临床分离志贺菌质粒介导喹诺酮类耐药基因的检测

目的 了解临床分离志贺菌质粒介导喹诺酮类耐药基因的种类、分布以及对抗菌药物的耐药性。方法 PCR法检测137株志贺菌的qnr、aac(6′)- Ib-cr和qepA基因,对阳性扩增产物进行测序分析并对阳性菌株做接合试验,采用琼脂倍比稀释法测定志贺菌、受体菌和接合子对喹诺酮类及其他抗菌药物的最低抑菌浓度(MIC)。PCR...     

Prevalence of plasmid-mediated quinolone resistance in Escherichia coli isolates in Wenzhou, Southern China, 2002-2008

A total of 514 consecutive clinical Escherichia coli isolates, irrespective of resistance background, were collected in the period 2002-2008 in Wenzhou, southern China, to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR). The dominant PMQR gene was aac(6')-Ib-cr, followed by qnr, whereas qepA was absent. A total of 253 (49.2%) of these isolates were aac(6')-Ib-positive. Subsequently, 134 of these isolates were sequenced and 42 (31.3%) found to harbor aac(6')-Ib-cr, 18 to harbor new aac(6')-Ib mutants, and 74 to harbor wild-type aac(6')-Ib. The genes qnrA, qnrB, and qnrS were found in 2 (0.4%), 6 (1.2%), and 14 (2.7%) of 514 isolates, respectively, with 2 isolates co-harboring qnrB and qnrS genes. Sequencing allowed us to identify qnrA1, qnrB4, qnrB6, and qnrS1 in 20 qnr-positive isolates, with qnrS1 being the most prevalent allele. The genes qnrC and qnrD were not found in any isolates. Interestingly, 35% of qnr-positive isolates and 16.7% of aac(6')-Ib-cr-positive isolates were susceptible to ciprofloxacin. PMQR genes are therefore present in both quinolone-resistant and -susceptible isolates and can also be transferred by conjugation experiments, thus suggesting a likely future increase in quinolone resistance.     

Molecular characteristics of extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines

β-Lactamases, including extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases, are major resistance mechanisms of Enterobacteriaceae. Emergence of plasmid-mediated quinolone resistance (PMQR) determinants in ESBL-producing isolates poses a global threat. The molecular characterisitcs of ESBL and PMQR determinants in the Philippines are not well characterized. In this study, we investigated ESBLs and AmpC β-lactamases in clinical isolates of Enterobacteriaceae from the Philippines, and analyzed the association between ESBL and PMQR genes. A total of 91 amoxicilin non-susceptible Enterobacteriaceae were collected at the Research Institute for Tropical Medicine of the Philippines from 2006 to 2008. AmpC- or ESBL-producing isolates were screened by detecting a zone diameter for cefoxitin ≤ 14 mm or cefpodoxime ≤ 20 mm, respectively. Possible ESBL-producing strains were assessed by the ESBL confirmation test of the Clinical and Laboratory Standards Institute. PCR and sequencing were performed to detect the ESBL and PMQR genes. The number of ESBL-producers and AmpC-producers confirmed phenotypically was 17 (18.7%) and 61 (67.0%), respectively. Of 17 phenotypic ESBL-producers, 14 isolates had ESBL genes, including 6 of Escherichia coli, 3 of Enterobacter cloacae, 2 of Enterobacter aerogenes, 2 of Klebsiella pneumoniae, and 1 of Klebsiella ozaenae. Among these isolates, there were 13, 4, and 12 with bla(CTX-M), bla(SHV), and bla(TEM), respectively. Of the bla(CTX-M)-positive isolates, bla(CTX-M-15) shows the highest prevalence, followed by bla(CTX-M-3) and bla(CTX-M-14). Of 14 ESBL-producers identified by PCR, 4, 6, and 7 isolates were positive for qnrB, qnrS, and aac(6')-Ib-cr, respectively. The frequency of aac(6')-Ib-cr positivity was significantly higher among CTX-M-15-producing isolates. Thus, we identified bla(CTX-M), aac(6')-Ib-cr, and qnr in ESBL-producing Enterobacteriaceae from the Philippines, and revealed a significant association between bla(CTX-M-15) and aac(6')-Ib-cr. Local epidemiological data are important for implementing appropriate antimicrobial therapy and effective infection control measures. Continuous monitoring of antimicrobial resistance genes in the Philippines will be required.     

广西生鲜畜禽产品源致病性大肠杆菌血清型、耐药性及耐药基因调查

目的 了解广西生鲜畜禽产品中致病性大肠杆菌血清型分布、耐药性以及β-内酰胺酶bla_TEM、bla_CTX-M基因和氟喹诺酮类抗生素耐药基因qnrA、oqxA、oqxB、aac(6')-Ib-cr的流行情况。方法 采用玻片凝集法对2015—2016年间分离自广西生鲜畜禽产品中的249株致病性大肠杆菌进行“O”抗原血清型鉴定;K-B纸片法对已定型菌株进行24种常见抗生素的敏感性试验;PCR方法检测bla_TEM、bla_CTX-M、qnrA、oqxA、oqxB、aac(6')-Ib-cr。结果 249株致病性大肠杆菌中136株可定型、96株未定型、17株自凝,分别占鉴定菌株的54.6%、38.6%、6.8%;136株已定型菌株分属25个血清型,以O8 (19.85%) 、O86 (8.82%) 、O6 (6.62%)、O28(6.62%) 、O124 (6.62%)为主要优势血清型;136株已定型菌株对24种抗生素均产生不同程度的耐药,耐药率从16.18%到100%,且全部为多重耐药,主要集中在12~18重耐药,共占多重耐药的72.06%;136株已定型菌株对6种耐药基因的检出率为qnrA 91.18%(124/136)、oqxA 80.88%(110/136)、oqxB 50.00%(68/136)、aac(6')-Ib-cr 35.29%(48/136)、bla_TEM 100.00%(136/136)、bla_CTX-M 23.53%(32/136)。结论 广西生鲜畜禽产品源致病性大肠杆菌O抗原血清型呈多样性分布,耐药情况严重,临床日益严重的耐药现象与耐药基因的普遍存在有重要的关系。     

48株鲍曼不动杆菌氨基糖苷类抗生素耐药基因检测

目的了解鲍曼不动杆菌氨基糖苷类抗生素耐药基因分布情况。方法收集蚌埠医学院第一附属医院2015年1月至12月临床分离的48株泛耐药鲍曼不动杆菌,VITEK 2Compact进行鉴定和药敏实验。PCR法检测12个氨基糖苷类修饰酶基因和3个甲基化酶基因以及外排泵基因adeB。结果在实验的16个基因中,共检出4种氨基糖苷类抗生素耐药基因aac(6′)-Ib、armA、adeB和ant(3″)-Ia,其中aac(6′)-Ib检出率为39.6%(19/48),armA基因检出率为89.6%(43/48),adeB检出率89.6%(43/48),ant(3″)-Ia检出率为10.4%(5/48),其余基因均未检出;存在两种以上耐药基因的共39株,检出率为81.3%(38/48)。结论 aac(6′)-Ib、armA基因以及外排泵adeB是介导我院鲍曼不动杆菌氨基糖苷类抗生素耐药的主要基因。     

Epidemiology of plasmid-mediated quinolone resistance in salmonella enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 225 isolates of Salmonella enterica serovar Typhimurium from food-producing animals collected between 2003 and 2007 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, namely qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')Ib-cr, in Japan. Two isolates (0.8%) of S. Typhimurium DT104 from different dairy cows on a single farm in 2006 and 2007 were found to have qnrS1 on a plasmid of approximately 9.6-kbp. None of the S. Typhimurium isolates had qnrA, qnrB, qnrC, qnrD, qepA and acc(6')-Ib-cr. Currently in Japan, the prevalence of the PMQR genes among S. Typhimurium isolates from food animals may remain low or restricted. The PFGE profile of two S. Typhimurium DT104 isolates without qnrS1 on the farm in 2005 had an identical PFGE profile to those of two S. Typhimurium DT104 isolates with qnrS1. The PFGE analysis suggested that the already existing S. Typhimurium DT104 on the farm fortuitously acquired the qnrS1 plasmid.     

下呼吸道感染患者铜绿假单胞菌分离株氨基糖苷类耐药相关基因分析

目的探讨分离自下呼吸道感染患者的氨基糖苷类耐药铜绿假单胞菌的耐药机制。方法从下呼吸道感染患者痰液中分离出52株对氨基糖苷类耐药的铜绿假单胞菌,PCR法检测6种氨基糖苷类修饰酶(AMEs)基因,并检测其中泛耐药菌的6种16S rRNA甲基化酶基因(以下简称甲基化酶基因)。对阳性产物测序加以证实。结果 52株铜绿假单胞菌中检出4种AMEs基因[aac(3)-Ⅱ、aac(6’)-Ⅰb、aac(6’)-Ⅱ和ant(2″)-Ⅰ],AMEs基因总检出率为92.3%。泛耐药菌中检出1种甲基化酶基因(rmtB)。16株高水平泛耐药菌中rmtB基因的检出率为81.3%。结论分离自下呼吸道感染患者的氨基糖苷类耐药铜绿假单胞菌中AMEs基因携带率高,其对氨基糖苷类耐药与aac(3)-Ⅱ、aac(6’)-Ⅰb、aac(6’)-Ⅱ和ant(2″)-Ⅰ有关;对氨基糖苷类高水平泛耐药主要与甲基化酶基因rmtB有关。     

外科住院患者感染耐甲氧西林凝固酶阴性葡萄球菌的耐药基因检测及耐药性分析

目的对南京市外科住院患者感染的耐甲氧西林凝固酶阴性葡萄球菌的耐药相关基因进行检测,以更好地控制病原菌耐药性,减少外科感染。方法从南京市多家医院外科住院患者各类临床标本分离耐甲氧西林凝固酶阴性葡萄球菌70株,经纯培养获单菌落后进行鉴定,并进行药敏试验,然后对耐药基因进行PCR测序,对PCR扩增产物进行电泳检测并记录。结果耐甲氧西林凝固酶阴性葡萄球菌对于头孢噻肟和红霉素100%耐药,对万古霉素耐药率为0,对其他抗菌药物的耐药率大小依次为:左氧氟沙星>诺氟沙星>氨苄西林>妥布霉素>环丙沙星>克林霉素>头孢曲松>庆大霉素>四环素>利福平。PCR检测耐药基因,3株只携带mecA 1种耐药基因。15株携带2种耐药基因,分别为:ermA+mecA、aac(6′)/aph(2′′)+mecA和aph(3′)-III+mecA和tetM+mecA。29株携带3种耐药基因,携带方式分别为:ermA+aac(6′)/aph(2′′)+mecA、aph(3′)-III+ermA+mecA、ermA+tetM+mecA、aac(6′)/aph(2′′)+aph(3′)-III+mecA、aac(6′)/aph(2′′)+tetM+mecA和tetM+aph(3′)-III+mecA。20株携带4种耐药基因,携带方式分别为:aac(6′)/aph(2′′)+tetM+ermA+mecA、aph(3′)-III+tetM+aac(6′)/aph(2′′)+mecA、aph(3′)-III+tetM+ermA+mecA和aph(3′)-III+aac(6′)/aph(2′′)+ermA+mecA。有3株携带5种耐药基因,即ermA+aph(3′)-III+tetM+aac(6′)/aph(2′′)+mecA。结论耐甲氧西林凝固酶阴性葡萄球菌对常用抗菌药物的耐药性普遍较高,5种耐药基因在菌株中出现的频率不同,但所有病原菌都存在mecA基因,因而该菌的耐药性可能与mecA基因有关。     

成都市人源沙门菌基因组特征分析

目的 基于全基因组测序技术,探究成都市人源沙门菌的基因组特征, 为监测与预防沙门菌感染提供资料。方法 收集35株成都市人源沙门菌(分离自腹泻患者粪便)进行全基因组测序。根据测序数据,进行血清型预测、耐药基因及可移动遗传元件预测、毒力因子注释及分布分析。结果 35株沙门菌分离株经全基因组测序,共预测到5种血清型,包括15株I 4,[5],12:i:-沙门菌(13株为ST34、2株为新ST型)、12株鼠伤寒沙门菌(ST19)、5株肠炎沙门菌(ST11)、2株德比沙门菌(ST40)与1株火鸡沙门菌(ST463)。35株沙门菌分离株共预测到10类42种不同的耐药基因,其中氨基糖苷类aac(6')-Iaa携带率为100%(35/35)。I 4,[5],12:i:-沙门菌的耐药基因、质粒及插入序列数目及种类多于其他血清型菌株。I 4,[5],12:i:-、鼠伤寒沙门菌和肠炎沙门菌的毒力因子数目大于其他血清型菌株。部分鼠伤寒沙门菌有更高的毒力质粒、质粒编码菌毛和血清抗性毒力因子分布。结论 成都市人源沙门菌不同血清型菌株之间耐药基因、可移动遗传元件、毒力因子分布差异明显,值得在转录组、蛋白组进一步探究其耐药与毒力机制。     

医院获得性鲍曼不动杆菌肺炎六例微生物学及临床观察

目的探讨医院获得性鲍曼不动杆菌肺炎患者临床微生物学特点和治疗转归。方法回顾性调查2005年1～8月白求恩国际和平医院经分子生物学方法证实的6例医院获得性产PER-1型超广谱13内酰胺酶（ESBL）鲍曼不动杆菌肺炎患者的临床资料。用脉冲场凝胶电泳检测6株鲍曼不动杆菌的同源性，聚合酶链反应分析相关耐药基因。结果6株鲍曼不动杆菌中5株仅对亚胺培南敏感，4株对美罗培南敏感。脉冲场凝胶电泳将其分为A1型2株，A2～如型各1株，携有β内酰胺酶blaPER-1基因，同时blaTEM-1型基因、耐消毒剂磺胺药物基因（qacEΔ1-sull）和1类整合子酶基因（intll）阳性，其中3株氨基糖苷类修饰酶基因aac（3）-I、aac（6’）-I同时阳性，2株aac（3）-I、ant（3”）-I同时阳性，1株aac（3）-I单独阳性。6例患者均患有严重的基础疾病，使用过呼吸机；检出鲍曼不动杆菌前15d内应用过广谱抗菌药物；经使用碳青霉烯类和（或）头孢哌酮／舒巴坦抗感染治疗，3例临床好转，3例死亡。结论6株产PER-1型ESBL鲍曼不动杆菌具有高度同源性，耐药基因复杂，其所引起的医院获得性肺炎临床预后差。     

Distribution of genes encoding resistance to aminoglycoside modifying enzymes in methicillin-resistant Staphylococcus aureus (MRSA) strains

Today Methicillin-Resistant Staphylococcus aureus (MRSA) have acquired multiple resistance to a wide range of antibiotics including aminoglycosides. So, this study was aimed to investigate the rate of aminoglycoside resistance and the frequency of aminoglycoside resistance mediated genes of aac(Ia)-2, aph(3)-IIIa and ant(4′)-Ia among MRSA strains. A total of 467 staphylococci isolates were collected from various clinical samples. S. aureus strains were identified by standard culture and identification criteria and investigating of presence of 16S rRNA and nuc genes. Cefoxitin disk diffusion, and oxacillin-salt agar screening methods were used to detect the MRSA strains with subsequent molecular identification for the presence of mecA gene. Antibiotic susceptibility of MRSA strains against aminoglycoside antibiotics was evaluated by using agar disk diffusion method. Multiplex PCR for the presence of aac(Ia)-2, aph(3)-IIIa and ant(4′)-Ia encoding genes for aminoglycosides were performed for MRSA strains. From total staphylococci tested isolates, 262 (56.1%) were identified as S. aureus, of which 161 (61.45%) were detected as MRSA and all comprised mecA gene. The resistance pattern of MRSA strains to aminoglycoside antibiotics were: gentamicin 136 (84.5%); amikacin 125 (77.6%); kanamycin 139 (86.3%); tobramycin 132 (82%); and neomycin 155 (96.3%). The frequency of aac(Ia)-2, aph(3)-IIIa, and ant(4′)-Ia genes among MRSA strains, were 64%, 42% and 11.8% respectively. In conclusion, as MRSA strains are of great concern in human infections, the results of present study could provide a useful resource for health sectors for choosing appropriate antibiotics for the effective treatment of infections due to MRSA strains.     

Prevalencia del determinante de resistencia plasmídica a quinolonas aac(6’)-Ib-cr en cepas de Escherichia coli y Klebsiella pneumoniae productoras de BLEE aisladas en diez hospitales de Chile

Introduction

The frequency of aac(6′)-Ib-cr gene in ESBL-producing strains of Klebsiella pneumoniae and Escherichia coli is unknown, in Chile.

Methodology

The aac(6′)-Ib and aac(6’)-Ib-cr genes were investigated using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and sequencing, in strains isolated from 10 Chilean hospitals between 2008-2009.

Results

The aac(6’)-Ib-cr gene was detected in 54% of K. pneumoniae and 74% of E. coli strains. The CIM₅₀ of CIP was higher among strains harboring aac(6’)-Ib-cr, 8 times higher in K. pneumoniae and 4 times higher in E. coli. Moreover, both aac(6’)-Ib and aac(6’)-Ib-cr were simultaneously found in 13 K. pneumoniae and 3 E. coli isolates.

Conclusion

This is the first report of aac(6’)-Ib-cr in ESBL-producing strains of K. pneumoniae and E. coli isolated from in-patients in Chilean hospitals located along an area of more than 2,800 Km.     

铜绿假单胞菌耐药基因分析及多位点序列遗传分型

目的 了解2011-2012年深圳市南山区医院病人,各医院、诊所内环境台面涂抹样、医护人员手涂抹样分离的铜绿假单胞菌,抗生素耐药基因分布及基因的遗传多样性。方法 采用聚合酶链反应技术检测铜绿假单胞菌的20种耐药基因:TEM、VEB、CARB、OXA、SHV、PER、GES、GTX、SPM、GIM、IMP、VIM、DHA、oprD、Aac(6')-Ⅰ、Aac(6')-Ⅱ、Aac(3')-Ⅰ、Aac(2″)-Ⅰ、qacE1-sull及Ⅰ类整合子基因。采用多位点序列分子分型方法 进行聚类和克隆分析。结果 检出11种耐药基因:TEM、SHV、IMP、DHA、Aac(6’)-Ⅰ、Aac(6')-Ⅱ、Aac(3')-Ⅰ、Aac(2″)-Ⅰ、qacE1-sull、Ⅰ类整合子及oprD基因,检出率分别为8.1%、6.4%、4.8%、9.7%、4.8%、14.5%、4.8%、56.5%,8.1%,8.1%,oprD基因缺失率为61.2%。52株铜绿假单胞菌检出耐药基因,形成19种耐药基因谱。多位点序列分型方法 将62株铜绿假单胞菌,分为39个ST型,5个克隆群,1个优势克隆群CC244,1个优势独特型ST856。结论 不同类型样本分离菌株携带耐药基因存在差异,部分病人分离株携带多种耐药基因。本研究铜绿假单胞菌具有遗传多样性,存在优势克隆群。     

First detection and sequence analysis of the bla-CTX-M-15 gene in Lebanese isolates of extended-spectrum-beta-lactamase-producing Shigella sonnei

The emergence in Shigella species of extended-spectrum beta-lactamases (ESBL) that impart resistance to third-generation cephalosporins is a growing concern world-wide. So far, however, ESBL-producing Shigella have only been reported seven times, albeit from seven different countries. In Lebanon, three ESBL-producing clinical isolates of S. sonnei were recovered from 30 cases of shigellosis diagnosed between July 2004 and October 2005. All three were found to be resistant to amoxycillin, cefotaxime, ceftazidime, aztreonam, trimethoprim/sulphamethoxazole, gentamicin, and kanamycin. Each harboured the bla-CTX-M gene, and the results of sequence analysis indicated this to be of the bla-CTX-M-15 type and encoded on a 70-kb plasmid, flanked by an insertion element (ISEcp1). The bla-TEM-1 gene was also detected on the chromosomes of two of the ESBL-producing isolates. Class-2 integrons containing dhfr1, aadA1 and sat1 genes were detected on the chromosomes of all three isolates but not on the plasmids. Fluoroquinolone-modifying factors [QnrA, QnrB, QnrS or AAC(6')-Ib-cr] were not detected. The results of RAPD analysis, combined with data on antimicrobial susceptibility, indicated that each isolate was unique. In conclusion, the emergence of ESBL-producing isolates of S. sonnei has been demonstrated for the first time in Lebanon. The resistance of these isolates to third-generation cephalosporins was mediated by the CTX-M-15 enzyme, which was plasmid-encoded.     

A 56-month prospective surveillance study on the epidemiology of aminoglycoside resistance in a Belgian general hospital.

In this survey, we studied the effect of extensive amikacin usage on the epidemiology of aminoglycoside resistance in a general hospital. The baseline resistance in the 12 months before amikacin was 5.8% for amikacin, 15.2% for gentamicin, 16.4% for tobramycin and 14.0% for netilmicin. During the following 44 months, amikacin was the aminoglycoside of first choice. In the first 2 years of this phase, resistance to amikacin did not change significantly. Later, amikacin resistance rose significantly, mainly due to the introduction of amikacin-resistant Enterobacter aerogenes strains. In general there was a significant decrease in resistance to gentamicin and tobramycin. Resistance mechanisms were examined in 380 strains. AAC(3)V, and AAC(6')I alone or coupled with ANT(2") or AAC(3) were the most prevalent enzymes. In the amikacin phase, we noticed a significant increase of strains harbouring the AAC(6')I enzyme, while strains with the AAC(3)V were less frequently isolated. Strains with permeability resistance did not become more prevalent during the period of extensive amikacin use.     

Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonellaenterica serovar Typhimurium and two were S.enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The β-lactamase-encoding gene, bla_TEM-1, was identified in five isolates and the extended-spectrum β-lactamase-encoding genes, bla_CMY-2 and bla_SHV-12, were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6′)-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6′)-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.     

The worldwide emergence of plasmid-mediated quinolone resistance

Fluoroquinolone resistance is emerging in gram-negative pathogens worldwide. The traditional understanding that quinolone resistance is acquired only through mutation and transmitted only vertically does not entirely account for the relative ease with which resistance develops in exquisitely susceptible organisms, or for the very strong association between resistance to quinolones and to other agents. The recent discovery of plasmid-mediated horizontally transferable genes encoding quinolone resistance might shed light on these phenomena. The Qnr proteins, capable of protecting DNA gyrase from quinolones, have homologues in water-dwelling bacteria, and seem to have been in circulation for some time, having achieved global distribution in a variety of plasmid environments and bacterial genera. AAC(6')-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Both mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels. Much remains to be understood about these genes, but their insidious promotion of substantial resistance, their horizontal spread, and their co-selection with other resistance elements indicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.     

Molecular diversity of class 1 integrons in human isolates of Aeromonas spp. from southern Taiwan

This work studies antimicrobial resistance and class 1 integrons of Aeromonas spp. in human isolates from southern Taiwan. PCR amplification and DNA sequence analyses were performed to characterize the gene cassette regions of the class 1 integron in 204 isolates of Aeromonas hydrophila, 36 isolates of A. sobria, 23 isolates of A. veronii, and 4 isolates of A. caviae. By using Southern hybridization with an intI1 probe to determine the presence of class 1 integrons in the 9 isolates of Aeromonas spp. harboring plasmid DNA, only 2 isolates, one A. veronii AV69 harboring 176-kb plasmid DNA, and one A. hydrophila AH207 harboring 149-kb plasmid DNA were identified. A conjugation experiment was carried out with 2 isolates of A. veronii AV69 and A. hydrophila AH207. Only one transconjugant of Escherichia coli AH207, containing 149-kb plasmids obtained from A. hydrophila AH207, was identified. ERIC-PCR analysis was performed to analyze the genetic relatedness in all isolates of Aeromonas spp. that carry class 1 integrons. The results of cluster analysis in this experiment revealed that none of these isolates were clonal, which may indicate that they were not related to the outbreak. Among the 267 isolates tested, class 1 integrons were detected in 37 isolates (13.9%) of Aeromonas spp. from humans. No class 2 or class 3 integrons were detected in this study. Gene cassette structures were identified in 30 (81.0%) of 37 isolates of Aeromonas spp. containing class 1 integrons. The gene cassette of dfrA12-orfF-aadA2 was the most prevalent in the gene cassette array (16.0%), followed by arr3-aacA4 (13.3%) and dfr2d-catB3-aadA1 (10.0%). Four novel arrays of gene cassettes were also identified, namely, dfr2d-catB3-aadA1, aadA1-aac(6')-II, aadA4a, and aac(6')-II-blaOXA-21-catB3. This is the first report of Aeromonas spp. isolates from humans.     

骨科患者术后创面感染鲍曼不动杆菌对喹诺酮类抗菌药物的耐药性分析

目的了解医院骨科患者手术后创面感染的鲍曼不动杆菌的耐药情况,以指导医生合理用药,有效控制鲍曼不动杆菌耐药性的进一步发展,预防和减少医院感染的发生。方法从骨科患者手术后创面感染标本分离鲍曼不动杆菌28株,作gyrA基因测序,并进行BLASTn比对;通过测定最小抑菌浓度(minimum inhibitory concentration,MIC)进行药敏试验,测定鲍曼不动杆菌的耐药性;选取经纯培养的鲍曼不动杆菌菌落置入含蛋白酶K溶液的离心管内,分别进行水浴,再离心,留取上清液,以此为模板PCR扩增与喹诺酮类抗菌药物耐药性相关的gyrA、qepA、qnrS、qnrA、qnrB和aac(6)-Ib基因,使用Chromas分析软件进行测序。结果鲍曼不动杆菌对诺氟沙星、氧氟沙星、培氟沙星、依诺沙星、环丙沙星、司帕沙星、左氧氟沙星、加替沙星、莫西沙星的耐药率分别为:96.43%、96.43%、96.43%、89.29%、85.71%、85.71%、82.14%、78.57%和78.57%。PCR检测28株鲍曼不动杆菌gyrA基因100%阳性,即都发生了gyrA基因突变,而其他相关基因检测均阴性。基因测序看出,其83位发生突变,即由TCA转变为TTA。结论本组分离骨科患者手术创面感染的鲍曼不动杆菌对喹诺酮类抗菌药物的耐药情况严重,其中对诺氟沙星、氧氟沙星、培氟沙星等的耐药率均在90%以上。临床抗感染治疗应根据耐药结果进行用药,并控制喹诺酮类抗菌药物的使用。     

A new subtype of 3′ region of cagA gene in Helicobacter pyloristrains isolated from Zhejiang Province in China

AIM: To isolate the subtypes of 3′ region of cagA gene in Helicobacter pylori (H pylon) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3′ region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3′ region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pyloriisolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes Ⅰ, Ⅱ, and Ⅲ according to their sizes. The sizes of subtypes Ⅰ, Ⅱ, and Ⅲ were 648-650bp, 705-707bp, and 815bp, respectively. Among the 132 cagA-positive H pylori strains, 123(93.2%) belonged to the group of subtype Ⅰ, 6 (4.5%) presented subtype Ⅱ, 1(0.8%) was subtype Ⅲ, and 2(1.5%) presented subtypes Ⅰ and Ⅲ both. The primary structure of subtype Ⅰ was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype Ⅱ possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3′ region of cagA gene which had not been reported before. The primary structure of subtype Ⅲ consisted of 4 repeats of R1, Ⅰ repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype Ⅰ strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype Ⅲ strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype Ⅱ strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%),respectively. There were no statistical differences in the distribution of subtypes of 3′ region of cagA gene among different H pylori-associated gastroduodenal diseases (x^2=11.544, P>0.05). CONCLUSION: There are three subtypes (Ⅰ,Ⅱ, and Ⅲ) of 3′ region of cagA gene in Hpylori strains isolated from Zhejiang Province, and subtype Ⅰ is predominant. Subtype Ⅱ is a newly found subtype of 3′ region of cagA gene. The result of this study does not support the view that the subtypes of 3′ region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.