Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA

The recent emergence and transmission of Neisseria gonorrhoeae isolates with reduced susceptibility to expanded-spectrum cephalosporins such as cefixime and ceftriaxone have been reported. The aim of this study was to determine the correlation of different polymorphisms in the penA, mtrR, porB1b (penB), and ponA genes of N. gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. Eighteen gonococcal isolates with reduced cefixime and ceftriaxone susceptibility (Cef(i)) and two susceptible isolates were characterized using serovar determination, antibiograms, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and sequencing of penA, mtrR, porB1b, and ponA alleles. For the Cef(i) isolates (n = 18), the MICs of cefixime and ceftriaxone ranged between 0.032 to 0.38 mug/ml and 0.064 to 0.125 mug/ml, respectively. These isolates were assigned five different serovars and six divergent NG-MAST sequence types. Eleven isolates (61%) with higher MICs of cefixime and ceftriaxone contained a nearly identical penA mosaic allele and previously described polymorphisms in mtrR (a single nucleotide [A] deletion in the promoter), penB (mutations in porB1b encoding loop 3 of PorB1b), and ponA (ponA1 polymorphism). The remaining seven Cef(i) isolates (39%), which had somewhat lower MICs of cefixime and ceftriaxone, contained an aspartic acid insertion (Asp-345a) in PBP 2 in conjunction with alterations of 4 to 10 amino acid residues in the C-terminal region of the transpeptidase domain of penA. In conclusion, an unambiguous association between penA mosaic alleles, in conjunction with genetic polymorphisms in mtrR, porB1b, and ponA, and greater reduced susceptibility to cefixime and ceftriaxone was identified.     

Trends in susceptibility of Neisseria gonorrhoeae to ceftriaxone from 1985 through 1991.

The antimicrobial susceptibilities of 16,441 gonococcal isolates from Seattle-King County were determined for ceftriaxone, cefoxitin, penicillin G, and tetracycline. From 1985 to 1989, ceftriaxone, in combination with doxycycline, was increasingly used for treatment of gonorrhea, and by 1989, it was used as therapy for > 80% of cases in Seattle-King County. MICs of ceftriaxone correlated significantly (P < 0.001) with those of the other beta-lactam antibodies included in this study. Geometric mean MICs of penicillin G for isolates that did not produce beta-lactamase increased from 1985 to 1991. The geometric mean MICs of cefoxitin, ceftriaxone, and tetracycline began to decline in 1987 but increased in 1990 and 1991. The percentage of strains with decreased susceptibility to ceftriaxone (MIC, 0.06 to 0.25 microgram/ml) rose from 0.3% in 1985 to 5.3% in 1987 but subsequently declined steadily to 2.6% in 1991, despite increased use of ceftriaxone as routine therapy for gonorrhea. Changes in patterns of antimicrobial susceptibility may be related not only to antimicrobial selection pressures but also to less well understood population shifts among Neisseria gonorrhoeae strains within a community.     

Amino acid substitutions in mosaic penicillin-binding protein 2 associated with reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae

The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.     

Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.     

淋球菌对抗菌药物敏感性分析

目的 监测佛山市2007～2008年度分离的淋病奈瑟菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最低抑菌浓度(MIC),纸片酸度法检测产β-内酰胺酶淋病奈瑟菌菌株.结果 108株淋病奈瑟菌中检出产β-内酰胺酶淋病奈瑟菌(PPNG)为43株(39.8%),四环素、环丙沙星和青霉素的耐药率分别为 89.8%(97/108)、93.7%(101/108)和95.4%(103/108);未发现对大观霉素和头孢曲松耐药菌株.青霉素、四环素和环丙沙星MIC50及MIC90均超过耐药标准,尤其是青霉素为甚,其MIC50及MIC90均超过耐药标准的2倍和大于16倍.结论 淋病奈瑟菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物,对青霉素、四环素和环丙沙星高水平耐药,不应作为淋病治疗的首选药物.     

DNA gyrase mutations in quinolone-resistant clinical isolates of Neisseria gonorrhoeae.

Eight quinolone-resistant clinical isolates of Neisseria gonorrhoeae were shown to carry mutations in their GyrA proteins. Six isolates had a single amino acid change of serine to phenylalanine at the position corresponding to Ser-83 in Escherichia coli. In addition to the change of serine to phenylalanine, two isolates had another change of aspartic acid to asparagine at the position corresponding to Asp-87 in E. coli.     

Multiplex TaqMan real-time PCR platform for detection of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone

A multiplex TaqMan real-time PCR platform was developed in this study for combined detection of opa and/or porA genes (identification of N. gonorrhoeae) and the key mutations (Ala501Val/Thr/Pro, and/or Gly545Ser) in penicillin-binding protein 2 (PBP2) associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs). Firstly, the specificities of the TaqMan probes/primers for the multiplex TaqMan real time PCR platform were confirmed by Basic Local Alignment Search Tool (BLAST) analysis. Then the multiplex PCR platform was performed on 77 isolates with decreased susceptibility to ceftriaxone (CRO) and 100 isolates with full susceptibility to CRO under universal optimized reaction conditions. As a result, based on cultivation-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing in vitro, the multiplex platform had a sensitivity of 100% and a specificity of 95.0% for identifying cultured isolates of Neisseria gonorrhoeae (N. gonorrhoeae, NG, GC) with decreased susceptibility to CRO. When directly screening N. gonorrhoeae with decreased susceptibility to CRO from clinical urogenital swabs, the multiplex platform offered a sensitivity of 96.1% and a specificity of 95.0%. Therefore, on the basis of sample culture and antimicrobial susceptibility testing in vitro, the multiplex TaqMan real time PCR platform has been proven to be a sensitivity of 100% and a specificity of 95.0% useful tool for screening cultured isolates of N. gonorrhoeae with decreased susceptibility to CRO, which can be finished within 2 days.     

Susceptibility of Neisseria meningitidis and Neisseria gonorrhoeae isolates to N-formimidoyl thienamycin.

A diverse collection of 123 meningococcal and 126 gonococcal isolates was tested for susceptibility to N-formimidoyl thienamycin (N-F-thienamycin; MK0787) and to penicillin G (PEN). All of the meningococci were susceptible to both of these, as well as to rifampin. Among gonococci, beta-lactamase-producing strains, which were resistant to PEN, were susceptible to N-F-thienamycin. Among non-beta-lactamase-producing strains, the minimal inhibitory concentrations of N-F-thienamycin and PEN were less than or equal to 1.28 micrograms/ml. Of these, the strains with PEN minimal inhibitory concentrations toward the higher end of the range were likely to be more susceptible to N-F-thienamycin, whereas the strains that were highly susceptible to PEN were likely to have higher minimal inhibitory concentrations of N-F-thienamycin.     

Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai

OBJECTIVES: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates. METHODS: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin. RESULTS: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin. CONCLUSIONS: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.     

In vitro susceptibility of Neisseria gonorrhoeae to 2-acetylpyridine thiosemicarbazones.

MICs of 20 2-acetylpyridine thiosemicarbazones and penicillin were determined for 25 clinical isolates of beta-lactamase-positive and beta-lactamase-negative Neisseria gonorrhoeae strains. The compounds most active against the beta-lactamase-producing strains were the N4,N4-disubstituted derivatives and the thiosemicarbazone derivatives of the 2-acetylpyridines, followed by the other compounds related to the 2-acetylpyridine thiosemicarbazones.     

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.     

Clonally related Neisseria gonorrhoeae isolates with decreased susceptibility to the extended-spectrum cephalosporin cefotaxime in Amsterdam, the Netherlands

From 2006 to 2008, Neisseria gonorrhoeae isolates were identified with decreased susceptibility to the extended-spectrum cephalosporin (ESC) cefotaxime among visitors of the Amsterdam sexually transmitted infections (STI) clinic, the Netherlands. Spread, clonality, and characteristics of 202 isolates were examined using antibiograms, conventional penA mosaic gene PCR, and N. gonorrhoeae multiple-locus variable-number tandem repeat analysis (NG-MLVA). A strictly defined subset was further characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and sequencing of ESC resistance determinants (penA, mtrR, and porB1b). Seventy-four N. gonorrhoeae isolates with a cefotaxime MIC of >0.125 μg/ml (group A), 54 with a cefotaxime MIC of 0.125 μg/ml (group B), and a control group of 74 with a cefotaxime MIC of <0.125 μg/ml (group C) were included. Fifty-three clonally related penA mosaic-positive isolates (penicillin-binding protein 2 type XXXIV) were identified in group A (n = 47 isolates; 64%) and B (n = 6 isolates; 11%). The 53 penA mosaic-positive isolates were predominantly NG-MAST ST1407 (87%) and contained an mtrR promoter A deletion (98%) and porB1b alterations G101K/A102N. All were assigned to the same NG-MLVA cluster that comprised in total 56 isolates. A correlation was found between decreased cefotaxime susceptibility and ST1407 that was highly prevalent among visitors of the Amsterdam STI clinic. The rapid spread of this strain, which also has been identified in many other countries, might be facilitated by high-risk sexual behavior and should be monitored closely to identify potential treatment failure. Quality-assured surveillance of ESC susceptibility on the national and international levels and exploration of new drugs and/or strategies for treatment of gonorrhea are crucial.     

Changing trends of antimicrobial susceptibility patterns of Neisseria gonorrhoeae in India and the emergence of ceftriaxone less susceptible N. gonorrhoeae strains

OBJECTIVES: To monitor the trend of antimicrobial susceptibility of Neisseria gonorrhoeae isolates from 2002 to 2006 in New Delhi, India under the Gonococcal Antimicrobial Susceptibility Programme and to document the emergence of any new antimicrobial resistance. METHODS: Antimicrobial susceptibility of 382 N. gonorrhoeae isolates from clinical cases in males and females to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone was determined by disc diffusion technique, using WHO reference strains as controls and WHO interpretative criteria. MICs were determined using Etests. RESULTS: A significant increasing trend of penicillin and ciprofloxacin resistance up to 2003 and 2004, respectively, and subsequent decrease in resistant strains with a concomitant increase in less susceptible strains, was observed. Tetracycline-resistant N. gonorrhoeae increased significantly from 6.7% in 2002 to 22.9% in 2005. Only one isolate was resistant to spectinomycin and nine isolates were less susceptible to ceftriaxone, during this 5 year period. A substantial proportion (23.3%) of strains were multiresistant. CONCLUSIONS: Emergence of ceftriaxone less susceptible N. gonorrhoeae isolates is a cause for concern, although treatment failure was not observed. An active, continuous and comprehensive programme for monitoring and surveillance of antimicrobial resistance needs to be established in many laboratories, and a search for new effective agents needs to be initiated to respond to the emergence of resistant isolates.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

耐氟喹诺酮淋病奈瑟菌基因突变研究

目的:研究淋病奈瑟菌的DNA旋转酶A亚单位(gyrA)和拓扑异构酶ⅣC亚单位(parC)基因突变与淋病奈瑟菌耐氟喹诺酮类药物的关系。方法:收集临床分离淋病奈瑟菌30株,用E试验法检测其对6种抗菌药的最低抑菌浓度(MIC),并用Nitrocefin纸片检测β内酰胺酶,PCR扩增22株淋病奈瑟菌喹诺酮耐药决定区(QRDR)相关gyrA和parC基因并测序分析。结果:大观霉素、头孢他啶、头孢曲松、四环素、青霉素和环丙沙星的耐药率分别为0、3.3%、3.3%、33.3%、66.7%和100%。β内酰胺酶检出率为17株(56.7%)。22株高水平耐氟喹诺酮菌(MIC≥4mg/L)均存在gyrA基因双位点突变和parC单(或双)位点突变。结论:淋病奈瑟菌对青霉素、四环素、环丙沙星分别有较高的耐药率。gyrA和parC基因突变在淋病奈瑟菌对氟喹诺酮类药物耐药中起重要作用。