89株山夫登堡沙门菌脉冲场凝胶电泳分子分型及耐药性分析研究

目的 分析2005-2011年杭州市下城区89株山夫登堡沙门菌的脉冲场凝胶电泳(pulse-field gel electrophoresis,PFGE)分子型别和耐药性,建立杭州市沙门菌指纹图谱数据库。 方法 89株山夫登堡沙门菌用XbaⅠ酶切、PFGE获得电泳图谱,使用BioNumerisc统计软件聚类分析比较同源性。采用K-B法测定菌株对抗生素敏感性。 结果 以相似度85%为判定标准,89株山夫登堡沙门菌可分为10个PFGE带型,其中68株集中在一个PFGE基因型内。不同年份基因型无明显规律性。89株山夫登堡沙门菌对所选11种抗生素敏感性85.19%。 结论 杭州市下城区2005-2011年分离的89株山夫登堡沙门菌有明显的克隆株优势带型存在,对抗生素敏感性较高。     

食源性沙门菌感染的实验室诊断及鼠伤寒沙门菌同源性分析

目的用改良的沙门菌分离鉴定方法提高肠道沙门菌的检出率,初步掌握上海市主要沙门菌流行状况及菌型分布,建立沙门菌脉冲场凝胶电泳(PFGE)分型数据库,以便在发生沙门菌病时快速溯源,为控制该病的传播提供技术支撑。方法用改良的沙门菌鉴定方法对可疑肠道粪便标本进行分离、培养和鉴定;用PFGE对鼠伤寒沙门菌进行DNA分子分型,分析试验菌株的同源性。结果用改良的沙门菌鉴定方法分离出的沙门菌中以肠炎沙门菌为主,占全部菌株的42.1%,其次为鼠伤寒沙门菌,占13.2%。PFGE分析的10株鼠伤寒沙门菌分成A、B 2个PFGE型,其中A型菌株6株,B型菌株4株。A型菌株有4个亚型。结论改良的沙门菌鉴定方法可以有效提高沙门菌的检出率;PFGE是一种研究鼠伤寒沙门菌分子分型的有效方法,鼠伤寒沙门菌的分型结果显示了相对集中的亲缘关系。     

脉冲场凝胶电泳技术应用于沙门菌鉴定的方法研究

目的利用脉冲场凝胶电泳(PFGE)技术和设备解决沙门菌鉴定困难的难题,探究简便、快速、准确的鉴定方法,确立积极有效检测鉴定手段和监控措施,防止食物中毒及传染病的暴发流行。方法根据测试菌不同,探讨PFGE技术实验条件,沙门菌的PFGE鉴定方法。结果经反复试验证实所建方法灵敏快速、准确可靠、分辨率高,实用性强。在没有图像分析仪的情况下,肉眼即可进行判断,可替代传统的血清学鉴定方法。结论脉冲场凝胶电泳基因分型及同源性鉴定方法分辨率高,应用性强,技术先进。     

东莞市腹泻患者沙门菌同源性及耐药性特征

目的了解广东省东莞市2007--2009年腹泻患者中沙门菌感染情况和菌株的血清型别、同源性及耐药性变化。方法对纳入研究的腹泻患者进行沙门菌检测,对分离到的菌株进行血清分型、药物敏感试验和脉冲场凝胶电泳（PFGE）分型。结果2007--2009年共检测760份腹泻患者粪便标本,分离到53株沙门菌,检出率为7．0％;共分得15种血清型,鼠伤寒和肠炎沙门菌居多;大多数沙门菌对常用的头孢菌素类和喹诺酮类抗菌药物敏感,除肠炎沙门菌、斯坦利维尔沙门菌和B型副伤寒沙门菌外,其余菌株的血清型无同一PFGE型别的菌株;用XbaI酶切11株肠炎沙门菌可分为PFGE—XbsⅠ 1～6型,其中PFGE-XbaI4型为优势型别。用SfiI和Notl酶对5株肠炎沙门菌进行再分型,综合用XbaI／SfiI／NotI3种酶的结果发现仍有2组菌的PFGE图谱是完全一致的。结论2007--2009年度广东省东莞市沙门菌的感染多数为散发病例,头孢菌素类和喹诺酮类抗菌药物是治疗沙门菌感染的首选药物。     

一起幼儿园肠炎沙门菌食物中毒事件的调查

本次事件共有81名儿童患病,罹患率为48.2%,主要症状为发热(100%)、腹泻(98.8%)、腹痛(97.5%)、呕吐(69.1%);从患者粪便和食物蛋炒饭中分离的肠炎沙门菌PFGE图谱完全一致,且与生鸡蛋涂抹样品中分离株的相似度为96.2%,提示为一起由鸡蛋中的肠炎沙门菌污染导致的食物中毒事件。     

不典型山夫登堡沙门菌腹泻株的分子溯源研究

上海市疾病预防控制中心(SCDC)于2006年始加入WHO全球非伤寒沙门菌的监测网络(GSS),通过临床腹泻病例的监测发现:2006年7-9月间,山夫登儇沙门菌血清型呈现过暴发的流行病学特征[1],其中一类罕见的H2S阴性山夫登堡沙门菌流行菌株占较大比例,为掌握山夫登堡沙门菌在本市的流行规律,本研究对2006-2007年GSS山夫登堡沙门菌腹泻菌株的耐药型谱、RP核糖体分型和脉冲凝胶电泳(PFGE)分子分型进行分析,了解不产硫化氢山夫登堡沙门菌的遗传学特征,为本市的非伤寒沙门菌监测和防治提供依据.     

2015年北京一起鼠伤寒沙门菌暴发流行的病原学检测分析

2015年5月北京一家大型企业发生聚集性腹泻疫情,流行病学调查后收集现场样本,经过实验室检测确认该次疫情为鼠伤寒沙门菌暴发流行。分离到的18株鼠伤寒沙门菌脉冲场凝胶电泳(PFGE)图谱带型一致,在北京市沙门菌PFGE数据库中未发现相同带型。药物敏感试验结果显示有一株菌株对12种抗生素耐药。这种耐药模式的出现提示需加强耐药监测。     

中国肠炎沙门菌脉冲场凝胶电泳分子分型数据库的分析

目的 了解近几年我国肠炎沙门菌临床分离株的流行病学及脉冲场凝胶电泳(PFGE)分子分型特点,为以实验室为基础的食源性疾病暴发的发现及疫情控制提供依据。 方法 根据PulseNet公布的沙门菌PFGE分型方案,对2004-2012年分离自我国13个省(直辖市)的981株肠炎沙门菌进行流行病学及PFGE分子分型分析。 结果 981株肠炎沙门菌经XbaⅠ酶切,PFGE获得126种带型,其分辨能力(D值)为0.8336。优势带型为JEGX01.CN0003、JEGX01.CN0001、JEGX01.CN0002、JEGX01.CN0005和JEGX01.CN0016。暴发相关带型是JEGX01.CN0003、JEGX01.CN0001和JEGX01.CN0150。肠炎沙门菌优势带型PFGE的共有条带分别为(0.8%)655、325、310、245、178和110 kbp。 结论 我国肠炎沙门菌临床分离株存在PFGE优势型别,PFGE对于肠炎沙门菌XbaⅠ单酶切分型能力有限,用于暴发确认或发现时需多种酶切联合使用,提高分辨能力。     

一起沙门菌食物中毒的实验室诊断与分析

目的 查明内蒙古某市某村发生食物中毒突发公共卫生事件的性质规模,调查可疑危险因素及其污染来源,控制疫情蔓延. 方法 制定病例定义,开展病例主动搜索,采集患者肛拭子及粪便样品、可疑食品及环境样品,进行病原菌的分离培养.分离菌株进行生化和血清学鉴定,PFGE分子分型和药敏检测. 结果 共出现43例感染病例,均参加了2014年5月6日的婚宴聚餐.19份检测样品中从7份患者肛拭子分离到肠炎沙门菌,志贺菌、致泻性大肠埃希菌、金黄色葡萄球菌及蜡样芽孢杆菌检测阴性.7株肠炎沙门菌XbaⅠ酶切后的PFGE谱型完全相同,说明感染菌株来自同一个克隆,即有共同的感染源头.PulseNet China数据库比对分析显示,本次疫情菌株的PFGE图谱与我国肠炎沙门菌分离株中常见的PFGE带型JEGX01.CN0002 完全相同. 结果 此次事件为一起肠炎沙门菌引起的食物中毒,感染菌株具有国内优势的PFGE带型.     

农村宴席沙门菌食物中毒脉冲场凝胶电泳溯源及药敏分析

目的对引起食物中毒的21株沙门菌进行表型分型以确定该菌型别,并进行脉冲场凝胶电泳分型溯源,查明事件暴发原因,了解菌株的耐药状况。方法 21株沙门菌采用全自动细菌鉴定系统进行生化鉴定,采用沙门菌属诊断血清对该菌株进行血清学鉴定。对21株沙门菌以限制性内切酶XbaⅠ酶切后进行脉冲场凝胶电泳,得到的图谱用生物分析软件BioNumerics进行同源性分析,微量肉汤稀释法对沙门菌进行耐药性检测。结果本起食物中毒的沙门菌经过生化复核、血清学检测确定为肠炎沙门菌。来源于患者的15株肠炎沙门菌和分离至食品的6株肠炎沙门菌指纹图谱经BioNumerics软件分析具有极高相似性。该肠炎沙门菌对青霉素类的氨苄西林、β-内酰胺类的氨苄西林/舒巴坦、头孢类的头孢唑啉、喹诺酮类的萘啶酸、氨基糖苷类的庆大霉素、大环内酯类的红霉素与阿奇霉素耐药。结论引起宴席食物中毒的病原为肠炎沙门菌。通过药敏检测发现该菌多重耐药。来源于患者的菌株和食品的菌株有高度同源性,结合流行病学调查结果判断为餐饮从业人员在加工食品环节中受到肠炎沙门菌污染所致。     

Class 1 integron-associated gene cassettes in Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in Brazil

OBJECTIVES: Two multiresistant Salmonella enterica subsp. enterica serovar Agona isolates from pig carcasses were investigated for antimicrobial resistance genes and their location with particular reference to the detection of class 1 integrons. METHODS: The two S. Agona isolates were investigated for their in vitro susceptibility to antimicrobial agents and their plasmid content. The resistance genes and class 1 amplicons were identified by PCR assays. Amplicons of class 1 integrons were cloned and sequenced. Transferability of resistance plasmids was confirmed by conjugation. RESULTS: Both S. Agona isolates carried conjugative plasmids of approximately 150 kb which harboured all resistance genes detected in the respective isolates. S. Agona 231 was resistant to chloramphenicol by catA1, to tetracycline and minocycline by tet(B), and to sulphonamides by sul1. In addition, it harboured a streptomycin resistance gene strA and a class 1 integron with a new aadA variant designated aadA23, which mediates resistance to streptomycin and spectinomycin. S. Agona 242 also carried the genes catA1, tet(B), and sul1. Moreover, it harboured a second sulphonamide resistance gene, sul2, and a class 1 integron with intact gene cassettes carrying new variants of the trimethoprim resistance gene dfrA15b or the chloramphenicol resistance gene cmlA4. The third gene cassette consisted of a truncated aadA2 gene. CONCLUSIONS: The results of this study show that large conjugative multiresistance plasmids are present in S. Agona from pigs. Analysis of the class 1 integrons revealed the presence of new variants of resistance genes so far not detected in Salmonella isolates.     

Tetracycline Resistance in Salmonella enterica subsp. enterica Serovar Dublin

The 47-kbp plasmid pGFT1 from Salmonella enterica subsp. enterica serovar Dublin mediated tetracycline resistance via a tet(A) gene located on an integrated copy of a Tn1721-analogous transposon. The integration site of the transposon was located within the reading frame of a fip gene. Plasmid pGFT1 was shown to be conjugative and to be able to replicate and express tetracycline resistance in Escherichia coli.     

Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis

OBJECTIVES: A Salmonella enterica serovar Infantis isolate of avian origin was investigated for the presence of the gene qnrS, its transferability and its association with other resistance genes. METHODS: The Salmonella Infantis isolate was investigated for its susceptibility to antimicrobial agents and its plasmid content. Hybridization experiments and PCR assays were performed to identify the resistance genes while transformation and conjugation studies were conducted to show their transferability. The quinolone resistance-determining regions of the genes gyrA, gyrB, parC and parE were sequenced. Moreover, extended sequence analysis was performed to gain insight into the structure and organization of the qnrS gene area. RESULTS: The Salmonella Infantis isolate exhibited the Asp87-->Tyr87 mutation in gyrA, but no resistance-mediating mutations in the other target genes. It carried a conjugative plasmid, pINF5, on which a qnrS gene was detected in close proximity to a Tn3-like, blaTEM-1-carrying transposon. Homology to the qnrS-carrying plasmid pAH0376 of Shigella flexneri was limited to the Tn3-qnrS region. Sequence analysis of an approximately 13.4 kb region of pINF5 identified truncated insertion sequences of types IS26 and IS2 as well as an internal segment of the CS12 fimbrial gene cluster of Escherichia coli up- and downstream of the qnrS gene. CONCLUSIONS: This is to the best of our knowledge the first report of a qnrS gene in a Salmonella isolate. The analysis of the regions flanking the qnrS gene suggested that this region developed in multiple steps that included not only the integration of insertion sequences and a Tn3-like transposon, but also interplasmid recombination events.     

Isolation of Salmonella enterica serovar Agona strains and their similarities to strains derived from a clone caused a serovar shift in broilers

Salmonella enterica serovar Agona strains isolated from human cases were compared to strains that were derived from a clone caused a serovar shift in broilers. Pulsed field gel electrophoresis (PFGE) analysis with XbaI or BlnI digestion showed that three of seven strains from human case strains and most of the 81 strains from broilers were clustered in single complex in a minimum spanning tree (MST) reconstructed from the PFGE data. All the strains from human cases and 22 randomly selected strains from broilers were also analyzed by whole genome sequencing (WGS). Analysis of single nucleotide polymorphism (SNP) in the S. Agona core genes showed that four strains from human cases and all the strains from broilers were clustered in a maximum likelihood phylogenetic tree (ML tree) and an MST. These results indicated that the strains derived from the clone caused the serovar shift had already spread to humans. PFGE analysis with XbaI showed that four strains from broilers did not cluster with the other strains in an MST, though all those strains clustered in an ML tree and an MST reconstructed from SNP data. Moreover, three strains from broilers did not cluster in an MST reconstructed from PFGE with BlnI digestion, though those strains clustered in an ML tree and an MST reconstructed from SNP data. Therefore, it was suggested that S. Agona strains derived from a particular clone could not be traced by PFGE analysis but can be investigated by WGS analysis.     

Antibiotic susceptibilities of Salmonella enterica serovar Typhi and S. enterica serovar Paratyphi A isolated from patients in Japan

The antibiotic susceptibilities of 62 strains of Salmonella enterica serovar Typhi and 37 strains of S. enterica serovar Paratyphi A were investigated with 18 antibiotics. Eighteen S. enterica serovar Typhi isolates and five S. enterica serovar Paratyphi A isolates were resistant to one or more antimicrobial agents, among which 10 S. enterica serovar Typhi isolates were nalidixic acid resistant and also showed decreased ciprofloxacin susceptibility.     

Integrons and transposons on the Salmonella enterica serovar typhimurium virulence plasmid

A virulence plasmid was identified in a multidrug-resistant Salmonella enterica serotype Typhimurium strain carrying the spvC, rck, and pefA virulence genes and two class 1 integrons linked to the Tn21 and Tn1696 transposons. A novel trimethoprim resistance gene, designated dfrA23, was also identified within the integron region. The association of multidrug resistance and virulence determinants represents an interesting example of virulence plasmid evolution.     

Salmonella enterica serovar typhi strains isolated in Korea containing a multidrug resistance class 1 integron

Six strains of Salmonella enterica serovar Typhi which were resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and gentamicin were isolated in Korea. This multidrug resistance was transferred by a conjugative plasmid of about 50 kb. The plasmid harbored a class 1 integron, which included six resistance genes, aacA4b, catB8, aadA1, dfrA1, aac(6')-IIa, and the novel blaP2, in that order. All of the isolates showed the same-size plasmids and the same ribotyping patterns, which suggests a clonal spread of these multidrug-resistant isolates.     

Effect of fluoroquinolone exposure on the proteome of Salmonella enterica serovar Typhimurium

OBJECTIVES: The physiological response of Salmonella enterica serovar Typhimurium to fluoroquinolone antibiotics was investigated using proteomic methods. METHODS: Proteomes were prepared from strain SL1344 following treatment of broth cultures with ciprofloxacin (0.03 and 0.008 mg/L; 2x and 0.5x MIC) and enrofloxacin (0.03 mg/L) and from a multiple antibiotic resistant (MAR) mutant. Protein expression was determined by two-dimensional HPLC-MS(n) and also after exposure to ciprofloxacin by two-dimensional gel electrophoresis (2D-GE). RESULTS: The number of proteins (mean +/- SD) detected by 2D-GE derived from control cultures of the wild-type strain was significantly (P < 0.05) reduced from 296 +/- 77 to 153 +/- 36 following treatment with ciprofloxacin (0.03 mg/L). Raised expression (P < 0.05) of 17 proteins was also detected, and increases of up to 8-fold (P < 0.0001) were observed for subunits of F1F0-ATP synthase, TolC and Imp. Analysis by two-dimensional HPLC-MS(n) provided higher proteome coverage with 787 +/- 50 proteins detected, which was reduced (P < 0.005) to 560 +/- 14 by ciprofloxacin (0.03 mg/L). Increased expression of 43 proteins was observed which included those detected by 2D-GE and additionally the efflux pump protein AcrB. The basal expression of the AcrAB/TolC efflux pump was elevated in the MAR mutant compared with the untreated wild-type and augmented following treatment with ciprofloxacin (0.03 mg/L). F1F0-ATP synthase and Imp were only elevated in the mutant when treated with ciprofloxacin. CONCLUSIONS: These studies suggest that increased expression of AcrAB/TolC was associated with resistance while other increases, such as in F1F0-ATP synthase and Imp, were a response to fluoroquinolone.     

Ciprofloxacin-resistant Salmonella enterica serovar Typhimurium strains are difficult to select in the absence of AcrB and TolC

It has been proposed that lack of a functional efflux system(s) will lead to a lower frequency of selection of resistance to fluoroquinolones and other antibiotics. We constructed five strains of Salmonella enterica serovar Typhimurium SL1344 that lacked efflux gene components of resistance nodulation cell division pumps (acrB, acrD, acrF, acrBacrF, and tolC) plus three strains that lack genes that effect efflux gene expression (marA, soxS, and ramA) and a hypermutable strain (mutS::aph). Strains were exposed to ciprofloxacin at 2x the MIC in agar, in the presence and absence of Phe-Arg-beta-naphthylamide, an efflux pump inhibitor. Mutants were selected from all strains except those lacking acrB, tolC, or acrBacrF. For strains from which mutants were selected, there were no significant differences between the frequencies of resistance. Except for mutants of the ramA::aph strain, two phenotypes arose: resistance to quinolones only and multiple antibiotic resistance (MAR). ramA::aph mutants were resistant to quinolones only, suggesting a role for ramA in MAR in S. enterica serovar Typhimurium. Phe-Arg-beta-naphthylamide (20 microg/ml) had no effect on the frequencies of resistance or ciprofloxacin MICs. In conclusion, functional AcrB and TolC in S. enterica serovar Typhimurium are important for the selection of ciprofloxacin-resistant mutants.