Identification of Quinolones/Fluoroquinolones Resistance Genes from Staphylococci Strains Isolated at the University Hospital of Brazzaville, Republic of the Congo

Staphylococci strains, like the majority of bacterial strains, have developed the resistance to several antibiotics, including Quinolones and Fluoroquin-olones In the Republic of the Congo, cases of resistance leading to treat-ment failures have been observed during the treatment of staphylococcal infections with antibiotics in hospitals. The objective of this study was to identify the Quinolone/Fluoroquinolone resistance genes from staphylo-cocci strains isolated in hospitals. A total of 51 strains of Staphylococci were isolated, including 16 (31.37%) community strains, and 35 (68.62%) clinical strains. 46 strains of Staphylococcus aureus (S. aureus) and 5 SCNs were identified. A total of 34 DNA fragments from different strains resistant to Quinolones/Fluoroquinolones, including 21 (61.67%) DNA fragments from clinical S. aureus and 13 (38.23%) from community SCN strains were analyzed by the molecular method (genotypic detection) by PCR. The genotypic results made it possible to identify the gyrA, grLA and norA genes and to show that these genes are involved in the resistance of the strains to the various antibiotics used. The grLA gene was the most identified gene with a frequency of 75%. The gyrA and grLA genes have been identified in Staphylococcus aureus and Coagulase Negative Staphy-lococci. The norA gene, on the other hand, has only been identified in Staphylococcus aureus. Two mechanisms are essentially involved in the resistance of Staphylococci to quinolones/Fluoroquinolones, the mecha-nism of resistance by efflux, which takes place thanks to a transmembrane protein coded by the norA gene and by point mutations (substitution and deletion of acids or nucleotides) observed within the protein and nucleic sequences of the chromosomal gyrA and grLA genes.

Genotypic Diversity and Characterization of Quinolone Resistant Determinants from Enterobacteriaceae in Yaoundé, Cameroon

Background: Enterobacteriaceae causes many types of infections which are often treated with quinolones and fluoroquinolone (Q/FQ). The resistance mechanisms to Q/FQ are usually associated with mutations in the quinolone resistance determining region which alter the conformation of target amino acid residues within the protein and in the qnr genes. This study aimed at determining the antimicrobial resistant profile of a sample of Enterobacteriaceae from Cameroon and the genetic diversity in quinolone-resistant isolates in view of implementing a better management, treatment, control and prevention of the transmission of these resistant strains. Methods: Identification and antimicrobial susceptibility testing was done using VITEK 2. The detection of plamid-mediated quinolone resistance (PMQR) genes was carried out using the conventional PCR method. Sequencing was done using the Applied Biosystem 3500 genetic analyser. DNA fingerprint was obtained using Pulsed-Field Gel electrophoresis. Results: Among 440 Enterobacteriaceae, the most prevalent genera were: Escherichia 178/440 (39.5%);Klebsiella 148/440 (33.6%);Enterobacter 35/440 (8%);Pantoea 28/440 (6.4%);Proteus 14/440 (3.2%) Salmonella 13/440 (3%). Ampicillin resistance showed the highest prevalence with 371/440 (81%) and Imipenem the lowest resistance 9/440 (2.1%). The ciprofloxacin resistance rate was 161/440 (36.6%). The detected plasmid mediated quinolone resistance (PMQR) genes were: qnrA, 2/161 (1.2%);qnrB, 31/161 (19.3%);qnrS, 13/161 (8.1%): Aac (6')Ib-cr, 84/161 (52.2%) and qepA, 3/161 (1.9%). There were several mutations in the parC gene of Klebsiella leading to S80D and S80N substitutions. Two pairs of Klebsiella peumoniae strains were phenotypically and genotypically identical with 100% similarity in the dendrogramme. Conclusion: This study showed that quinolone resistance was high. The PMQR genes contributing to this resistance were diverse. This high PMQR indicates that there has been an unknown circulation of these genes in our community. To avoid the rapid dissemination of these PMQR genes continuous surveillance of antimicrobial resistance should be carried out not only in humans but also in animals to monitor the evolution of these genes.

mcr-1基因阳性禽源性大肠埃希菌耐药基因研究

目的调查鲁中地区禽源性大肠埃希菌mcr-1基因的携带情况,了解mcr-1基因阳性禽源性大肠埃希菌对常用β-内酰胺类药物、氨基糖苷类药物、喹诺酮类药物耐药基因的携带情况,掌握其耐药性。方法收集2020年4月至2021年10月鲁中地区3家大规模养殖场302份健康活鸡、鸭肛拭子新鲜粪便,对分离的大肠埃希菌用聚合酶链反应(PCR)检测mcr-1基因的携带率。对mcr-1阳性大肠埃希菌,采用BD凤凰hoenix TM 100 NMIC/ID-4复合板鉴定菌种及进行药敏试验,采用PCR检测各种β-内酰胺类药物耐药基因、氨基糖苷类修饰酶耐药基因、16S rRNA甲基化酶耐药基因和质粒介导的喹诺酮类药物耐药基因。结果302份样品中共分离出291株禽源性大肠埃希菌,其中27株携带mcr-1基因,mcr-1基因携带率为9.3%。27株mcr-1基因阳性禽源性大肠埃希菌中:超广谱β-内酰胺酶(ESBL)基因CTX-M-14、CTX-M-15、TEM-1携带率分别为100.00%(27/27)、70.37%(19/27)、74.07%(20/27);质粒介导AmpC酶耐药基因CMY-2携带率为14.81%(4/27);未检出bla SHV、bla DHA、OXA等β-内酰胺类药物相关耐药基因;未检出碳青霉烯酶基因;16S rRNA甲基化酶耐药基因rmtB及氨基糖苷类修饰酶耐药基因aac(6′)-Ⅰb-cr、ant(3″)-Ⅰ的携带率分别为25.93%(7/27)及25.93%(7/27)、92.59%(25/27);喹诺酮类药物耐药基因qnrS的携带率为81.48%(22/27),未检出qnrA、qnrB基因。对多黏菌素B、头孢噻肟、头孢西丁、左氧氟沙星、复方磺胺甲噁唑表现出了100.00%耐药,对头孢吡肟、头孢他啶、氨曲南、哌拉西林/他唑巴坦、阿米卡星和庆大霉素的耐药率分别为85.19%、33.33%、62.96%、14.81%、25.93%和77.78%,未出现对碳青霉烯类药物耐药的菌株。结论禽源性大肠埃希菌mcr-1基因的携带率为9.3%,是引起多黏菌素耐药的主要耐药机制。mcr-1基因阳性禽源性大肠埃希菌同时携带多种耐药基因,表现出多重耐药的特征。

Emerging bedaquiline resistance:A threat to the global fight against drug-resistant tuberculosis

Bedaquiline resistance is increasingly observed in the treatment of rifampicin-resistant tuberculosis(TB),yet standardized regimens for managing bedaquiline-resistant TB are lacking.Studies indicate a high proportion of bedaquiline-resistant cases have previously been treated for TB,and often involve strains resistant to quinolones.Regular monitoring of the culture status in patients receiving bedaquiline resistance treatment is advised.Methods such as experimental evolution,protein modeling,genome sequencing,and phenotypic analysis have been instrumental in identifying the mechanisms of bedaquiline resistance.Specifically,variants in the Rv0678 transcriptional repressor of the MmpS5-MmpL5 efflux system are linked to this type of resistance.Bayesian probability estimates show promise in determining the genotypic–phenotypic association for bedaquiline resistance,suggesting potential utility in clinical practice.Future research should explore the practical application of Bayesian probabilities in managing bedaquiline resistance.Sequencing-based technologies are anticipated to play a vital role in the early detection and management of drug-resistant TB strains.

Drug-resistant genes carried by Acinetobacter baumanii isolated from patients with lower respiratory tract infection

Background Acinetobacter baumanii （A. baumanii ） remains an important microbial pathogen resulting in nosocomialacquired infections with significant morbidity and mortality. The mechanism by which nosocomial bacteria, like A. baumanii, attain multidrug resistance to antibiotics is of considerable interest. The aim in this study was to investigate the spread status of antibiotic resistance genes, such as multiple 13-1actamase genes and aminoglycoside-modifying enzyme genes, from A. baumanii strains isolated from patients with lower respiratory tract infections （LRTIs）. Methods Two thousand six hundred and ninety-eight sputum or the bronchoalveolar lavage samples from inpatients with LRTIs were collected in 21 hospitals in the mainland of China from November 2007 to February 2009. All samples were routinely inoculated. The isolated bacterial strains and their susceptibility were analyzed via VITEK-2 expert system. Several kinds of antibiotic resistant genes were further differentiated via polymerase chain reaction and sequencing methods. Results Totally, 39 A. baumanii strains were isolated from 2698 sputum or bronchoalveolar lavage samples. There was not only a high resistant rate of the isolated A. baumanfi strains to ampicillin and first- and second-generation cephalosporins （94.87%, 100% and 97.44%, respectively）, but also to the third-generation cephalosporins （ceftriaxone at 92.31%, ceftazidine at 51.28%） and imipenem （43.59%） as well. The lowest antibiotic resistance rate of 20.51% was found to amikacin. The OXA-23 gene was identified in 17 strains of A. baumanii, and the AmpC gene in 23 strains. The TEM-1 gene was carried in 15 strains. PER-1 and SHV-2 genes were detected in two different strains. Aminoglycoside-modifying enzyme gene aac-3-1a was found in 23 strains, and the aac-6＂lb gene in 19 strains, aac-3-1a and aac-6＂lb genes hibernated in three A. baumanfi strains that showed no drug-resistant phenotype. Conclusions A. baumanii can carry multiple drug-resistant genes at the same time and result in multi-drug resistance. Aminoglycoside-modifying enzyme genes could be hibernating in aminoglycoside sensitive strains without expressing their phenotype.

Comparison of extended spectrum β-lactamasesproducing Escherichia coli with non-ESBLsproducing E.coli:drug-resistance and virulence

The virulent factors of Escherichia coil （E.cofi） play an important role in the process of pathopoiesis. The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-1actamases （ESBLs）-producing E.coli and non-ESBLs-producing E.cofi to provide a reference for physicians in management of hospital infection. From October 2010 to August 2011,96 drug-resistant strains of E. coli isolated were collected from the specimens in Qingdao Municipal Hospital, Qingdao, China. These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group. Drug sensitivity tests were performed using the Kirby-Bauer （K-B） method. Disinfectant gene, qacEAl-sull and 8 virulence genes （CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1） were tested by polymerase chain reaction （PCR）. Among the 96 E.coli isolates, the ESBLs-producing E.coli comprised 46 （47.9%） strains and the non-ESBLs-producing E.cofi consisted of 50 （52.1%） strains. The detection rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA,VT1, est, bfpA, elt, and CNF1 in 46 ESBLs-producing E.coli isolates were 89.1%, 76.1%, 6.5%, 69.6%, 69.6%, 89.1%, 10.9%, 26.1%, 8.7%, and 19.6%, respectively. In the non-ESBLs-producing E.cofi strains, the positive rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1 were 62.0%, 80.0%, 16.0%, 28.0%, 64.0%, 38.0%, 6.0%, 34.0%, 10.0%, and 24.0%, respectively. The difference in the detection rates of multiple drug-resistant strain, hlyA and VT1 between the ESBLs-producing E.cofi strains and the non-ESBLs-producing E.cofi strains was statistically significant （P〈0.05）. The positive rate of multiple drug-resistant strains is higher in the ESBLs-producing strains than in the non-ESBLs-producing strains. The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains. Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.

耐碳青霉烯类肺炎克雷伯菌对喹诺酮类药物的耐药特性及机制研究

背景 耐碳青霉烯类肺炎克雷伯(carbapenem-resistant Klebsiella pneumoniae,CRKP)对喹诺酮类药物的耐药机制及对新一代喹诺酮类药物西他沙星的耐药特性目前国内未见相关研究。目的 研究CRKP对喹诺酮类药物的耐药特性及耐药机制。方法 收集解放军总医院临床分离非重复耐碳青霉烯类肺炎克雷伯菌29株,采用微量肉汤稀释法检测CRKP对喹诺酮类药物的敏感度及外排泵抑制剂对CRKP耐药性的影响;通过棋盘法检测西他沙星联合其他抗菌药物对CRKP的体外抗菌活性;采用Sanger测序检测喹诺酮耐药决定区(quinolone resistance determining region,QRDR)突变;全基因组测序检测质粒介导的喹诺酮耐药基因(plasmid mediated quinolone resistance,PMQR)、外排泵、多位点序列分型等分子病原学特征。结果 29株CRKP对西他沙星、莫西沙星、环丙沙星的耐药率分别为90%、93%、93%,西他沙星MIC50值是莫西沙星的4倍、环丙沙星的8倍。西他沙星对blaKPC、blaNDM、blaOXA-48阳性的CRKP的敏感度分别为4.7%、66.6%、0。联合药敏结果表明西他沙星与黏菌素、依拉环素、替加环素联合用药对CRKP的协同、部分协同及相加作用分别为70%、20%、6%。blaKPC、blaNDM、blaOXA-48阳性的CRKP的QRDR突变率分别为95%、0、100%;质粒介导的PMQR携带率为83%;RND家族外排泵在CRKP菌株中普遍携带,外排泵抑制剂使CRKP对西他沙星的敏感度由10%上升至83%,耐药率由90%降至7%。解放军总医院优势传播菌株为ST11型blaKPC-2阳性菌株。结论 CRKP对喹诺酮类药物耐药率高,西他沙星与黏菌素的协同效果最好。QRDR突变和外排泵过表达是CRKP对喹诺酮类药物主要的耐药机制,产NDM酶CRKP的QRDR未见突变并且对喹诺酮类药物敏感性较好;ST11型blaKPC-2阳性菌株是解放军总医院优势流行菌株。

临床分离气单胞菌喹诺酮类的耐药机制研究

目的探讨临床分离气单胞菌属细菌(Aeromonas spp.)喹诺酮类药物耐药机制。方法收集从北京友谊医院2017年腹泻患者粪便标本中分离的气单胞菌,采用微量肉汤稀释法检测其对喹诺酮类药物的敏感性,提取菌株核酸进行PCR扩增,测序分析气单胞菌喹诺酮耐药决定区(quinolone-resistance-deter mining region,QRDR)及喹诺酮耐药质粒相关(plasmid-mediated quinolone resistance,PMQR)基因。结果共收集111株气单胞菌,其中7株(6.3%)对萘啶酸(NAL)和环丙沙星(CIP)均耐药,54株(48.6%)对NAL耐药但对CIP敏感,50株(45.0%)对NAL和CIP均敏感。QRDR测序结果显示,NAL耐药菌株gyrA基因第83位发生丝氨酸(Ser)突变,突变为异亮氨酸(Ile)、缬氨酸(Val)或酪氨酸(Tyr)。对NAL-CIP耐药的菌株中,除了gyrA基因突变外,parC基因第87位Ser突变为Ile。CIP敏感的104株菌株中,仅有7株parC基因第87位Ser突变。PMQR基因检测结果显示,QnrS基因检出率为7.2%(8株),aac(6′)-Ib-cr基因检出率为6.3%(7株),且QnrS和aac(6′)-Ib-cr基因检出主要集中在CIP耐药菌株中,CIP耐药菌株PMQR基因检出率为85.7%。结论气单胞菌喹诺酮低水平耐药主要与gyrA基因第第83位突变或QnrS基因有关。gyrA基因第83位突变和parC基因第87位突变Ser87Ile联合PMQR基因存在时,可能与气单胞菌对喹诺酮的高水平耐药相关。

朱鹮大肠杆菌分离鉴定及耐药性与喹诺酮类耐药基因分子特征分析

【目的】掌握浙江省德清县驯养朱鹮大肠杆菌的耐药性、耐药基因与分子特征,为治疗朱鹮大肠杆菌感染提供基础数据。【方法】采集该地驯养朱鹮新鲜粪便样本,采用分离培养、形态观察、生化鉴定及16S rDNA序列分析鉴定大肠杆菌;进而采用肉汤稀释法检测环丙沙星、庆大霉素、丁胺卡那等7种抗菌药对源自不同朱鹮的大肠杆菌分离株菌株的最小抑菌浓度(minimal inhibitory concentration,MIC);选择代表性菌株,通过PCR和测序鉴定其携带qnrS 1、gyrA、gyrB等9种喹诺酮类耐药基因的情况,并分析其蛋白关键氨基酸位点突变与耐药性的关系,采用接合转移分析耐药质粒水平转移情况及其与耐药性的关系,应用卡方检验(Chi-square test)和费歇尔精确检验(Fisher exact test)分析分离株耐药表型与朱鹮年龄、耐药基因的相关性。【结果】本研究共采集了98只朱鹮的粪便,经分离鉴定均获得了大肠杆菌;源自不同朱鹮的98株大肠杆菌对喹诺酮类环丙沙星呈现高度耐药(耐药率为65.3%,64/98),对其余6种药物高度敏感(敏感率均>90%),朱鹮年龄与环丙沙星耐药性极显著相关(P<0.01);选取的31株分离株喹诺酮类耐药基因marR、gyrA、parC和gyrB的突变株占比在3.2%~80.6%之间,携带质粒耐药基因qnrS 1的菌株占比22.6%(7/31);耐药基因阳性菌株占比93.5%(29/31)并分布于6个耐药基因型,其中,marR/gyrA/parC 10株、marR/qnrS 16株、marR 6株、gyrA/parC 3株、marR/gyrA 3株、marR/gyrB/qnrS 11株;环丙沙星耐药表型与耐药基因显著相关(P=0.026),gyrA和parC单基因或联合突变均与喹诺酮耐药显著相关(P=0.038);大肠杆菌分离株qnrS 1基因一次接合成功率为28.6%(2/7),并可导致受体菌对喹诺酮类药物的耐药性增加。【结论】本研究鉴定了朱鹮群体大肠杆菌耐药性,并明确了朱鹮源大肠杆菌对喹诺酮类耐药的主要原因是gyrA和parC单基因或联合突变并携带可水平转移的耐药基因qnrS 1,本研究结果为人工驯养朱鹮救护和耐药性监测提供参考依据,提示应重视朱鹮大肠杆菌对喹诺酮类药物的耐药性。

Drug-resistant gene based genotyping for Acinetobacter baumannii in tracing epidemiological events and for clinical treatment within nosocomial settings

Background Acinetobacter baumannfi has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis （PFGE） and amplified fragment length polymorphism （AFLP） in Acinetobacter baumannfi genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak. Methods From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing （DRGT） were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP. Results Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and β-1actamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance. Conclusion Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.

PMQR与耐碳青霉烯类肠杆菌科细菌喹诺酮耐药机制的关系

目的 分析质粒介导的喹诺酮耐药基因(PMQR)在耐碳青霉烯类肠杆菌科细菌(CRE)中的分布,探讨喹诺酮类药物的耐药机制。方法 收集2021年7月至2022年9月该院临床分离非重复的43株CRE菌株,采用全自动微生物分析系统进行菌株鉴定和药敏试验,聚合酶链反应(PCR)技术检测PMQR、碳青霉烯酶和β内酰胺酶耐药基因,进行CRE菌株的喹诺酮耐药机制分析。结果 31株耐碳青霉烯类大肠杆菌对喹诺酮类药物的耐药率为93.55%~96.77%。12株耐碳青霉烯类肺炎克雷伯菌对喹诺酮类药物的耐药率为66.67%~83.33%。43株CRE对β内酰胺酶阻滞剂类药物的耐药率均在79.07%~100.00%;对多黏菌素和替加环素100.00%敏感。对氨基糖苷类的阿米卡星和妥布霉素耐药率较低。43株CRE中,88.37%携带有1个或2个PMQR,其中acc(6′)Ib-cr检出率为83.72%,qnrS检出率为69.77%,qnrB检出率为2.33%,没有检测到qnrA和qepA。碳青霉烯酶耐药基因以NDM(88.97%)和KPC(32.56%)为主。β内酰胺酶耐药基因以SHV(83.23%)和CTX-M15(67.57%)为主。结论 PMQR在CRE菌株中的检出率很高,与CRE的喹诺酮类药物耐药机制有关,可能与NDM、KPC、CTX-M15共同作用导致多药耐药。

喹诺酮类药物及细菌对其耐药性机制研究进展

本文从喹诺酮类抗菌药物的结构、进入细菌体内的方式及喹诺酮类药物的作用机理进行简明阐述。针对喹诺酮类药物的广泛使用而导致的细菌对喹诺酮类药物的耐药性逐渐增加的原因入手,阐述了细菌对喹诺酮类药物耐药性——从染色体突变到质粒介导的喹诺酮类耐药性的几种分子机制及研究进展,为研究新型喹诺酮类抗菌药物提供依据。

大肠埃希菌与克雷伯菌属细菌qnr基因的检测

目的 了解我院临床分离大肠埃希菌和克雷伯菌属细菌qnr基因的存在情况。方法 用聚合酶链反 应(PCR)对227株大肠埃希菌和36株克雷伯菌属细菌进行qnr基因检测。药敏检测分别采用K B法、break point法。结果 227株大肠埃希菌和36株克雷伯菌属细菌中有6株细菌检出qnr基因(2株大肠埃希菌、1株产 酸克雷伯菌、3株肺炎克雷伯菌)。其中产酸克雷伯菌为突变株,已在基因库注册,授权号:AY675584。6株qnr 基因阳性菌株均为产超广谱β 内酰胺酶(ESBLs)且多重耐药菌株。结论 qnr基因阳性菌株有严重的多重耐药 性,我院克雷伯菌属细菌中qnr基因的携带率高于大肠埃希菌。

水产动物源嗜水气单胞菌药物敏感性及QRDR基因突变分析

为了解临床分离的59株嗜水气单胞菌对喹诺酮类药物的耐药性及其靶基因gyrA和parC编码的喹诺酮类耐药决定区(QRDR)的突变情况。采用纸片琼脂扩散法测定15种抗菌药物对水产动物源嗜水气单胞菌的耐药情况,采用PCR法扩增gyrA和parC,并通过测序分析其靶基因突变情况。结果表明:24株(40.7%)嗜水气单胞菌对喹诺酮类药物耐药,其中对萘啶酸、诺氟沙星、氧氟沙星、环丙沙星的耐药率依次为40.7%、16.9%、20.3%、8.5%。敏感菌株QRDR靶位点未发生突变,24株喹诺酮类耐药菌株中gyrA基因编码的第83位氨基酸均存在Ser→Ile突变;19株菌parC基因编码的第87位氨基酸也发生突变,其中18株为Ser→Ile突变,1株为Ser→Arg突变,有5株未检测到突变;ParC亚基氨基酸突变的菌株其GyrA亚基均同时发生氨基酸突变。表明临床分离的嗜水气单胞菌对喹诺酮类药物耐药程度不一,其中萘啶酸的耐药最为严重,诺氟沙星、氧氟沙星和环丙沙星敏感性较高。喹诺酮类耐药菌株GyrA亚基QRDR氨基酸突变,可能是引起萘啶酸耐药的主要原因。临床分离嗜水气单胞菌QRDR区域的突变具有随机性,但靶位点GyrA的Ser83→Ile以及ParC的Ser87→Ile是最主要的突变方式,另外喹诺酮类药物耐药性的产生可能还与其他耐药机制存在关联。

产DHA-1型AmpC酶和TEM-1型β-内酰胺酶及携带Ⅰ类整合子的鲍氏不动杆菌

目的分析鲍氏不动杆菌临床株(编号0524)的β-内酰胺酶基因(BLA)、质粒介导耐氟喹诺酮基因(qnr)、氨基糖苷类修饰酶基因(AMEs)、消毒剂与磺胺类耐药基因(qacE△1-sul1)和Ⅰ类整合子酶基因(intⅠ1)存在情况。方法对0524株应用K-B法检测其耐药表型,应用三维试验检测其产生的β-内酰胺酶表型,应用聚合酶链反应及序列分析方法分析其BLAs、AMEs、qnr、qacE△1-sul1和intⅠ1基因类型。结果0524株鲍氏不动杆菌对第三代头孢菌素、头孢西丁、环丙沙星、阿米卡星耐药,对头孢吡肟、亚胺培南、头孢哌酮/舒巴坦敏感,产生AmpC酶,携有blaDHA-1基因,同时blaTEM-1型基因,acc(3)-Ⅰ、aac(6′)-Ⅰ、ant(3″)-;ⅠqacE△1-sull和intⅠ1均阳性,其他受试基因均阴性。结论0524号菌株产生DHA-1型AmpC酶和TEM-1型的广谱β-内酰胺酶,且携带Ⅰ类整合子、消毒剂与磺胺类耐药基因和3种氨基糖苷修饰酶基因,其耐药机制复杂。

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%(118/231)和45.9%(106/231);gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。

质粒介导的克雷伯菌耐喹诺酮类药物机制研究

目的了解华南地区qnr基因介导的克雷伯菌耐喹诺酮类药物的情况并研究其耐药机制。方法收集临床分离的克雷伯菌株共200株,其中产酸克雷伯菌6株。PCR方法筛查基因,琼脂稀释法测MIC,质粒结合试验,聚合酶链反应(PCR)通用引物扩增各组基因及测序。结果200株细菌中筛查到带qnr基因的菌株共2株(其中产酸克雷伯菌1株),均对环丙沙星敏感。进一步确证实验中证实2菌株都含qnr基因。2菌株经结合实验后在选择平板上都有菌生长,而且对环丙沙星的MIC值均有30倍以上的的提高。2株菌均有gyrASer83→Tyr突变,产酸克雷伯菌株有parCSer80→Ile突变。2株菌质粒PCR扩增的qnr产物测序结果与NCBI中qnr基因比对吻合率达100%。结论华南地区已有质粒介导的耐喹诺酮类菌株存在,它们能够跨种属转移,值得临床医生重视。

乌鲁木齐牛羊肉源沙门氏菌对喹诺酮类药物的耐药状况及相关基因分析

目的:研究牛羊肉源沙门氏菌对15种抗生素的药敏性以及对喹诺酮类药物的相关耐药基因,更好地了解沙门氏菌耐药性的产生和传播途径,为预防与控制沙门氏菌疾病提供基础信息。方法:用琼脂稀释法测定沙门氏菌的药敏性,用聚合酶链式反应和基因序列测定法确定耐药沙门氏菌中与喹诺酮类抗生素耐药相关的喹诺酮类抗性决定区基因突变及质粒携带的耐药基因。结果:30株沙门氏菌中对甲氧苄啶、氯霉素、萘啶酮酸、四环素、磺胺异甲二唑、链霉素、甲氧嘧啶/磺胺异恶唑、阿莫西林、氨苄西林的耐药率分别为100%、86.7%、66.7%、60.0%、50.0%、33.3%、26.7%、6.7%、6.7%,对环丙沙星、头孢曲松、庆大霉素、卡那霉素、头孢西丁、阿米卡星均表现为敏感;qnr B、qnr A、qnr S、aac(6′)-Ib-cr基因的检出率分别为5.0%、45.0%、0%、5.0%;30株沙门氏菌发生gyr A基因突变的菌株数为14株,主要突变类型是Ser83Phe,发生par C基因突变的菌株数为25株,主要突变类型是Thr57Ser。结论:乌鲁木齐牛羊肉源沙门氏菌的耐药情况较为严重,对喹诺酮类药物的耐药状况应当予以关注,其耐药突变基因及质粒携带的耐药基因在一定程度上是导致沙门氏菌耐药的重要原因。

动物源大肠杆菌PMQR基因流行性检测

【目的】检测分离自广东省散养型养殖场的动物源大肠杆菌中oqxAB基因及其它质粒介导喹诺酮类耐药基因(PMQR)的流行情况。【方法】采用PCR方法检测oqxA、oqxB、qnr、qepA和aac(6′)-Ib-cr基因;琼脂平板稀释法对PMQR阳性菌株进行18种抗菌药物的最小抑菌浓度测定。【结果】qnrB、qnrS、aac(6′)-Ib-cr、oqxA、oqxB的检出率依次为10.49%、18.88%、31.47%、44.8%和48.9%,但未检测到qnrA、qnrC、qnrD和qepA。oqxA与oqxB的检出率较高,且oqxA与oqxB常常同时存在。多数菌株同时携带两个或两个以上的PMQR基因。PMQR阳性菌对18种兽医临床常用抗生素耐药率较高。【结论】PMQR基因在广东省兽医临床传播广泛,广东省大肠杆菌对临床常用抗菌药物耐药较为严重,药敏谱呈多样化,多重耐药株比例较高。

人型支原体对喹诺酮类药物耐药机制的初步研究

目的探讨人型支原体(mycoplasma hominis,MH)对喹诺酮类药物的耐药机制。方法泌尿生殖道分泌物中分离的对3种喹诺酮类药物耐药的10株MH,采用PCR方法检测其DNA螺旋酶基因序列,与对喹诺酮类药物敏感的标准菌株ATCC23114及基因库中的野生型菌株PG21基因序列对比,分析MHDNA螺旋酶II保守区突变位点与菌株耐喹诺酮类药物的关系。结果与野生株(PG21)比较,对司帕沙星耐药的7株Mh均检出GyrA氨基酸残基S153→L变异,对应于大肠杆菌则为S83→L,4号株还同时检出对应于大肠杆菌S56→I变异。对司帕沙星敏感的Mh菌株及标准菌株则未发现GyrA中氨基酸残基变异。结论 Mh菌株GyrA基因编码的对应于大肠杆菌S83→L氨基酸残基变异与Mh耐司帕沙星相关。