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.

Detection of Shigella gyrA mutations and correlations between gyrA mutations and quinolones resistance

118 clinical strains of Shigella were serotyped, in which 116 strains were tested to be S.flexneri. The susceptibilities of the S.flexneri strains to quinolones were measured by the disk-diffusion method. It was found that most S.flexneri strains were susceptible to norfloxacin and ciprofloxacin, but resistant to nalidixic acid. To study the correlation between gyrA mutations and quinolones resistance, a fragment within the gyrA gene was amplified by PCR. The SSCP (Single-Strand Conformation Polymorphism) analysis was applied to detect mutations in PCR products of different strains. The mutations were then confirmed by DNA sequencing. Altogether, two types of mutation were revealed, in which one type was single mutation (C42-T), and the other was double mutations (C42-T and A54-G). By statistical analysis, C42-T (encoding Ser83-Leu substitution) was shown to have correlation with nalidixic-acid resistance in the clinical strains of Shigella, while A54-G (encoding Asp87-Gly substitution) was shown to have correlations with both norfloxacin resistance and ciprofloxacin resistance.

Molecular Detection of Mutations within the Quinolone Resistance-Determining Regions in Non Typhoidal Salmonella Isolates from Malaysia

Introduction: The efficacy of chemotherapy in bacteraemia caused by non-typhoidal Salmonella (NTS) is compromised by antibiotic resistance. Objective: This study was undertaken to describe the mechanism of resistance among clinical NTS isolates. Materials & Methodology: Thirty of NTS were isolated from blood (n = 19), stool (n = 10) and bronchioalveolar lavage (BAL;n = 1) respectively. These isolates were tested for susceptibility testing by disc diffusion method against ampicillin, gentamicin, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin and ceftriaxone. Epsilometer tests (E-test) for nalidixic acid and ciprofloxacin were performed for nalidixic acid resistant isolates by disc diffusion method. DNA sequencing was carried out on six of the nalidixic acid resistant Salmonella Enteritidis isolates to identify mutations within quinolones resistance determining regions (QRDR) of gyrA, gyrB, parC and parE genes. Results: Resistance rates of NTS isolates from blood, stool, and BAL were respectively 37%, 20% and 0% for ampicillin, 79%, 40% and 0% for tetracycline, 32%, 40% and 0% for co-trimoxazole, 37%, 10% and 100% for nalidixic acid. Eight isolates were resistant to nalidixic acid and had exhibited reduced susceptibility towards ciprofloxacin by E-test. Mutation within QRDR was detected in gyrA gene (n = 6;Asp 47 → His [3], Asp 51 → Asn [1], Asp 73 → Gly [1], and Gly 48 → Asp [1]) and double mutation was detected in parE gene (n = 3;Gly 48 → Asp [3], Glu 82 → Ser [3]). Out of six isolates, three isolates were found to have both gyrA and parE gene mutations. Conclusions: There was no mutation observed in gyrB and parC gene. Mutation in gyrA gene was sufficient to induce decreased susceptibility to ciprofloxacin. Variation in amino acid sequences are novel, while detection of other gene mutation was uncommon.

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.

Distribution of gyrA mutations in fluoroquinolone-resistant Helicobacter pylori strains

AIM:To investigate the resistance of Helicobacter pylori(H.pylori) to ciprofloxacin(CIP),levofloxacin(LVX) and moxifloxacin(MOX) in the Beijing area and to elucidate the resistance mechanisms.METHODS:Seventy-nine H.pylori clinical strains,isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009,were tested for their susceptibility to CIP,LVX and MOX using the E-test method.H.pylori strain 26695 was included in the susceptibility testing as a control strain.According to the minimal inhibitory concentration(MIC) values,a strain was classified as resistant to CIP,LVX or MOX when the MIC was > 1 μg/mL.We amplified by polymerase chain reaction(PCR) and sequenced the quinolone resistance-determining regions of the gyrA and gyrB genes from 29 quinolone-resistant and 16 quinolone-susceptible H.pylori strains selected at random.RESULTS:In this study,the resistance rates of H.pylori to CIP,LVX or MOX were 55.7%(44/79),and the primary resistance rates were 26.6%(21/79).Patients with secondary resistance had received LVX in previous eradication treatments,but not MOX or CIP.Forty-five strains,including 29 CIP,LVX or MOX-resistant strains(MIC:1.5-32 μg/mL) and 16 susceptible strains,were selected randomly from the 79 strains and used in PCR analysis.Among these 45 strains,27 resistant strains had mutations in the gyrA gene,including 11 strains with mutations corresponding to Asp-91(MIC:2-32 μg/mL),one of which also had a mutation corresponding to Val-150,and 16 strains had mutations at Asn-87(MIC:4-32 μg/mL),three of which also had mutations corresponding to Arg-140 or Val-150.In addition,Arg-140,Val-150 or Ala-97 mutations were separately detected in three susceptible strains.Analysis of the gyrB gene showed that one strain of low resistance had a mutation corresponding to Ser-457 that coexisted with an Asp-91 mutation.There was a significant difference in the occurrence of mutations in the gyrA gene between CIP,LVX and MOX-resistant and-susceptible strains(P < 0.05),but 2 resistant strains were found to possess no quinolone resistance-determining region mutations.CONCLUSION:Resistance is primarily mediated through point mutations in gyrA.Whether other mechanisms are responsible for resistance in strains without mutations in the QRDR should be detected.

Molecular characterization of antimicrobial multi-drug resistance in non-typhoidal Salmonellae from chicken and clam in Mangalore, India

Salmonella enterica has been documented as one of the leading causes of salmonellosis throughout the world and is most commonly associated with the consumption of contaminated food products. Thus, this research was aimed at studying the antimicrobial susceptibility pattern and detection of quinolone resistance in Salmonella spp isolated from food of animal origin. Thirty-six Salmonella isolates comprising 8 from poultry and 28 from seafood（clams） were identified, serotyped and characterized for their antimicrobial susceptibility against 10 different antibiotics. Plasmid DNA was isolated from all the isolates by alkaline lysis, quinolone resistant non-typhoidal S. Weltevreden were examined for mutation in the DNA gyrase coding gene. Among the 36 Salmonella isolates, 20 were S. weltevreden（8 from poultry and 12 from seafood） and 16 were S. Typhimurium（from seafood）. All the isolates showed multiple resistance to nalidixic acid, tetracycline, co-trimoxazole and nitrofurantoin, but, interestingly, the isolates were 100% susceptible to ampicillin, chloramphenicol and gentamicin. Resistant isolates from the study carried the genes responsible for resistance to respective antibiotics. The strain S130 isolated in the study showed single point mutation,Asp87Gly, at position 87 in quinolone resistance determining region. It revealed mutation in quinolone resistance determining region as a cause for quinolone resistance in non-typhoidal Salmonellae. The occurrence of genes accountable for plasmid mediated resistance to quinolones（viz., qnrA, qnrB and qnrS） in plasmid of non-typhoidal Salmonellae isolates provides evidence for plasmid mediated quinolone resistance.

Molecular mechanism of the qnrA genemediated quionlone resistance in Gram-negative bacteria

To explore the prevalence of the plasmid-mediated quinolone resistance gene qnrA in Gram- negative bacteria and to investigate its molecular genetic background and resistance profile in isolates harboring this gene,a total of 629 nalidixic acid-resistant isolates of non-repetitive Gram-negative bac- teria were collected from clinical specimens between April 2004 and April 2006 and these isolates were screened for qnrA gene by PCR using specific primers combined with DNA sequencing.The extended spectrumβ-lactamase(ESBL)or AmpC-producing isolates were distinguished by the phenotypic confir- matory test combined with DNA sequencing,and the antibiotics susceptibility test for qnrA-positive iso- lates was carried out by Kirby-Bauer and E-test method.To detect the location of the qnrA gene,plas- mid conjugation and Southern hybridization were performed and the integron structure containing the qnrA gene was cloned by PCR strategy and sequenced by primer walking.It was demonstrated that the incidence of the qnrA-positive strains in nalidixic acid-resistant bacteria was 1.9%(12/629),in which the detection rates for Klebiesiella pneumoniae.Enterobacter cloacae,Enterobacter aerogenes, Citrobacterfreundii and Salmonella choeraesuis were 2.2%(3/138),17.1%(6/35),9.1% (1/11),12.5%(1/8),and 14.3%(1/7),respectively.The qnrA gene was found to be embedded in the complex su/1-type integron located on plasmids with varied size(80-180 kb).Among them,4 qnrA-positive isolates carried integron In37 and 8 isolates carried a novel integron,temporarily desig- nated as InX.All the qnrA-positive isolates were ESBL-producing and transferable for the muhi-drug resistance.It is concluded that the plasmid-mediated drug-resistance mechanism exists in the quinolone resistant strains of isolates from hospitals in Guangdong area,but the incidence was rather low.Never- theless,it is still possible that the horizontal transfer of the resistant qnrA gene might lead to the spreading of drug-resistance.

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。

无锡地区2021—2023年肺炎支原体药敏分析

目的回顾性分析无锡地区2021年1月—2023年11月成人感染肺炎支原体对抗菌药物的敏感性,为临床用药提供参考依据。方法回顾性分析2021年1月—2023年11月来上海交通大学医学院附属瑞金医院无锡分院就诊患者呼吸道感染的104株肺炎支原体,对14种常见抗菌药物的耐药性。结果104株肺炎支原体中,耐乙酰螺旋霉素57株(54.8%),耐莫西沙星45株(43.3%),耐强力霉素34株(32.7%),耐美满霉素26株(25.0%),耐环丙沙星16株(15.4%),耐克林霉素14株(13.5%);耐依托红霉素、加替沙星、交沙霉素、左氧氟沙星、阿奇霉素、克拉霉素、红霉素及罗红霉素均在9株以下。结论对成人呼吸道感染的肺炎支原体,临床应以大环内酯类和喹诺酮类抗菌药物进行治疗,但应避免使用大环内酯类中的乙酰螺旋霉素和喹诺酮类中的莫西沙星和环丙沙星。

集中带量采购政策对注射用喹诺酮类药物处方行为和细菌耐药性影响分析

目的 研究集中带量采购政策对注射用喹诺酮类药物使用情况的影响和细菌耐药情况的变化。方法 提取南京鼓楼医院2017年1月—2019年10月和2020年1月—2022年10月注射用喹诺酮类药物的使用数据,采用中断时间序列模型分析集采政策对其产生的影响;基于集采政策相关的调查问卷分析本院医务人员对政策的看法及产生相应行为的原因;利用相应时间段细菌耐药率、检出量进行相关性分析。结果 第三批集采政策实施后,莫西沙星使用量呈下降趋势;第五批集采政策实施后,莫西沙星和环丙沙星的使用量呈上升趋势,且相较于对照组变化存在显著性差异;左氧氟沙星使用量呈持续下降趋势。问卷调查显示,喹诺酮类药物作为替代药优先选择,与第五批集采后喹诺酮类药物使用量的上升一致。喹诺酮类药物耐药率受政策影响不明显,与喹诺酮类药物用量无高度相关性。结论 喹诺酮类药物在第三批集采政策实施后使用量下降,在第五批实施后也未出现异常增长现象。相关病原菌耐药率与药物使用量无高度相关性,政策对耐药率变化的影响还需长期监测。

混合抗生素对大肠杆菌耐药性的联合诱导效应

群体感应抑制剂(quorum sensing inhibitors,QSIs)作为一类新型抗生素,具有广阔的应用前景,目前已被使用在水产养殖、污水治理和农作物病害防治等领域中,其与传统抗生素极有可能在环境中共存。因此,有必要探究QSIs与传统抗生素对细菌耐药性的联合效应。以往的研究往往采用单位法探究固定浓度下混合抗生素的联合耐药效应,但单位法无法在较大浓度范围评价其联合耐药风险。该研究以RP4质粒接合转移表征细菌耐药性的传播,将独立作用模型(independent action,IA)应用到联合耐药作用的判别中,以喹诺酮(quinolones,QNs)作为传统抗生素的代表,探究了QSIs与QNs对大肠杆菌(Escherichia coli)RP4质粒接合转移的联合效应。结果表明,15组QSIs-QNs混合物均能诱导E.coli产生接合转移效应,混合物的最大促进率在23.32%~73.07%。对比IA曲线与混合物对E.coli接合转移的剂量-效应曲线的位置关系,发现QSIs-QNs对E.coli的联合接合作用均表现出浓度依赖特征,即混合物促进E.coli接合转移频率时的联合作用模式随浓度增加由协同作用转变成拮抗作用。通过QSIs-QNs对E.coli的联合毒性实验,推测测试混合抗生素浓度依赖的联合接合作用与其联合毒性作用密切相关。该研究能够为今后探索QSIs与传统抗生素联合暴露的环境风险评估提供参考。

食源性沙门氏菌携带质粒介导的喹诺酮类耐药基因的液相芯片检测技术研究

目的:建立应用新型液相芯片技术检测食源性沙门氏菌携带的质粒介导喹诺酮类耐药(PMQR)基因中4种基因:qnrS、aac(6')-Ib-cr、oqxA、oqxB的方法。方法:针对食源性沙门氏菌携带的qnrS、aac(6')-Ib-cr、oqxA、oqxB四种PMQR基因,设计对应引物和微球,采用液相芯片技术对7株标准菌株进行特异性实验;对4株各含1种PMQR基因的食源性沙门氏菌进行重复性和灵敏度实验;然后检测来自食源性风险监测的71株耐喹诺酮类沙门氏菌,和普通PCR进行对比实验。结果:成功建立了液相芯片技术检测食源性沙门氏菌携带的qnrS、aac(6')-Ib-cr、oqxA、oqxB四种PMQR基因的方法,携带qnrS基因的耐药株检出限为5 CFU/mL、携带aac(6')-Ib-cr基因的耐药株检出限为25 CFU/mL、携带oqxA和oqxB基因的耐药株检出限为10 CFU/mL。所有阳性判定结果荧光中位值(MFI)均≥5倍阴性对照组。重复性实验变异系数(CV)均小于5%,特异性实验结果特异性100%,阴性菌株无阳性信号反应。qnrS检出率29.6%(21/71)、aac(6')-Ib-cr 35.2%(25/71)、oqxA 28.2%(20/71)、oqxB 23.9%(17/71),方法比对的结果符合率为100%。结论:实验建立的液相芯片技术检测食源性沙门氏菌质粒介导喹诺酮类耐药基因qnrS、aac(6')-Ib-cr、oqxA、oqxB的方法具有灵敏度高、特异性好、稳定性强、结果准确的特点,可以为食源性沙门氏菌PMQR基因的检测以及耐药性的监控提供技术支撑。

肺炎克雷伯菌的喹诺酮类耐药株gyrA基因研究

目的分析肺炎克雷伯菌的喹诺酮类耐药株gyrA基因的耐药机制。方法收集经Phoenix 100全自动细菌鉴定系统鉴定分离的肺炎克雷伯菌的喹诺酮类耐药株43株,采用聚合酶链反应(PCR)方法进行gyrA基因检测,扩增产物纯化后测序并进行序列分析。结果43株肺炎克雷伯菌的喹诺酮类耐药株均发生gyrA基因突变,gyrA基因的PCR扩增均得到目的片段,扩增产物长度为441 bp。结论肺炎克雷伯菌的喹诺酮类耐药株gyrA基因突变非常普遍,监测其突变频率及特点对研究喹诺酮类耐药机制和指导临床合理用药非常必要。

患病牦牛和藏猪源沙门菌对氨基糖苷类、喹诺酮类和β-内酰胺类药物敏感性试验

为了解川西北高原牦牛和藏猪源沙门菌对氨基糖苷类、喹诺酮类和β-内酰胺类抗生素的耐药情况,本研究采用沙门菌选择培养基对采集的样本进行沙门菌分离,利用PCR扩增保守基因invA的方法进行沙门菌鉴定。采用微量肉汤释法对分离鉴定的沙门菌进行氨基糖苷类(庆大霉素、大观霉素、卡那霉素、妥布霉素、链霉素和阿米卡星)、喹诺酮类(沙拉沙星、环丙沙星、达氟沙星、恩诺沙星和萘啶酸)和β-内酰胺类(头孢噻呋)抗生素敏感性实验。试验结果显示,从198份肛拭子样本中分离鉴定得到24株沙门菌,分离率为12.12%。24株沙门菌对庆大霉素的耐药率最高,为66.7%,对链霉素和阿米卡星敏感。对大观霉素、萘啶酸、沙拉沙星、环丙沙星、达氟沙星、卡那霉素、妥布霉素、头孢噻呋和恩诺沙星耐药率分别为33.3%、33.3%、29.2%、25.0%、25.0%、16.7%、16.7%、16.7%和4.2%。分离的24株沙门菌牦牛和藏猪源沙门菌耐药性严重并存在多重耐药现象,临床上应强调加强沙门菌的耐药性检测以合理选用抗菌药物。

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。

牛源无乳链球菌对喹诺酮耐药的基因特征

采用微量肉汤稀释法测定左氧氟沙星、诺氟沙星和环丙沙星3种喹诺酮类药物对110株奶牛隐性乳腺炎无乳链球菌(分离自中国10省区21个规模化奶牛养殖场)的最小抑菌浓度(MIC),通过PCR检测gyrA、gyrB、parC和parE基因的喹诺酮耐药决定区(QRDR),并分析其突变位点.结果表明:110株牛源无乳链球菌对左氧氟沙星、诺氟沙星和环丙沙星的耐药率分别为85.5%、98.2%、94.5%;左氧氟沙星耐药菌株对其他2种喹诺酮类药物耐药,且均发生了QRDR基因突变,氨基酸突变位点有GyrA的Ser81Leu、Glu85Lys,ParC的Ser79Phe、Asp83Tyr,ParE的Asp437Asn,其中,耐药菌基因最主要的突变类型是GyrA(Ser81Leu)+ParE(Asp437Asn),该突变类型对喹诺酮类药物呈中高度耐药;ParC的Ser79Phe或Asp83Tyr突变类型对喹诺酮类药物的敏感性下降,ParC的Ser79Phe突变与GyrA的Ser81Leu或Glu85Lys突变联合时,对喹诺酮类药物高度耐药.本研究结果对兽医临床上喹诺酮类药物的耐药控制及合理使用提供了一些依据.

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。

耐喹诺酮类药物革兰阴性菌的适应性研究

随着广谱高效喹诺酮类药物的过度应用,革兰阴性菌对喹诺酮类药物的耐药率逐年上升,已严重威胁到人类健康。革兰阴性菌对喹诺酮类药物的抗性是通过耐药基因突变或者获得外源抗性基因实现的。耐药性的维持通常会对细菌造成一定的适应性代价,表现为细菌生长速率、毒性和传播的减弱,但耐药菌可通过补偿突变缓解适应性代价,甚至增强耐药菌适应性,严重阻遏临床疗效。而且耐药菌株的适应性增强,导致细菌即使在缺乏抗菌剂使用的条件下,其耐药率也增加,导致无法通过减少抗菌剂的使用减弱菌株耐药率。所以本文分别对革兰阴性菌的染色体和PMQR基因介导的耐喹诺酮药物的机制对其适应性变化的影响进行综述,并对耐药菌适应性的研究方法进行总结,以期为耐喹诺酮类药物革兰阴性菌的新型治疗方案、相关的肠道致病菌抑制剂的开发和未来与之相关的研究提供一定参考。

两种结核分枝杆菌氟喹诺酮类药物耐药性检测方法比较及不一致原因初探

目的:评估荧光PCR熔解曲线法(简称“熔解曲线法”)对结核分枝杆菌氟喹诺酮类药物(FQs)耐药性的诊断价值,分析其与表型药物敏感性(简称“药敏”)试验结果不一致的原因。方法:采用简单随机抽样的方法,随机选取2019年1月至2020年6月期间来自重庆市39个区(县)经菌种鉴定和比例法药敏试验确定为耐多药结核病的126例患者的临床分离株,分别采用微量肉汤稀释法和熔解曲线法对左氧氟沙星(Lfx)和莫西沙星(Mfx)进行表型药敏及分子药敏试验。以表型药敏试验结果为标准,评价熔解曲线法对FQs耐药的检测效能,并采用全基因组测序(WGS)对表型药敏和分子药敏试验不一致的结果进行分析。结果:以表型药敏试验结果为标准,熔解曲线法对FQs耐药检测的敏感度和特异度分别为94.5%(86/91;95%CI:87.1%~98.0%)和100.0%(35/35;95%CI:87.7%~100.0%)。两种方法结果不一致者有12株,不一致率为9.5%(12/126)。表型药敏试验耐药而熔解曲线法敏感者5株,WGS显示2株未见gyrA耐药基因相关突变,2株存在gyrB基因突变(该4株菌的最低抑菌浓度值,Lfx为1~2μg/ml,Mfx均为0.5μg/ml),1株发现gyrA基因Asp94Ala突变,突变频率为15.3%。表型药敏试验敏感而熔解曲线法耐药7株,其中5株发现gyrA基因Ala90Val突变,1株发现gyrA基因Asp94Ala突变,1株发现gyrA基因Asp94Asn突变,该7株菌对Lfx和Mfx的最低抑菌浓度范围分别为1~2μg/ml和0.25~0.5μg/ml。结论:熔解曲线法对FQs耐药的检测效能较好,FQs异质性耐药菌的比例及低水平耐药相关突变是影响FQs熔解曲线法检测效能及导致其与表型药敏试验结果不一致的主要原因。

多色探针熔解曲线分析法评估结核分枝杆菌阳性患者对不同抗结核药物耐药性的诊断效能

目的:分析多色探针熔解曲线分析法(MMCA)评估结核分枝杆菌阳性患者对利福平、乙胺丁醇、链霉素、异烟肼、喹诺酮耐药性的诊断效能。方法:选取2023年1—6月泉州市各县(市)区结核定点医院收治的100例结核分枝杆菌阳性患者的痰培养标本,经过培养与分离获取100例结核分枝杆菌株,以改良罗氏比例法药敏试验为金标准,通过MMCA检测结核分枝杆菌对利福平、乙胺丁醇、链霉素、异烟肼、喹诺酮的耐药性,记录其符合率、敏感度、特异度、阳性预测值、阴性预测值。结果:MMCA检测利福平耐药性的符合率为94.00%,敏感度为83.33%,特异度为95.45%,阳性预测值为71.42%,阴性预测值为97.67%。MMCA检测乙胺丁醇耐药性的符合率为97.00%,敏感度为87.50%,特异度为97.83%,阳性预测值为77.78%,阴性预测值为98.90%。MMCA检测链霉素耐药性的符合率为98.00%,敏感度为85.71%,特异度为98.92%,阳性预测值为85.71%,阴性预测值为98.92%。MMCA检测异烟肼耐药性的符合率为90.00%,敏感度为76.47%,特异度为92.77%,阳性预测值为68.42%,阴性预测值为95.06%。MMCA检测喹诺酮耐药性的符合率为94.00%,敏感度为81.82%,特异度为95.51%,阳性预测值为69.23%,阴性预测值为97.70%。结论:MMCA评价结核分枝杆菌对利福平、乙胺丁醇、链霉素、异烟肼、喹诺酮的耐药性具有较好的特异度与敏感度,且便于检测,有助于对耐药结核杆菌的快速筛查。