguishable to the previously described globally transmitted strain, i.e. displayed serovar IB‐4 (Bpyvut), similar antibiograms, ST210 (n = 10)/ST292 (n = 3) and contained an identical single nucleotide pip gene deletion. Wherever high reliance is placed on PIP activity for N. gonorrhoeae species confirmation, changes in the diagnostic strategies may need to be considered and/or monitoring of the PIP activity is crucial.     

Comparison of microscopy, culture and in-house PCR and NASBA assays for diagnosis of Neisseria gonorrhoeae in Russia

This study aimed to assess the laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia. In total, 334 consecutive symptomatic patients were enrolled. Cervical and urethral specimens from women (n=286) and urethral specimens from men (n=48) were analyzed by microscopy, culture and two in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed using porA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positive predictive values of 100%. Compared to the PCR (most sensitive method in the present study), in women the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. In men, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. The negative predictive values of microscopy, culture, and NASBA were 97.3%, 97.3%, and 99.6% in women, and 97.8%, 95.7%, and 100% in men, respectively. According to the PCR, the prevalences of N. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated Russian NAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoea in Russia is suboptimal and crucially requires validation, improvements and quality assurance.     

淋病奈瑟菌opa基因分型方法的流行病学应用

目的调查我国4个不同城市淋病奈瑟菌opa基因型的分布状况,并考察实验结果与临床资料之间的一致性,从而了解淋病的传播路线及传播方式。方法研究于2006年在我国沿海地区4个不同城市共收集330株淋病奈瑟菌,其中中国医学科学院皮肤病研究所（南京）采集86株,山东省皮防所（济南）采集80株,福建省皮防所（福州）采集94株,广西壮族自治区皮防所（南宁）采集70株。对330株淋球菌进行opa基因分型,并应用GelCompar分型软件对各菌株opa基因扩增片段的酶切产物条带进行聚类分析。研究还收集了这些菌株相应的临床和流行病学资料,其中包括淋病患者的性接触信息。结果 330株淋球菌opa基因经过TaqI与HpaII两种限制性内切酶酶切后共产生了309个opa型,其中292个opa型各包含一株淋球菌,14个opa型各包括两株淋球菌,2个opa型各包括3株淋球菌,1个opa型包括4株淋球菌。在292个各包括一株淋球菌的opa型中,有18个opa型（18株淋球菌）两两之间形成9个opa型相似对。在这4个城市中,只包括一株菌的opa型占各自城市opa型总数的比率分别为：南京90.7%,济南77.5%,福州89.4%,南宁97.1%。流行病学资料显示19例淋病患者存在明显的性接触史：包括8对两两之间存在性接触的淋病患者及一个三人组淋病患者。其中有8组（七对与一个三人组）淋病患者间的性接触信息通过opa基因分型研究中的8个opa簇证实,而余下的一对淋病患者性接触信息则通过研究中的一个opa型相似对所印证。结论 Opa分型是一种效率极高的淋球菌基因分型方法,能够很好地应用于淋球菌感染的流行病学调查研究中。用这种技术所得到的实验结果比患者提供的性接触信息更贴近实际情况,并能让我们更好地了解淋球菌的传播状况。     

Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain

A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002-2003. In conclusion, a precise, i.e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.     

The Neisseria gonorrhoeae population in Sweden during 2005-phenotypes, genotypes and antibiotic resistance

In Sweden, the gonorrhoea incidence has significantly increased since an all-time low in 1996. We aimed to phenotypically and genotypically characterise N. gonorrhoeae isolates (n=180) transmitted in Sweden during 2005. All isolates were susceptible to cefixime, ceftriaxone, and spectinomycin. However, 2%, 50% and 75% displayed intermediate susceptibility or resistance to azithromycin, ciprofloxacin and ampicillin, respectively. The isolates were assigned to 28 different serovars using Genetic Systems monoclonal antibodies (Mabs) (discriminatory index, 91.0%) and 46 different serovars using Pharmacia Mabs (index, 94.4%). Furthermore, they displayed 95 porB sequences (index, 97.8%) and 95 N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types (STs) (index, 98.0%). 51 (54%) of these STs have not been previously described. 14 ST clusters, comprising between 3 and 15 isolates, were identified that indicate the existence of several transmission chains. The high number of unique STs (n=63) may be associated with import of strains from abroad, local emergence of new STs, incomplete epidemiological surveillance, and/or suboptimal diagnostics, including contact tracing. Overall, the Swedish N. gonorrhoeae population was remarkably diversified. Comprehensive knowledge regarding transmission, phenotypes (including antibiotic resistance), but also in many cases highly discriminative and precise genotypic characteristics of the N. gonorrhoeae strains circulating in our societies, is crucial.     

Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae

The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.     

淋病球菌单克隆抗体的制备和特性的研究

应用杂交瘤技术筛选出２株淋球菌特异性单克隆抗体Ｎｇ４（ＩｇＧ２ａ）和Ｎｇ１８（ＩｇＧ３）有可能用于淋病的实验诊断．２株单抗的腹水效价可达１０７，抗原识别位点为淋球菌外膜蛋白约３５ｋＤ的糖蛋白成分．在免疫荧光实验中，２株单抗可与不同型淋球菌标准株反应，不与有关的细菌和真菌交叉。     

淋病奈瑟菌NspA原核表达及其抗原性研究

目的 研究淋病奈瑟菌表面蛋白A(NspA)的抗原性及其在淋病快速诊断技术中的应用价值.方法 用PCR技术克隆淋病奈瑟菌NspA基因,构建其原核表达载体,在大肠杆菌中表达重组NspA,表达产物复性后免疫小鼠,Western blot、ELISA分别检测免疫血清与淋病奈瑟菌细胞裂解液中NspA及与淋病奈瑟菌全细胞的结合能力.结果 NspA基因可在大肠杆菌中表达,纯化并复性的重组NspA(rrNspA)可在小鼠体内刺激产生高效价特异性抗体,抗rrNspA抗体可与淋病奈瑟菌完整细胞及细胞裂解液中NspA特异性结合.结论 获得的rrNspA及抗rrNspA抗体在建立淋病奈瑟菌抗体或抗原快速检测方法中具有潜在的应用价值.     

广州地区质粒和染色体介导的淋球菌对青霉素和四环素耐药株趋势分析

目的监测2000—2005年间广州地区质粒和染色体介导的淋球菌对青霉素和四环素的耐药性及耐药株流行趋势。方法采用琼脂稀释法检测淋球菌对青霉素和四环素抗菌药物的最低抑菌浓度（MIC），用纸片酸度法测定质粒介导的产青霉素酶淋球菌（PPNG）。结果6年来共测定631株临床分离的淋球菌，检出PPNG132株（20．9％），阳性率从2000年的17．1％上升到2005年的23．7％（Χ^2=0．955，P〉0．05）；质粒介导的高度耐四环素淋球菌（TRNC）222株（35．2％），阳性率从2000年的20％上升到2005年的46．1％（Χ^2=11．94，P〈0．05）。染色体介导的青霉素耐药率从2000年的76％上升到2005年的98．3％（Χ^2=12．94，P〈0．05），染色体介导的四环素的耐药率介于70．7％-85．7％（X2=3．246，P〉0．05）。在2000—2005年期间，青霉素的MIC50和MIC90由1mg／L和2mg／t,上升到4mg／L和〉32mg／L，四环素的MIC50和MIC90由1mg／L和2mg／L上升到2mg／L和4mg／L，且都超过了耐药标准。结论广州地区近6年来质粒介导的淋球菌耐药株PPNG和TRNG增长速度较快。呈逐年上升趋势；染色体介导的淋球菌对青霉素和四环素耐药比率很高。     

Evidence for a reserpine-affected mechanism of resistance to tetracycline in Neisseria gonorrhoeae

The presence of a reserpine-affected mechanism of tetracycline resistance was investigated in 17 Neisseria gonorrhoeae clinical isolates. To establish this fact the MIC of tetracycline in the presence and absence of reserpine was determined, and, in addition, mechanisms of tetracycline resistance were analyzed by PCR. The results showed that reserpine affects the MIC of tetracycline at least 4-fold in all isolates, including those containing the tetM gene. An inhibitory effect of reserpine against the MtrCDE efflux system was ruled out by using strains either with an inactive or with an unrepressed MtrCDE system. The results suggest the presence of a constitutive system of resistance to tetracycline, by a possible efflux pump, which may be inhibited by reserpine. Further studies are required to determine the exact nature of the action of reserpine on the MIC of tetracycline.     

Mutations in gyrA,gyrB, parC,and parE in quinolone-resistant strains of Neisseria gonorrhoeae

Mutations in the genes for the subunits GyrA and ParC of the target enzymes DNA gyrase and topoisomerase IV are important mechanisms of resistance in quinolone-resistant bacteria, including Neisseria gonorrhoeae. The target enzymes also consist of the subunits GyrB and ParE, respectively, though their role in quinolone-resistance has not been fully investigated. We sequenced the quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE in 25 ciprofloxacin-resistant strains from Bangladesh (MIC 4-->32 mg/l) and 5 susceptible strains of N. gonorrhoeae. All the resistant strains had three or four mutations. Two of these were at positions 91 and 95 of gyrA. Fourteen strains had an additional mutation in parC at position 91, and 17 strains had an additional mutation in parE in position 439. No alterations were found in gyrB. The five susceptible strains had identical DNA sequences. Data indicate that the mutations detected in the QRDR of gyrA and parC may be important in the development of quinolone resistance. According to transformation experiments we assume that the alteration in parE is not related to a high degree of quinolone resistance. There was no correlation between ciprofloxacin MICs and pattern or number of mutations in the target genes.     

佛山地区淋球菌流行株对抗生素敏感性测定

目的了解佛山地区2007年分离的淋球菌流行株对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的耐药性及产β-内酰胺酶淋球菌（PPNG）和四环素高度耐药淋球菌（TRNG）的流行状况。方法采用琼脂稀释法测定最小抑菌浓度（MIC）,纸片酸度法检测β-内酰胺酶。结果98株淋球菌检出PPNG34株（34．7％）,TRNG63株（64．3％）。青霉素、环丙沙星和四环素的耐药率分别为96．9％、96．9％和94．9％,未发现大观霉素及头孢曲松耐药株。青霉素、四环素和环丙沙星的MIC50及MIC50均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC50均超过耐药标准的8倍和32倍。结论大观霉素及头孢曲松是治疗淋病的首选药物。     

Incidence, epidemiology and evolution of reduced susceptibility to ciprofloxacin in Neisseria gonorrhoeae in Korea

Objective: To verify the decrease of susceptibility to ciprofloxacin in Neisseria gonorrhoeae , determine the size of the recently reported new β-lactamase plasmid and explain the high prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG).
Methods: Gonococci were isolated from prostitutes in Korea. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. Plasmid was isolated by an alkaline lysis method. Patterns of Nhe l-digested genomic DNA were compared after pulsed-field gel electrophoresis (PFGE).
Results: The minimum inhibitory concentration of ciprofloxacin for 50% of the isolates rose from 0.015 mg/L in 1993 to 0.12 mg/L in 1996. The proportion of PPNG remained at 70% or over during the 5-year period. The size of a novel β-lactamase plasmid, first reported in 1994, was determined to be approximately 3.2 MDa, and 48% of the PPNG isolates contained it. Twelve of 50 isolates had the same PFGE pattern and nine others another pattern.
Conclusion: The rapid decrease of fluoroquinolone-susceptible gonococci suggests that in the near future the drug may become less useful for gonorrhea treatment. The new 3.2-MDa plasmid may have been introduced as a result of the recent increase in overseas travel. The PFGE pattern suggests that high prevalence of PPNG may be due to dissemination of a few resistant clones among the high-risk groups.     

Cefixime and ceftriaxone susceptibility of Neisseria gonorrhoeae in Italy from 2006 to 2010

Neisseria gonorrhoeae resistance to cephalosporins, the currently recommended treatment, and treatment failures with cefixime have been reported worldwide. The purposes of the present study were (i) to examine the susceptibility of N. gonorrhoeae isolates isolated in Italy from 2006 through 2010 to cefixime (n = 293) taking into account both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical And Laboratory Standards Institute (CLSI) criteria for categorization; (ii) to determine the contribution to decreased/resistant susceptibility of mutations in the penA, mtrR, ponA and porB1b genes in a subsample of isolates; and (iii) to genotype the isolates showing decreased susceptibility or resistance to cefixime, by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and by pulsed-field gel electrophoresis (PFGE) to identify the predominant genotypes. Minimum inhibitory concentrations (MICs) were determined by the E-test and agar dilution method on 293 isolates and results were interpreted according to both EUCAST 2010 (MIC R >0.12 mg/L) and CLSI 2008 (MIC R >0.25 mg/L) criteria. All isolates showed full susceptibility to ceftriaxone, whereas those with a MIC for cefixime ≥0.125 mg/L were on the increase from 2008 through 2010. The same penA gene alterations were found among isolates with MICs close to the EUCAST breakpoint as the resistant ones, and they belong to ST1407. Seven isolates, belonging to various sequence types, showed a different por allele, though similar to the por 908 allele present in ST1407. PFGE divided strains ST1407 into two main groups confirming their genetic relationship.     

不孕不育者生殖道淋球菌、沙眼衣原体和解脲支原体的检测结果分析

目的了解在不孕不育患者中生殖道淋球菌（NC）、沙眼衣原体（CT）、解脲支原体（UU）感染的情况。方法应用实时荧光定量PCR方法对465例不孕不育患者生殖道3种病原体基因进行定量测定。结果阳性检出268例，总阳性率为57．64％，其中UU的阳性检出率最高，占43．87％；重叠感染中以UU＋CT的阳性检出率最高，占3．44％。结论不孕不育患者生殖道中3种病原体感染率不尽相同，尤以UU感染率最高，是造成不孕不育的重要原因。     

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.     

Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone

Context: Gonorrhoea is one of the most common sexually transmitted infections (STI) in developing countries and is a global health problem. Aims: To analyze the trend of antimicrobial susceptibility of Neisseria gonorrhoeae isolates over the years, in a tertiary care hospital of North India. Settings and Design: The study population comprised males with urethritis and females with endocervicitis attending STI clinic of our hospital. Materials and Methods: In our STI laboratory, all gonococcal isolates are subjected to antimicrobial susceptibility testing by disc diffusion method as per CLSI guidelines. ß-lactamase production is determined by chromogenic cephalosporin test. Minimum Inhibitory Concentration (MIC) for ceftriaxone is determined by E-test. Statistical Analysis Used: Data were expressed as percentages. The differences in percentages were tested for statistical significance by using χ² test and P values were determined. Results: The percentage of penicillinase producing N. gonorrhoeae (PPNG) increased from 8% in 1995-96 to 20% in 2004-05 and 88% in 2011-2013. Quinolone-resistant N. gonorrhoeae (QRNG) showed a significant increase from 12% in 1995-96 to 98.3% in 2004-05, while 84% isolates were found to be QRNG by 2011-2013. In January 2013 we detected our first gonococcal isolate with decreased susceptibility to third-generation cephalosporins; Ceftriaxone, Cefixime and Cefpodoxime (MIC for ceftriaxone = 0.19 μg/ml). Conclusions: The results of our study highlighted an alarming increase in the percentage of PPNG and QRNG strains over the years. Emergence of N. gonorrhoeae isolates with decreased susceptibility to third-generation cephalosporins is a cause of concern and thus emphasises the importance of antimicrobial susceptibility testing.     

Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus

Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.     

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping

PURPOSE: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. METHODS: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. RESULTS: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciprofloxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identified. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. CONCLUSIONS: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.     

Increasing Incidence of High-Level Tetracycline-Resistant Neisseria gonorrhoeae due to Clonal Spread and Foreign Import

PurposeThe detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea.ResultsThe incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted.ConclusionThe increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import.