Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China

Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniazid （INH） resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance. Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide （RIFO） assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA （-15C/T） and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism （RFLP） method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five （91.5%） of 71 RIF resistant and 52 （92.9%） of 56 multidrug-resistant （MDR, i.e. resistant to at least RIF and INH） strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315 AGC〉ACC and inhA-15C〉T mutations were found in 40 （60.6%） and 10 （15.2%） of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into ＂modern＂ （76.6%） and ＂ancestral＂ （23.4%） group. There is no significant difference between ＂ancestral＂ and ＂modern＂ group in prevalance of drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of I NH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.     

Relationship between antimicrobial resistance and aminoglycoside- modifying enzyme gene expressions in Acinetobacter baumannii

Background Acinetobacter baumannii is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by multi-drug resistance Acinetobacter baumannii is very difficult to treat. This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycoside-modifying enzymes including N-acetyltransferases and O-phosphotransferases in Acinetobacter baumannii. Methods Bacterial identification and antimicrobial susceptibility test were performed by Phoenix^TM system in 247 strains of Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multi-drug resistant Acinetobacter baumannii were detected by agar dilution. Four aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer. Results The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50%. Imipenem and meropenem showed high antibacterial activities with resistance rates of 3.2% and 4. 1%. MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multi-drug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 93.3%, respectively. But their resistance rates to tobramycin, netilmicin and neomycin were 86.7%, 93.3% and 46.7%, respectively. Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected. Their positive rates were 93.3%, 20. 0% and 20. 0%, respectively. These three genes existed simultaneously in No. 19 strain. Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 97.9% and 99.7% identities with GenBank genes (AY307113, S68058 and AY307114). Conclusion Multi-drug resistant Acinetobacter baumannii strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.     

Antibiotic Resistance of Probiotic Strains of Lactic Acid Bacteria Isolated from Marketed Foods and Drugs

Objective To identify the antimicrobial resistance of commercial lactic acid bacteria present in microbial foods and drug additives by analyzing their isolated strains used for fermentation and probiotics. Methods Antimicrobial susceptibility of 41 screened isolates was tested with disc diffusion and E-test methods after species-level identification. Resistant strains were selected and examined for the presence of resistance genes by PCR. Results Distribution of resistance was found in different species. All isolates were susceptible to chloramphenicol, tetracycline, ampicillin, amoxicillin/clavulanic acid, cephalothin, and imipenem. In addition, isolates resistant to vancomycin, rifampicin, streptomycin, bacitracin, and erythromycin were detected, although the incidence of resistance to these antibiotics was relatively low. In contrast, most strains were resistant to ciprofloxacin, amikacin, trimethoprim/sulphamethoxazole, and gentamycin. The genes msrC, vanX, and dfrA were detected in strains of Enterococcus faecium, Lactobacillus plantarum, Streptococcus thermophilus, and Lactococcus lactis. Conclusion Antibiotic resistance is present in different species of probiotic strains, which poses a threat to food safety. Evaluation of the safety of lactic acid bacteria for human consumption should be guided by established criteria, guidelines and regulations.     

Detection of the mutations in katG315 and inhA-15 of Mycobacterium tuberculosis strains isolated from Chinese patients

The situation of tuberculosis and M. tuberculosis （MTB） drug resistance is severe in China.Among the MTB isolates, 27.8% were resistant to one primary antituberculosis drug at least. Isoniazid （INH） is one of the first-line anti-TB drugs. The rates of primary resistance and acquired resistance to INH were 11.0% and 31.0%, respectively. MTB strains resistance to INH is mainly caused by the alterations in several genes encoding the molecular targets as follows： catalase peroxidase （katG）,     

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective： To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR- SSCP to clinical application. Methods： A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test（AST）. Results： The gene muta- tion rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% （30/52）, 65.38% （32/52） and 40.38% （21/52）. The rate of reversion was 78.85%（41/52） and the result of drag-resistant genes was invariable. The results of AST showed that there were 40 （76.92%） multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21 （40.38%） and that of two-drug resistant was 19（36.54%）, but only 12（23.08%） strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene （P 〉 0.05）. Conclusion： PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.     

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.     

Antimicrobial resistance and molecular epidemiological characteristics of clinical isolates of Staphylococcus aureus in Changsha area

Background  Increasing prevalence of Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus (MRSA) has been reported in China. In this study, we investigated the drug resistance characteristic, genetic background, and molecular epidemiological characteristic of S. aureus in Changsha.

Methods  Between January 2006 and December 2008, 293 clinical isolates of S. aureus were collected from 11 hospitals in Changsha and identified by the Vitek-2 system. All the isolates were verified as MRSA by PCR amplification of both femA and mecA genes. K-B disk method was used to test drug sensitivity of S. aureus to antibiotics. Pulsed-field gel electrophoresis (PFGE) was performed for genotypic and homologous analysis of 115 isolates randomly selected from the original 293 clinical S. aureus isolates.

Results  S. aureus was highly resistant to penicillin, ampicillin, erythromycin, and clindamycin with resistant rates of 96.6%, 96.6%, 77.1%, and 67.2% respectively. All the isolates were susceptible to tecoplanin, vancomycin, and linezolid. MRSA accounted for 64.8% (190/293) of all the S. aureus strains. The 115 S. aureus isolates were clustered into 39 PFGE types by PFGE typing, with 13 predominant patterns (designated types A to M) accounting for 89 isolates. The most prevalent PFGE type was type A (n=56, 48.7%) and 100.0% of type A strains were MRSA. PFGE type A included 13 subtypes, and the most prevalent subtype was subtype A1 (46.4%, 26/56). Strains with PFGE type A were isolated from eight hospitals (8/11), and both subtypes A1 and A4 strains were isolated in a university hospital.

Conclusions  Clinical isolates of S. aureus in Changsha were resistant to multiple traditional antibiotics. There was an outbreak of PFGE type A MRSA in this area and the A1 subtype was the predominant epidemic clone. Dissemination of the same clone was an important reason for the wide spread of MRSA.     

Prevalence of Resistance to Macrolide, Lincosamide, and Streptogramin Antibiotics in Staphylococcus Epidermidis Isolates from a Chinese Hospital

To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin B （MLSs） antibiotics in Staphylococcus epiderrnidis caused nosocomial infection in the Chinese multicenter hospital, we tested the antibiotic susceptibility of 126 clinical isolates of S. epidermidis to the macrolide erythromycin, the lincosamide clindamycin and the streptogramin quinupristin/dalfopristin. The isolates were mostly resistant to macrolide and lincosamide, but were susceptible to quinupristin/daltbpristin. The resistance phenotypes of erythromycin-resistant isolates were determined by the double-disc test with erythromycin and clindamycin, which showed most exhibited constitutive MLS phenotype （cMLSB）. Among the cMLSB isolates, the methicillin resistant S. epidermidis （MRSE） proportion appeared high, whereas high methicillin susceptible S. epidermidis （MSSE） proportion was found in the inducible MLSB phenotype （iMLS~）. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the presence of the relative genes （ermA, ermB, ermC and msrA） to MLSB resistance was identified by PCR. The ermC was shown as the most frequent determinant to the resistance. No specific endemic strain was found by PFGE analysis. Our results indicate that the MLSB resistance in S. epidermidis caused nosocomial infection is prevalent in the hospital and MLSB antibiotics should be used judiciously. The high resistant rate to MLSB is largely due to antibiotic selective pressure.     

美罗培南联合头孢哌酮钠舒巴坦钠及米诺环素治疗大面积烧伤合并泛耐药鲍曼不动杆菌感染

Pan-drug resistant Acinetobacter baumannii （A.baumannii） （PDRAB） is resistant to all currently-available antimicrobials （including carbapenems）, withthe exception of colistin （polymyxin）. Recently, PDRABinfection has become a serious medical problem. Themechanisms of A. baumannii drug resistance includelow permeability of the outer membrane to antibiotics,an antibiotic efflux pump, and an extended-spectrum13-1actamase, etc. In addition, drug resistance can beeasily acquired by incorporating genetic elements such asplasmids, transposons, and integrons. Multidrug-resistantA. baumannii （MDRAB; ie, strains with resistance to ≥3classes of drugs） strains are being increasingly reportedworldwide. Infections due toare associated with increasedCarbapenems, which used tosuch resistant microbesmorbidity and mortality.be the antimicrobials ofchoice, have become increasingly ineffective against suchmulti-resistant strains and no longer constitute salvagetherapy for A. baumannii infections. In the last few years,PDRAB infections have been encountered several timesduring the treatment of patients with extensive burns. A.baumannii is susceptible to polymyxin B, which is notavailable in China. Therefore, the nine PDRAB infectedpatients were successfully treated with high doses ofmeropenem, cefoperazone-sulbactam and minocycline.     

泛耐药鲍曼不动杆菌医院获得性感染相关因素分析

目的 研究泛耐药鲍曼不动杆菌(PDRAB)医院获得性感染的临床因素、菌株耐药性与分子溯源,分析不同株系流行特征与感染相关因素。方法 收集北京同仁医院2009年1月至2011年1月9次医院感染流行分离的PDRAB60株及临床资料;纸片琼脂扩散法检测其对12种抗生素的耐药表型;分别应用重复序列PCR( REP-PCR)和基质辅助激光解析电离飞行时间质谱仪(MAIDI-TOF-TOF MS)对流行菌株进行基因水平和蛋白水平的溯源和鉴定。结果 除2株菌对亚胺培南、美罗培南敏感外,其余抗生素均耐药。60株PDRAB REP-PCR检测分为REP-A～L共12型,MALDI-TOF-TOF MS分为a～e5个类型。与MALDI-TOF-TOFMS相比,REP-PCR分型结果更为精细准确。呼吸机的携带与交叉传播可能是造成2009年7-10月呼吸科PDRAB大流行的主要原因;医护人员手传播是造成2010年1-2月外科重症监护病房PDRAB感染的关键因素;超级传播者鼻咽部或呼吸道PDRAB对周边环境的污染与传播是造成其余7次PDRAB流行的主要原因;急诊科是本院PDRAB获得性感染的传染源。结论 早期识别和隔离超级传播者、加强环境器械消毒和医护人员手卫生、规范抗生素的使用是控制医院获得性感染的关键要素。MALDI-TOF-TOF MS方便、价廉、快速鉴定病原菌及分型优势有望成为微生物鉴定分型发展的方向。     

海口地区鲍曼不动杆菌医院感染分布特征及耐药性分析

目的 研究海口地区医院感染鲍曼不动杆菌的医院感染分布特征及其对常见抗生素的耐药性.方法 采用回顾性调查方法,分析海口地区四家医院临床分离的570株鲍曼不动杆菌的标本来源、感染科室分布及耐药状况.结果 570株鲍曼不动杆菌来自痰标本427株(74.91%),脓液48株(8.60%),尿液42株(7.37%);药物敏感试验...     

鲍曼不动杆菌所致医院感染的调查研究

为调查鲍曼不动杆菌在医院感染中的流行情况,对９８年从我院分离的４５株鲍曼不动杆菌的分布及药敏作回顾性研究,结果发现鲍曼不动杆菌在医院各科分布广泛,其中从ＩＣＵ中检出２７株,占６０％,从上呼吸道分离出３７株,占８４．４％,药敏结果显示对头拖拉院及增效磺胺耐药,对广谱青霉素／酶抑制剂,三代头孢,氨基甙类抗生素的耐药率较低,尤其是亚胺培南极少发现耐药株,可用于鲍曼不动杆菌的治疗。我们认为鲍曼不动杆菌是新近发现的重要条件致病菌,易引起ＩＣＵ病人的爆发流行,在医院感染中的地位不容忽视。     

衡阳地区鲍曼不动杆菌基因分型及医院感染分布情况

目的研究鲍曼不动杆菌医院感染分子流行病学的情况,为控制医院感染提供直接、可靠的参考依据。方法对衡阳地区2011年1月～12月临床送检18 000份标本中分离出的480例鲍曼不动杆菌的药敏试验结果等进行回顾性分析,了解鲍曼不动杆菌医院感染现状;建立随机扩增多态性DNA(RAPD)基因分型方法对其中140株多重耐药鲍曼不动杆菌进行基因分型。结果 480例鲍曼不动杆菌标本主要来自ICU、呼吸内科、烧伤科等;标本分布以痰标本最高占65.0%,其次为伤口分泌物标本占20.0%,中段尿10.0%,其它类型标本占5.0%;480株鲍曼不动杆菌的多重耐药情况非常严重,对亚胺培南/美罗培南耐药率分别为36.0%和38.0%,对其它大多数抗菌药物的耐药率为80%以上;140株多重耐药鲍曼不动杆菌用RAPD分型方法分为8种类型:A～H,其中A、B、C 3型共占70.0%。结论鲍曼不动杆菌耐药性呈上升趋势,且具有多重耐药性;衡阳地区医院感染多重耐药鲍曼不动杆菌可能存在院内的克隆传播。     

嗜麦芽窄食单胞菌医院感染特点及耐药性分析

目的分析近2年我院发生的嗜麦芽窄食单胞菌(SMA)医院感染的特点及耐药性,为控制感染和临床合理使用抗菌药物提供依据。方法采用美国临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,检测从40例感染患者中分离的50株SMA对24种抗生素的耐药情况,通过脉冲场凝胶电泳(PFGE)全DNA指纹技术对菌株进行分型。结果 SMA感染主要发生在下呼吸道,50岁以上的患者居多,且多数为混合感染。SMA对头孢吡肟/舒巴坦、加替沙星、左氧氟沙星、氧氟沙星、敏感性高,而对多数抗菌药物耐药,对头孢曲松、头孢呋辛、头孢西丁的耐药率达到了100.0%,对亚胺培南、氨曲南、四环素、哌拉西林和庆大霉素耐药率分别为92.3%、88.9%、82.4%、75.0%和70.0%。多数SMA(17/21)没有同源性,仅一对同源菌株来自同一病房,另一对同源菌株来自医院的不同病房。结论患者被医院环境中自然存在的SMA定植可能是SMA基因组的多样性和低同源性的原因。头孢吡肟/舒巴坦、加替沙星、左氧氟沙星、氧氟沙星、头孢哌酮/舒巴坦是治疗SMA感染的有效药物。     

检出鲍曼不动杆菌临床分布及耐药性分析

目的了解检出鲍曼不动杆菌的耐药性及临床分布特征，为诊治提供参考。方法用琼脂扩散法（K—B法）进行药物敏感性检测，按表型确证试验检测ESBLs。结果检出的鲍曼不动杆菌对IPM近100％敏感，对AMX／CA、CAZ／CA、CAZ、CIP敏感率达80％以上，对CZ、FOX几乎全部耐药，ESBLs检出率为16％。本组结果感染者以呼吸内科住院者为多，标本来源以下呼吸道标本最多见。结论鲍曼不动杆菌耐药率高，可普遍存在于医院环境中，应加强院内感染控制，防止暴发流行。     

重症监护病房鲍曼不动杆菌耐药性分析及对策

目的分析ICU病房鲍曼不动杆菌（ABA）耐药情况和临床特点,为预防和控制ABA的感染提供依据。方法对本院2009年1月～2010年5月ICU病房患者送检标本中分离的鲍曼不动杆菌的病区分布情况与耐药性进行回顾性分析。结果 ABA对常用药都很耐药,仅对亚胺培南、左氧氟沙星的耐药率较低为42.4%和46.3%,泛耐药株检出率达7.8%。结论 ABA在ICU病房的感染较为严重,而且耐药性很高。必须采取相应的防治和监控措施,预防ABA的高耐药性和多重耐药性造成的医院感染。     

多重耐药鲍曼不动杆菌中仅黏菌素敏感菌株的分子流行病学研究

目的从基因水平了解多重耐药鲍曼不动杆菌（MDR-AB）中仅黏菌素敏感鲍曼不动杆菌（COS-AB）的流行情况，为控制其流行暴发提供实验数据。方法收集并分析上海瑞金医院2004年6月至2005年5月136例COS—AB临床感染患者的临床资料，同时收集2004年5月至2004年12月临床分离的鲍曼不动杆菌66株，其中COS-AB33株，非黏菌素敏感鲍曼不动杆菌（non-COS-AB）33株，采用随机引物PCR（RAPD）和脉冲场凝胶电泳（PFGE）等分子流行病学方法和统计学方法分析其流行情况。结果证实2004年6月至2005年5月出现COS-AB的暴发流行，经RAPD（引物ERIC2、272）分型均为一个型别，而PFGE分型为两个型别，其中烧伤科COS-AB为PFGEB型，为单一克隆的科室内流行；而烧伤科以外的其他科室为A型，为科室间流行，且主要集中于外科系统。结论COS-AB交叉感染严重，应严格消毒隔离措施，加强对COS-AB在环境及患者中流行情况的严密监测。用PFGE对鲍曼不动杆菌进行分型更为精确。     

伤寒沙门菌PFGE分子分型及耐药性研究

目的对深圳市2004-2011年伤寒沙门菌进行脉冲场凝胶电泳(PFGE)分子分型及耐药性分析,为临床用药和追踪传染源提供依据。方法采用K-B法对42株伤寒沙门菌进行20种抗菌药物敏感性测试,并用PFGE对其进行分子分型。结果 42株伤寒沙门菌株对氨苄西林、阿莫西林/克拉维酸、氨苄西林/舒巴坦、头孢噻吩、头孢他啶、头孢曲松、头孢吡肟、头孢西丁、阿米卡星、庆大霉素、卡那霉素、环丙沙星、左旋氧氟沙星、复方新诺明、甲氧苄啶、氯霉素和四环素17种药物的敏感率均超过90%,对萘啶酸耐药率最高达61.5%。42株伤寒沙门菌株可分为SZ0001-SZ0028共28个PFGE型别,其中流行优势型为SZ0023型别。结论结合药物敏感试验结果和PFGE分子分型结果可以判断伤寒疫情的优势株型。     

云南省鼠伤寒沙门氏菌PFGE分子分型及耐药情况

目的 了解云南省鼠伤寒沙门氏菌PFGE分子分型及耐药状况, 为防控由鼠伤寒沙门氏菌引起的疾病提供科学依据.方法 根据Pulse Net China公布的沙门氏菌PFGE分型技术进行分子分型.分析得出药敏板MIC值, 根据CLSI的相应标准获得出S、I、R结果.结果 云南省68株鼠伤寒沙门氏菌呈53种PFGE带型.68株鼠伤寒沙门氏菌对复合磺胺 (SXT) 的耐药率最高, 为45.59%, 对亚胺培南 (IMP) 最敏感.其中1株菌对14种抗生素中11种抗生素耐药.结论 云南省鼠伤寒沙门氏菌分子分型呈多样性.复方磺胺是云南地区鼠伤寒沙门氏菌最耐药抗菌素.地区鼠伤寒沙门氏菌多重耐药不典型.