Characterization of Salmonella Enterica Serotype Typhimurium from Outpatients of 28 Hospitals in Henan Province in 2006

Objective To characterize the diarrheal patients with Salmonella typhimurium （S. typhimurium） infections and to set up the first baseline for S. typhimuri_um pulsed-field gel electrophoresis （PFGE） patterns in Henan province, thus laying a foundation for comprehensive surveillance of Salmonella in human as well as foods. Methods S. typhimurium isolates recovered from outpatients with diarrhea in Henan province from May to October of 2006 were characterized. Antimicrobial susceptibility tests of 8 antimicrobial agents and PFGE were carried out to analyze the S. typhimurium isolates. Results Twenty-four （0.9%） S. typhimurium isolates were identified from 2661 stool specimens of diarrheal cases. Eighty-eight percent of isolates showed resistance to at least one antimicrobial agent. The resistance to chloramphenicol （79%） was most common. Fifty-eight percent of isolates were resistant to ciprofloxacin. All the 14 ciprofloxacin-resistant isolates were resistant to more than five antimicrobial agents. Thirty-three percent of S. typhimurium isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline （R-type ACSSuT）. Eight antimicrobia-resistant phenotypes were found among the 24 isolates in 16 PFGE patterns. Conclusion The rate of multidrug-resistant S. typhimurium is relatively high in S. typhimurium PFGE patterns of Henan province. Multidrug-resistant S. typhimurium should be considered a public health threat.     

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.     

Molecular Characterization and Correlation with β-lactam Resistance of Streptococcus pneumonia Isolates in Hangzhou,China

Penicillin-binding proteins(PBPs) are the target of β-lactam antibiotics(the major treatment for Streptococcus pneumoniae infections),and mutations in PBPs are considered as a primary mechanism for the development of β-lactam resistance in S.pneumoniae.This study was conducted to investigate the mutations in the PBPs of clinical S.pneumoniae isolates in Hangzhou,China,in correlation with β-lactam resistance.Results showed that 19 F was the predominant serotype(7/27) and 14 of the S.pneumoniae isolates were resistant to both penicillin G and cephalosporin.Genotyping results suggested that β-lactam-resistant isolates primarily exhibited single-site mutations in both the STMK and SRNVP motifs of pbp1 a in combination with double-site mutations in the STMK motif of pbp2 x,which might be the primary mechanisms underlying the β-lactam resistance of the isolates in this study.     

Molecular Epidemiological Characteristics of Streptococcus pyogenes Strains Involved in an Outbreak of Scarlet Fever in China, 2011

Objective To investigate molecular characterization of streptococcus pyogenes isolates involved in an outbreak of scarlet fever in China in 2011. Methods Seventy-four 5treptococcol pyogenes involved in an outbreak of scarlet fever were isolated from pediatric patients in the areas with high incidence in China from May to August of 2011. Emm genotyping, pulsed-field gel electrophoresis （PFGE）, superantigen （SAg） genes and antimicrobial susceptibility profiling were analyzed for these isolates. Results A total of 4 different emm types were identified. Emm12 was the most prevalent type which contained four predominating PFGE patterns corresponding to four different virulence and superantigen profiles. Emm12（79.7%） and emml （14.9%） accounted for approximately 94% of all the isolates. The speA gene was all negative in emm12 isolates and positive in emml isolates. All strains were resistant to erythromycin, and 89.4% of them were resistant to erythromycin, tracycline, and clindamycin simultaneously. Conclusion Several highly diversified clones with a high macrolide resistance rate comprise a predominant proportion of circulating strains, though no new emm type was found in this outbreak. The data provide a baseline for further surveillance of scarlet fever, which may contribute to the explanation of the outbreak and development of a GAS vaccine in China.     

Comparison of the Effects of Three Different Anti-fungus Drugs on Candida Albicans of Murine Vaginal Mucosa

To compare the therapeutic effects of three different anti-fungal drugs (i.e., terbinafine, fluconazole and intraconazole) in the treatment of experimental vaginitis caused by Candida albicans (C. albicans) in mice, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animal had been pretreated with estradiol. Mice were divided at random into different groups and then respectively treated with terbinafine, flucona- zole and intraconazole given by gastrogavage. The burden of the fungus in the vaginal lavage fluids in the mice of the different groups was measured dynamically at different time points after the begin- ning of the drug treatment. The fungal burdens in the vaginal lavage fluids taken at different time points from the mice treated with terbinafine were significantly higher than those taken at corre- sponding time points from mice treated with fluconazole or itraconazole (P<0.01). The fungal bur- dens in the vaginal lavage fluids taken from mice 1 week after the beginning of the treatment with terbinafine remained at a relatively high level. A dramatic drop in the fungal burden was noted in the vaginal lavage fluids taken on the 2nd day of the treatment from mice treated with itraconazole or flu- conazole group and the fungal burden on the 3rd day of the treatment in these mice were at a very low level, suggesting that fluconazole or itraconazole were highly effective for the treatment. How- ever, the difference in the therapeutic effect between the two drugs was not significant (P>0.05). Itraconazole or fluconazole, but not terbinafine, is very effective for the treatment of fungal vaginitis caused by C. albicans in mice.     

淋病奈瑟菌对环丙沙星耐药性的基因研究

目的 探讨临汾地区淋病奈瑟菌对环丙沙星的耐药性及其与gyrA基因喹诺酮类耐药决定区(QRDR)之间的关系.方法 采集33例淋球菌感染标本,通过药敏试验筛选出对环丙沙星耐药菌株,设计对应于淋球菌gyrA上QRDR区基因片段的引物,经过聚合酶链式反应(PCR)扩增其目的 基因片段,并对扩增产物进行DNA序列测定.结果 33株淋病奈瑟菌临床分离株对环丙沙星全部耐药;所有耐药菌株均经PCR扩增出大小为225 bp(对应于QRDR)的基因片段;测序结果显示耐药菌株都发生了91位点突变(Ser-91→Phe),其中单独91位点突变的有3株,发生91和95双位点突变的有30株,95位点突变类型有三种:Asp-95→Gly, Asp-95→Asn, Asp-95→Ala,以Asp-95→Gly最为常见,有19株,占gyrA 95位点突变株的63.3%.结论 临汾地区淋病奈瑟菌对环丙沙星的耐药情况很严重;gyrA基因突变在淋病奈瑟菌对环丙沙星耐药中起着重要作用. Abstract： Objective To investigate the occurrence of ciprofloxacin(cip)-resistance among Neisseria gonorrhoeae strains in Linfen region in 2006 and determine the frequency and patterns of mutations in gyrA gene in the ciprofloxacin resistance isolates. Methods A total of 33 gonococcal isolates were collected in Linfen region for Neisseria gonorrhoeae. In vitro, susceptibility tests of ciprofloxacin were performed in 33 isolates, gyrA gene were amplified by polymerase chain reaction(PCR)assay and the PCR products were directly sequenced in order to see if there were mutations in gyrA gene. Results The 33 clinical isolates demonstrated 100% resistance to ciprofloxacin. The gyrA gene containing quinolone resistance-determining region (QRDR)were amplified by PCR in all clinical isolates. The mutations had amino acid substitutions of Ser to Phe at codon 91(33 strains),and Asp to Gly,Asn,Ala at codon 95 of gyrA respectively. The most common gyrA alteration at codon 95, Asp-95 to Gly was found in 19 strains (63.3 %). Conclusions The resistant strains of Neisseria gonorrhoeae to ciprofloxacin in Linfen region are serious. The results from this study suggested that mutations in gyrA gene play an important role in the development of fluoroquinolone resistance of Neisseria gonorrhoeae.     

Analysis of genital Candida albicans infection by rapid microsatellite markers genotyping

Background Candida albicans （C. albicans） infection, often occurring in genital candidiasis, has increased dramatically recently. Developing an efficient C. albicans typing method may contribute to understanding its epidemiological characteristics and guiding efficient treatment. We used rapid microsatellite genotyping assay for interstrain differentiation of C. albicans isolates and explored some characteristics of its spread. Methods DNA was extracted from C. albicans isolates from gentalia, recta and mouths of 39 female cases and 27 male cases of genital candidiasis. Three fluorescent primers for the microsatellite markers in conserved genes （CDC3, EF3 and HIS3） of Co albicans were used to amplify the isolates DNA by PCR. Fluorescent signals were read with an automatic sequencer and analyzed with GeneScan software. Results Analysis of the three microsatellites markers showed 18 gene allelic associations in genital C. albicans infected patients： 10 allelic associations in female and 11 allelic associations in male, of which 3 allelic associations shared by both genders covered 71% of infections. The most dominant allele association of pathogenic strains for both genders was 116：124, 122：131,160：200 that covered about 50% of infection. Gentalia and recta shared the same strains in 80% of female patients, but in only 3.8% of male patients. There were 2.7% female patients, but no males, with same strain in both gentalia and mouths. Five of seven genital Co albicans infected couples had the same allelic associations of which 4 were the dominant pathogenic C. albicans susceptible for both genders. Conclusions The predominant allelic association of the pathogenic strain in genital C. albicans infection is 116：124, 122：131, 160：200. Vaginal pathogenic strains are probably maintained from the rectal reservoir. Pathogenic strains of male patients are probably from frequent sexual intercourse. The aggressiveness of some strains varies with gender.     

Characterization of human metapneumoviruses isolated in Chongqing, China

Background Human metapneumovirus （hMPV） has recently been recognized as a notable respiratory pathogen in children. However, no isolation processes and only a limited understanding of hMPV epidemiology present in Chinese children are documented by far. Methods Nasopharyngeal aspirates from 86 hospitalized children with acute respiratory tract infections from December 2004 to July 2005 were inoculated onto Vero-E6 cells for hMPV isolation. Total RNA was extracted from infected cells with a cytopathic effect and then subjected to a RT-PCR amplification of the N and F genes of the hMPV. Nucleotide sequences of amplified F gene products were examined using a variation analysis with the MegAlign program and the phylogenetic tree construction using the neighbor-joining algorithm with a Phylip package. Results Six strains of hMPV were isolated from the samples during winter, spring and summer. The most common symptoms were coughing and cyanosis, and the diagnoses were bronchiolitis, bronchopneumonia, infantile asthma, or upper respiratory tract infection. All isolates were in the A2 genetic sublineage and shared a high percentage of homology with the F gene in the nucleotide （99.8%--100%） and amino acid （99.3%--100%） sequences. Conclusions This report indicates that hMPV is an important viral agent for acute respiratory tract infections present in Chongqing, China. Knowledge of phylogeny and genes will benefit the studies on the treatment and prophylaxis of hMPV infection.     

Sequence analysis and genotypes of glutamate rich protein of Plasmodium falciparum isolates from different malaria endemic areas in China

To sequence the gene encoding glutamate rich protein (GLURP) and identify the genotypes of geographically different Plasmodium falciparum (P. f ) isolates from China. Methods The gene of R2 repeat region of GLURP was amplified by nested polymerase chain reaction and cloned into T-vector. The nucleotide sequence of GLURP gene was determined by automatic sequencer (Dideoxy termination method) and analyzed by DNA Star software. Results At least 7different GLURPgenotypesranging from 600 bp to 1 500 bp were found in Yunnan and Hainan provinces. R2 region of GLURP gene consisted of several repeat units. Each repeat unit was composed of 19-20 residues which were shown to be highly conserved. GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. Conclusion GLURP gene is highly structure conserved and size polymorphic, and so is useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.     

Resistant mechanisms of Candida albicans to fluconazole

ＯｂｊｅｃｔｉｖｅＴｏｄｅｔｅｃｔｔｈｅｒｅｓｉｓｔａｎｔｍｅｃｈａｎｉｓｍｓｏｆＣａｎｄｉｄａａｌｂｉｃａｎｓｔｏｆｌｕｃｏｎａｚｏｌｅ（ＦＣＺ）ａｔｍｏｌｅｃｕｌａｒｂｉｏｌｏｇｙｌｅｖｅｌ,ｓｉｎｃｅｔｈｅｒｅｓｉｓｔａｎｃｅｍｅｃｈａｎｉｓｍ...     

光滑假丝酵母菌氟康唑耐药机制研究

目的探讨光滑假丝酵母菌CDR1、CDR2、SNQ2和ERG11基因mRNA表达差异以及ERG11基因突变在氟康唑耐药形成中的作用。方法采用罗丹明6G试验比较氟康唑耐药组与敏感组的外排泵作用,Rea1—TimePCR检测外排相关基因CDR1、CDR2、SNQ2和ERG11表达情况。同时,对ERG11基因进行PCR扩增和测序,比较耐药组和敏感组的突变位点。结果耐药组光滑假丝酵母菌外排泵功能明显强于敏感组,差异有统计学意义（P〈0．01）。耐药组的CDR1和CDR2mRNA表达水平显著高于敏感组（P〈0．01,P〈0．05）;但两组间SNQ2和ERG11mRNA表达水平比较差异无统计学意义（P〉0．05）。检测到ERG11基因4个突变位点,均为同义突变,其中3个突变位点在耐药组和敏感组中均出现,1个突变位点只在敏感组中出现。结论外排泵功能在光滑假丝酵母菌氟康唑耐药过程中起重要作用。ERG11基因序列存在多态性,但未发现与氟康唑耐药相关的突变位点。     

肾移植术后患者白念珠菌分离株的唑类药物敏感性观测

目的 了解肾移植术后患者分离的白念珠菌对唑类药物的敏感性。方法 参照美国国家临床实验标准委员会(NCCLS)公布的M27-A方案微量稀释法，对我院近年的19株肾移植术后患者的白念珠菌临床分离株进行氟康唑和伊曲康唑药物的敏感性观测。结果 19株临床分离株中，发现氟康唑耐药株7株，敏感株8株，剂量依赖的敏感菌株4株；伊曲康唑耐药株9株，敏感株8株，余2株为剂量依赖的敏感菌株。结论 目前肾移植患者人群白念珠菌分离株存在严重的唑类药物耐药，而且氟康唑和伊曲康唑之间也存在交叉耐药。     

白假丝酵母ERG11基因突变分析

目的探讨白假丝酵母氟康唑作用的靶酶编码基因(ERG11)突变与氟康唑耐药性的关系。方法选择从尿道、阴道、口咽部、呼吸道、血液和前列腺液分离的10株氟康唑耐药白假丝酵母菌和3株敏感菌株,以抽提的白假丝酵母菌基因组DNA为模板,对ERG11基因序列进行PCR扩增,PCR产物直接DNA序列测定,最后应用BLAST和ClustalW软件对测序结果进行比较。结果13株白假丝酵母菌的ERG11基因序列(以已知序列第1个ATG起始密码的A作为第1个记数单位)共有21个位点发生突变,其中17个同义突变位点和4个错义突变位点;在第348bp位点和第383bp位点,耐药菌株和药物敏感株均可发生D116E和K128T变异;在靶酶活性中心血色素结合区和疏水端螺旋区的对应基因片段,耐药株在第1309bp位点可致V437I变化;在第1320bp位点耐药菌株发生碱基突变,形成A、C基因杂合子,有可能导致N440K变化。结论(1)ERG11基因的D116E和K128T位点变异可能与白假丝酵母菌耐药无关;(2)ERG11基因的V437I和N440K位点的改变,可能与耐药形成有关。     

菌丝相和酵母相白念珠菌ERG11基因部分序列差异性的研究

目的研究白念珠菌菌丝相和酵母细胞氟康唑作用的靶酶编码基因(ERG11)碱基序列上的差异,从而探讨两相细胞之间的差异性.方法分别抽提7株来自同一母体、依次对氟康唑逐渐耐药的白念珠菌菌丝和酵母相的基因组DNA,根据ERG11编码序列设计一对引物,对ERG11基因的第403位点至第701位点的298的碱基序列进行PCR扩增,经PCR产物直接测序比较两相ERG11基因碱基序列上的差异.结果7株白念珠菌的菌丝相与酵母相细胞间的ERG11基因碱基序列在该片断上无差异.结论菌丝相和在酵母相白念珠菌ERG11基因的部分碱基序列是无差异的.     

Alcohol dehydrogenase I expression correlates with CDR1, CDR2 and FLU1 expression in Candida albicans from patients with vulvovaginal candidiasis

Background The most critical mechanism governing drug resistance in Candida albicans (C.albicans) involves efflux pumps,the functionality of which largely depends on energy metabolism.Alcohol dehydroge...     

白念珠菌人工诱导耐药株唑靶酶三磷酸鸟苷环水解酶Ⅰ功能区的序列分析

目的:研究羊毛甾醇14α-脱甲基酶(14-DM)DNA序列中三磷酸鸟苷(GTP)环水解酶Ⅰ功能区域基因突变与白念珠菌对氟康唑耐药性的关系.方法:体外采用氟康唑结合阿苯达唑进行人工耐药性诱导1株白念珠菌标准菌株和2株临床分离对氟康唑敏感株,将获得的3株人工诱导耐药白念菌株和另外2株临床分离对氟康唑耐药的白念菌株,通过PCR扩增目的片段,并克隆到pMD-18T载体上,测序分析诱导前后基因序列碱基变化.结果:经体外人工耐药性诱导后,标准白念珠菌菌株和临床分离敏感白念珠菌菌株GTP环水解酶Ⅰ功能区域多处碱基突变,部分碱基变化引起了氨基酸的改变,与临床分离耐药白念珠菌在碱基变化及其导致的编码氨基酸变化相似.结论:14-DM GTP环水解酶Ⅰ功能区域基因突变与白念珠菌的耐药性有相关性.     

阴道白假丝酵母菌的耐药性和ERG11基因突变关系研究

目的探讨ERG11基因突变与白假丝酵母菌对吡咯类药物敏感性下降的关系。方法收集VVC患者的白假丝酵母菌60株,用科玛嘉念珠菌显色培养基作菌种鉴定,纸片扩散法进行药敏试验,选取所有中度敏感株和耐药株用聚合酶链反应扩增(PCR)ERG11基因,将PCR产物进行测序分析和比对分析。结果 60株白假丝酵母菌对咪康唑、酮康唑和氟康唑敏感率分别为36.7%、56.1%和80%;将38株中度敏感株和耐药株进行ERG11基因分析后共发现29个有义突变和25个同义突变,其中,有义突变中发现多个新突变位点。结论有些新发现的突变位点可能与耐药相关;多位点有义突变可能降低菌株对药物的敏感性。     

过氧化氢对5种唑类药物体外抗耐药白假丝酵母菌的协同作用

目的考察过氧化氢(H2O2)对5种唑类药物:氟康唑(FLC),伊曲康唑(ICZ),酮康唑(KCZ),咪康唑(MCZ)和硫康唑(SCZ)体外抗耐药白假丝酵母菌作用的影响。方法利用8株对氟康唑耐药,2株对氟康唑敏感的白假丝酵母菌,采用微量液基稀释法测定药物单用时的最低抑菌浓度MIC80值,采用棋盘式微量液基稀释法测定两药联用时的MIC80值,在此基础上计算部分抑菌浓度指数(FICI)即协同指数,依据FICI值判断H2O2与唑类药物联用是具有协同、无关还是拮抗作用。结果对所有耐药菌株,两药联用的FICI值均小于0.5,H2O2与唑类药物联用显现出协同作用;对2株敏感菌株,两药联用的FICI值大于0.5、小于2,显示为无关作用。结论 H2O2和唑类药物(氟康唑,伊曲康唑,酮康唑,咪康唑,硫康唑)联用有协同抗耐药白假丝酵母菌作用。     

复发性外阴阴道假丝酵母菌病致病菌的药敏及其基因分布

目的：探讨复发性外阴阴道假丝酵母菌病（ RVVC）病原体的药敏情况及其基因CDR1、CDR2、ERG11、CaMDR1和FLU1的分布状况。方法采取60例RVVC患者的白带标本,于科玛嘉念珠菌显色平板上培养,Vitek-2全自动细菌鉴定仪进行菌株鉴定,纸片法进行药敏试验;以 PCR 扩增法检测 CDR1、CDR2、ERG11、CaMDR1和FLU1基因分布。结果60份白带标本分离出60株假丝酵母菌,其中白色假丝酵母菌、光滑假丝酵母菌、热带假丝酵母菌、克柔假丝酵母菌、乳酒假丝酵母菌分别占73．3％、15．0％、6．7％、3．3％、1．7％;假丝酵母菌株对咪康唑的耐药率最高为21．7％,其次是5-氟尿嘧啶为13．3％,对氟康唑、伊曲康唑、两性霉素B的耐药率分别为8．3％、6．7％、5．0％。 PCR耐药基因组检测CDR1、CDR2、ERG11、CaMDR1和FLU1阳性率分别为63．3％、61．7％、58．3％、63．3％、1．7％。44株白色假丝酵母菌株对咪康唑耐药株11株、敏感株29株、中介4株,其中耐药株CDR1、CDR2、ERG11、CaMDR1阳性表达率分别为81．8％、72．7％、72．7％、72．7％,高于敏感株的34．5％、31．0％、27．6％、37．9％（P均＜0．05）,FLU1阳性率差异无统计学意义（P＞0．05）。结论假丝酵母菌对临床常用的抗真菌药物存在一定的耐药性,耐药基因CDR1、CDR2、ERG11、CaMDR1在多种假丝酵母菌株中均有表达。