淋病奈瑟菌对阿奇霉素的敏感性及mtrR基因突变分析

目的 探讨淋病奈瑟菌对阿奇霉素的敏感性及其相关耐药基因mtrR的突变特征.方法 收集该院2018—2020年检出的43株淋病奈瑟菌菌株,采用琼脂稀释法检测淋病奈瑟菌对阿奇霉素的最小抑菌浓度(MIC),采用PCR和测序技术分析阿奇霉素非敏感组(对阿奇霉素非敏感的菌株11株)和阿奇霉素敏感组(随机抽取的10株对阿奇霉素敏感的菌株)淋病奈瑟菌mtrR基因的突变特征.结果 43株菌株中检出对阿奇霉素非敏感的菌株共11株(25.6％).mtrR基因启动子区域A碱基缺失突变及编码区突变在阿奇霉素非敏感组、阿奇霉素敏感组间的差异无统计学意义(P>0.05),但阿奇霉素非敏感组菌株在mtrR基因编码区可见多个位点突变.结论 临床应根据淋病奈瑟菌的药敏报告结果选择抗菌药物,mtrR基因编码区多个位点的突变可能导致淋病奈瑟菌对阿奇霉素的耐药性升高.     

某地区淋球菌对6种抗生素耐药性的结果分析

目的了解广州地区淋球菌对6种抗生素的耐药性及产青霉素酶的淋球菌(PPNG)和质粒介导的耐四环素的淋球菌(TRNG)的流行状况。方法用琼脂稀释法测定青霉素、四环素、环丙沙星、阿奇霉素、头孢曲松、大观霉素的最低抑菌浓度(MIC);用纸片碘量法检测β-内酰胺酶。结果 88株淋球菌检出PPNG 34株(38.6%)、TRNG 51株(60%)、环丙沙星耐药株87株(98.9%),同时检出PPNG、TRNG、环丙沙星高度耐药(MIC≥161 mg/L)菌株16株,占18.2%。阿奇霉素耐药株24株(27.2%),未发现头孢曲松、大观霉素的耐药菌株。结论推荐大观霉素和头孢曲松作为广州地区治疗淋病的首选药物,加强淋球菌耐药性的连续性监测十分重要。     

淋球菌临床分离株青霉素耐药性与β-内酰胺酶基因相关性探讨

目的　了解淋球菌临床分离株青霉素耐药性与β 内酰胺酶基因存在的相关性。方法　对2003年2~12月分离的128株淋球菌临床分离株用PCR法检测其特异的隐蔽质粒 CPPB基因和青霉素耐药菌的β 内酰胺酶基因;用琼脂稀释法测定其对青霉素的最小抑菌浓度(MIC)。结果　128株临床分离菌中CPPB基因阳性者125株,占97.7%。其中MIC≥1 mg/L的青霉素耐药菌86株,占CPPB基因阳性者的68.8%;MIC≥8 mg/L的高耐药菌59株,占耐药菌株总数的68.6%。86株耐药菌中β 内酰胺酶基因PCR扩增阳性者62株,占 72.1%,其中 58 株(67.4%)为高耐药菌。结论　研究表明,目前淋球菌临床分离株耐药情况较为严重,如果用青霉素治疗淋病需首先进行耐药检测。用PCR技术扩增CPPB基因有助于淋病的确诊。淋球菌的耐药性与β 内酰胺酶基因存在一定关系。β 内酰胺酶基因的检测对快速判断淋球菌是否高度耐受青霉素有临床指导意义。     

parC基因突变致环丙沙星不敏感化脓性链球菌及其同源性研究

目的 研究环丙沙星不敏感化脓性链球菌的耐药机制及其同源性.方法 对2012年3月北京地区分离自猩红热患者的48株化脓性链球菌采用稀释法检测环丙沙星及临床常用7种抗生素的MIC.对环丙沙星MIC ≥4 mg/L的13株化脓性链球菌检测氟喹诺酮染色体介导的耐药基因gyrA,gyrB,parC,parE的突变,同时以环丙沙星MIC≤0.25 mg/L的4株化脓性链球菌做比对.采用脉冲场凝胶电泳(PFGE)对分离自北京不同地区的菌株进行同源性分析.结果 化脓性链球菌对左氧氟沙星、氨苄西林和青霉素的敏感率均为100％.对四环素、红霉素和克林霉素的耐药率分别为91.7％ (44/48)、91.7％ (44/48)和89.6％ (43/48).环丙沙星、左氧氟沙星和莫西沙星的MIC50分别为2 mg/L、1 mg/L和≤0.25 mg/L;MIC90分别为4 mg/L、2 mg/L和0.5 mg/L.48株化脓性链球菌中有12株对环丙沙星MIC为4 mg/L,1株MIC为8 mg/L,菌株来自北京朝阳区.对这13株耐药基因检测结果显示,12株在parC基因上出现79位丝氨酸突变为苯丙氨酸或酪氨酸(Ser79Phe/Tyr),10株在parC基因的121位出现丙氨酸突变为缬氨酸(Ala121Val).发现1株在parC基因上Ser79Phe突变合并parE基因上371位丝氨酸突变为亮氨酸(Ser371Leu)的化脓性链球菌,但该菌株对环丙沙星的MIC未显著增高(MIC=4 mg/L).17株试验菌株的PFGE结果显示为7个克隆,其中克隆A均为环丙沙星MIC≥4 mg/L的菌株,主要分布在朝阳区、大兴区、丰台区、顺义区和石景山区,占MIC≥4 mg/L菌株的69.2％.克隆C菌株也为环丙沙星MIC ≥4 mg/L菌株,均分布在怀柔区.克隆B、D、E、F和G分别分散在不同地区.结论 parC基因突变是北京地区分离的化脓性链球菌对环丙沙星MIC略有升高的主要原因,PFGE分析显示化脓性链球菌感染在北京部分地区有小范围流行.     

肺炎支原体对大环内酯类抗生素耐药性及耐药机制研究

目的 了解肺炎支原体(mycoplasma pneumoniae,MP)对大环内酯类抗生素的耐药情况及耐药机制.方法 对370份咽拭子标本进行MP分离培养,应用套式PCR扩增MP种特异16S核蛋白体RNA(16SrRNA)基因对临床分离株进行分子鉴定;通过药物敏感实验测定MP分离株对大环内酯类药物的MIC并筛选出耐药株;设计套式PCR扩增红霉素作用靶位23S核蛋白体RNA(23SrRNA)基因,扩增产物进行全自动DNA测序,测得序列与NCBI已登录的MP标准株M129(登录号X68422)23SrRNA基因作比对.结果 370份临床标本中分离MP 50株,分离阳性率为13.5%.50株中敏感株4株,耐药株46株(占92%).耐药菌株的红霉素、阿奇霉素、交沙霉素MIC值均升高.4株敏感株和肺炎支原体国际标准株FH的23SrRNA基因序列与基因库的MP基因序列相同,46株耐药株的23SrRNA基因发生点突变,41株突变位点在23SrRNA V区中心环的2063位,其中40株发生了A→G的点突变,1株发生了A→C的点突变;另5株突变位点在2064位,A→G.结论 MP对大环内酯类抗生素耐药率高,耐药性的分子基础是23SrRNA基因的点突变,其中2063位点突变占主导地位.23SrRNA基因发生点突变的肺炎支原体对红霉素、阿奇霉素及交沙霉素的MIC值均升高.     

淋球菌mtrR基因突变与多重耐药的研究

[目的]研究淋球菌多重耐药调节基因mtrR突变与淋球菌多重耐药的关系。[方法]纸片扩散法检测52株淋球菌对4种抗生素的敏感性；PCR扩增mtrR基因中最常发生突变的250个碱基进行SSCP（单链构象多态性）分析；根据SSCP的结果，选取11株标本，扩增其mtrR基因，将其核苷酸序列与标准敏感株ATCC19424进行比较。[结果]52株细菌中有8株对4种抗生素都敏感：对一种抗生素耐药的有19株，只有一株mtrR基因发生点突变；对两种抗生素耐药的有16株，其中有两株mtrR基因发生了点突变：对3种抗生素和4种抗生素耐药的株菌分别是7株和2株，它们的mtrR基因都发生了点突变。在这11株标本中，45gly（GGC→GAC）asp突变为5株，105位his（CAC→TAC）tyr的突变为6株。[结论]mtr基因突变介导淋球菌的多重耐药；mtrR基因45gly（GGC→GAC）asp和105位his（CAC→TAC）tyr的突变与淋球菌多重耐药的关系密切。     

淋球菌临床分离株青霉素耐药性与β-内酰胺酶基因相关性探讨

目的了解淋球菌临床分离株青霉素耐药性与β-内酰胺酶基因存在的相关性。方法对2003年2～12月分离的128株淋球菌临床分离株用PCR法检测其特异的隐蔽质粒CPPB基因和青霉素耐药菌的β-内酰胺酶基因；用琼脂稀释法测定其对青霉素的最小抑菌浓度(MIC)。结果128株临床分离菌中CPPB基因阳性者125株，占97．7％。其中MIC≥1mg／L的青霉素耐药菌86株，占CPPB基因阳性者的68．8％；MIC≥8mg／L的高耐药菌59株，占耐药菌株总数的68．6％。86株耐药菌中β-内酰胺酶基因PCR扩增阳性者62株，占72．1％，其中58株(67．4％)为高耐药菌。结论研究表明。目前淋球菌临床分离株耐药情况较为严重，如果用青霉素治疗淋病需首先进行耐药检测。用PCR技术扩增CPPB基因有助于淋病的确诊。淋球菌的耐药性与β-内酰胺酶基因存在一定关系。β-内酰胺酶基因的检测对快速判断淋球菌是否高度耐受青霉素有临床指导意义。     

上海市某教学医院淋病奈瑟菌耐药特征及耐药机制

目的了解同济大学附属东方医院南院淋病奈瑟菌(简称淋球菌)的耐药特征、耐药质粒型别和耐药机制,为淋球菌的防控提供参考依据。方法收集2016年9月—2017年9月同济大学附属东方医院南院142株淋球菌临床分离株,采用纸片扩散法进行淋球菌对7种临床常用抗菌药物的体外药物敏感性试验;采用头孢硝噻吩法检测菌株β-内酰胺酶;对β-内酰胺酶阳性菌株的质粒进行分子分型,分析其流行病学特征;扩增头孢菌素非敏感菌株penA基因并测序,与参考菌株比对,分析不敏感的机制。结果淋球菌对青霉素、头孢曲松、头孢克肟、四环素、环丙沙星、大观霉素和阿奇霉素的非敏感率分别为100%、1.4%、17.6%、99.3%、99.3%、0%和8.5%。有54株(38%)淋球菌产β-内酰胺酶,其中携带亚洲型、非洲型和多伦多型质粒的淋球菌分别有26株(48%)、27株(50%)和1株(2%)。25株头孢菌素非敏感菌株中,药物作用靶蛋白[青霉素结合蛋白2(PBP2)]均发生了突变。突变点主要集中在311～582氨基酸位点,有12株突变位点较少(≤10个),但存在A501T/V、A516G、G542S、P551S/T关键位点突变;另外13株突变位点较多(50～60个),但无关键突变位点。结论治疗淋球菌感染经验用药可首选头孢曲松和大观霉素,阿奇霉素和头孢克肟可作为备选药物,青霉素、四环素和环丙沙星已经不适用于淋球菌感染的治疗。淋球菌对β-内酰胺类抗菌药物耐药,以携带非洲型质粒和亚洲型质粒为主,多伦多型质粒少见。淋球菌对头孢菌素不敏感的机制较复杂,药物作用靶蛋白突变多样。     

金黄色葡萄球菌临床株对利奈唑胺耐药性及耐药机制

目的了解金黄色葡萄球菌(金葡菌)临床株对利奈唑胺耐药性及耐药机制。方法收集2011-2014年浙江省人民医院900株金葡菌,采用纸片扩散法测定利奈唑胺药敏,对筛选到的耐药菌株采用琼脂稀释法测定常用药物的最低抑菌浓度(MIC),同时对其耐药基因cfr、optr A、23S r RNA基因第5功能区进行PCR扩增和序列分析。对cfr或optr A基因阳性的菌株进行基因周围序列分析和菌株多位点序列分型(MLST)。结果临床分离金葡菌对利奈唑胺耐药率为0.1%(1/900),仅发现1株耐药株,该菌株为耐甲氧西林金葡菌(MRSA),对利奈唑胺MIC为8 mg/L、头孢西丁48 mg/L、青霉素32 mg/L、环丙沙星128 mg/L、克林霉素32 mg/L、氯霉素128 mg/L、万古霉素0.75 mg/L、替考拉宁0.5 mg/L。除万古霉素和替考拉宁,对其他抗菌药物均耐药。PCR结果显示该菌株cfr基因为阳性,optr A阴性,无23S rRNA突变。该菌株携带的cfr基因位于"Tn4001-like转座子-cfr-orf1-ISEnfa4"复合转座子中,并定位于一个39 504 bp的质粒上。该菌株的MLST分型为ST5。结论临床分离金葡菌对利奈唑胺耐药率低,发现1株cfr基因介导的利奈唑胺耐药株,cfr基因位于质粒上一个常见的复合转座子中。     

武陵山区淋病奈瑟菌对7种抗菌药物的耐药情况及分子生物学特征分析

目的通过监测武陵山区淋病奈瑟菌的抗菌药物耐药情况及分子生物学特征,了解该地区是否存在头孢曲松耐药菌株及相关流行病学信息。方法回顾性分析2018年4月1日至2019年3月31日该地区培养分离出的122株淋病奈瑟菌的临床资料。药敏试验参考欧洲药敏试验委员会(EUCAST)标准,采用K-B法和琼脂稀释法检测淋病奈瑟菌对头孢曲松和阿奇霉素等7种抗菌药物的敏感性。采用多抗原序列分型法(NGMAST)对头孢曲松低敏感性及耐药菌株进行基因分型,并用MEGA6构建POR和TBPB基因系统发育树。结果 122株淋病奈瑟菌株对头孢曲松、头孢泊肟、头孢克肟、壮观霉素、阿奇霉素、四环素和环丙沙星的敏感率分别为98.36%、92.62%、90.16%、81.15%、78.69%、37.70%和12.29%。2株头孢曲松耐药菌株最低抑菌浓度(MIC)分别为1.000μg/mL和0.250μg/mL,NG-MAST型分别为ST2987型和ST14155型。结论该研究发现1株高水平头孢曲松耐药的淋病奈瑟菌,但未发现流行病学同源耐药菌株。武陵山区淋病奈瑟菌耐药形势严峻,应严控头孢曲松耐药菌株传播。     

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.     

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.     

Azithromycin Resistance and Its Mechanism in Neisseria gonorrhoeae Strains in Hyogo,Japan

Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala→Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection.     

High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure

Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management.     

Is Neisseria gonorrhoeae Initiating a Future Era of Untreatable Gonorrhea?: Detailed Characterization of the First Strain with High-Level Resistance to Ceftriaxone

Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.     

Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates in Kyoto and Osaka,Japan, 2010 to 2012: Intensified Surveillance after Identification of the First Strain (H041) with High-Level Ceftriaxone Resistance

In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 μg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.     

Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity

In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen(i)) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to beta-lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen(i) isolates comprised an MIC of >0.094 microg/ml for penicillin G and an MIC of >0.064 microg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen(s) or Pen(i). In comparison with the wild-type penA sequence, two specific Pen(i) sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.     

Acquired macrolide resistance genes and the 1 bp deletion in the mtrR promoter in Neisseria gonorrhoeae

The presence of macrolide-lincosamide-streptogramin B resistance genes erm(B), erm(C) and erm(F), the macrolide resistance mef(A) gene, and the DNA sequence of a 13 bp repeat in the promoter region of the mtrR gene, were determined in 62 Neisseria gonorrhoeae isolates collected between 1992 and 1999 in Seattle, Washington, USA. Eleven isolates with erythromycin and azithromycin MICs of < or =0.06 mg/L, had no acquired genes or deletions in the 13 bp repeat region. Among 44 isolates with erythromycin MICs 1.0-16.0 mg/L, and azithromycin MICs 0.06-4.0 mg/L, 16 carried the 1 bp deletion in the mtrR promoter region alone, nine carried one or more of the four acquired macrolide resistance genes alone, and 14 carried both acquired macrolide resistance genes plus the 1 bp deletion in the mtrR promoter region. Three isolates with erythromycin MICs > or = 8 mg/L, and azithromycin MICs of 4.0 mg/L, carried only erm genes. Five isolates with MICs of 1-2 mg/L did not carry the 1 bp deletion, or any of the acquired resistance genes examined. Our data suggest that the 1 bp deletion in the mtrR promoter region is not found in all erythromycin-resistant (MIC > or = 1.0 mg/L) N. gonorrhoeae.     

The spectrum of antibiotic resistance in human and veterinary isolates of Escherichia coli collected from 1984-86 in northern India.

This study was undertaken to assess the spectrum of drug resistance prevalent in Escherichia coli isolates from human and animal populations in Northern India. Three hundred and two isolates of Escherichia coli isolated from various infections of humans (47 from diarrhoea; 101 from urinary tract infection) and veterinary animals (17 from poultry septicaemia; 75 from bovine diarrhoea; 14 from ovine diarrhoea and 48 from equine metritis) were studied for their susceptibility to ampicillin, cephaloridine, amoxycillin, cloxacillin, oxytetracycline, doxycycline, chloramphenicol, gentamicin, and trimethoprim-sulphamethoxazole. 63.2% of the isolates (37.7%, human; 25.5%, animal) were resistant to one or more drugs, of which about 41% isolates were multiresistant. Resistant isolates had a wide range of MIC values from 12 to 3200 mg/L, irrespective of origin. Most of the isolates (43.5%) were resistant to ampicillin (MIC greater than 16 mg/L) followed by oxytetracycline (MIC greater than 5 mg/L) (36.4%). Only 9.3% isolates were resistant to trimethoprim-sulphamethoxazole (MIC greater than 21 mg/L). The E. coli isolated from animals tended to be resistant to fewer antibiotics than those isolated from man. 99% of the isolates from human urinary tract infection were resistant compared to only 48.9% of the isolates from diarrhoea. Most of the resistant isolates from animals were recovered from diarrhoea, followed by septicaemia and metritis. This study shows that a high frequency of multiresistant strains are prevalent in both human and animal bacterial populations of Northern India. Since the exchange of these strains among both populations is possible, they pose a great risk in both the selection and the spread of resistance.     

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.