河南省26例甲型副伤寒沙门菌聚集性病例的病原学研究及溯源分析

目的 分析2015年3-7月份河南省登封市分离自临床诊断为副伤寒聚集性病例的甲型副伤寒(S.Paratyphi A)沙门菌病原学特征及分子流行病学关联。方法 采集副伤寒病例患者的静脉血8~10 mL,双相血培养瓶37 ℃培养4~7 d,沙门菌科玛嘉选择性培养基分离培养,系统生化鉴定,丹麦SSI分型血清进行沙门菌O抗原及H1/2相鞭毛诱导血清凝集试验。根据PulseNet China病原体分子分型网络实验室公布的沙门菌脉冲场凝胶电泳(PFGE)操作指南与美国临床实验室标准化协会(CLSI)沙门菌K-B法药敏测试方案对其进行XbaI/BlnI双酶切PFGE分子分型与聚类分析及8类13种抗生素药敏测试。结果 从29例病人血培养物中共分离到26株甲型副伤寒沙门菌,其经XbaI与BlnI限制性双酶切和PFGE电泳后带型完全一致(PTYA1);药敏测试结果显示24株甲型副伤寒沙门菌耐药表型一致。结论 病原学诊断及PFGE分子分型结果证实了26例甲型副伤寒沙门菌感染病例间可能存在高度的聚集性与分子流行病学关联,为进一步的追踪溯源和调查取证提供了技术支撑。     

脉冲场凝胶电泳在肠炎沙门菌食源性疾病溯源中的应用

目的对2006年广州地区食源性疾病中分离的肠炎沙门菌进行分子分型,探讨广州地区肠炎沙门菌的分子型别和多态性,为食源性疾病溯源及致病菌数据库的建立提供依据。方法采用限制性内切酶XbaI,对2006年分离到的菌株进行PFGE分子分型,使用BioNumericsVersion4.0软件(使用Dice系数和UPGMA法)对菌株进行聚类分析,并与深圳市的肠炎沙门菌PFGE型别进行比较。结果所有74株肠炎沙门菌均得到一致的PFGE克隆型,表明两次不同的食源性疾病均由同一PFGE型引起。广州与深圳的肠炎沙门菌PFGE图谱的比较表明,两地食源性疾病分离株具有很近的亲缘关系。结论PFGE分子分型与流行病学资料紧密结合可增强对肠炎沙门菌食源性疾病的溯源和预警。     

非脱羧勒克菌脉冲场凝胶电泳分子分型方法建立

目的利用不同的内切酶和电泳条件评价非脱羧勒克菌的脉冲场凝胶电泳(PFGE)实验方法,建立一系列非脱羧勒克菌的PFGE方法。方法 2013—2015年从甘肃省某婴儿奶粉加工企业车间的环境和原料中分离非脱羧勒克菌,利用溶菌酶和蛋白酶K对其裂解后,选用不同的内切酶对胶块内的核酸进行酶切,脉冲场电泳后评价分析电泳图谱,得到最佳实验方案。结果经过几种内切酶的对比试验后,发现SpeⅠ酶在本试验方案中反应稳定,酶切后条带大约为17~21条,且分布均匀,更适合用于图谱分析。结论本研究确定了非脱羧勒克菌的PFGE方法,可以为非脱羧勒克菌在食品、药品等加工过程的污染以及医院感染监测,提供溯源技术手段。     

河南省一起斯坦利沙门菌聚集性病例的病原学研究及溯源分析

目的 分析2013年1-7月份河南省2地市3家医院分离自婴幼儿患者粪便中的斯坦利(S.stanley)沙门菌的病原学特征及分子流行病学关联,为食源性疾病聚集性及暴发病例的调查及院感防控提供新的思路和模式。方法 采集河南省3家医院婴幼儿住院病例的腹泻粪便,SBG增菌,沙门菌科玛嘉选择性培养基分离培养,API20E肠杆菌科系统生化板条鉴定后使用丹麦SSI分型血清进行沙门菌O抗原及H1/2相鞭毛诱导血清凝集试验。根据PulseNet China病原体分子分型网络公布的沙门菌脉冲场凝胶电泳(PFGE)操作指南与美国临床实验室标准化协会(CLSI)沙门菌K-B法药敏测试方案,对其进行PFGE分子分型与聚类分析及17种抗生素药敏测试。结果 分离自15位婴幼儿住院病例粪便中的沙门菌经鉴定均为斯坦利沙门菌(S.stanley),药敏测试显示其对环丙沙星、左氧氟沙星、复方新诺明、四环素、亚胺培南等8种抗生素敏感,对氨苄西林、头孢噻肟、头孢吡肟、阿莫西林等9种抗生素耐药,其中12株菌耐药表型完全一致。15株斯坦利沙门菌经XbaI与BlnI限制性双酶切和PFGE电泳后分为STN1和STN2两种带型,其中STN1带型包含14株菌,与另1株(STN2)具有流行病学关联度的菌株带型差异度较大。STN1经“PulseNet China”数据库比对证实为斯坦利沙门菌河南省独有带型。结论 病原学诊断及PFGE分子分型结果证实了15例斯坦利沙门菌感染病例间可能存在高度的分子流行病学关联和聚集性,为进一步的追踪溯源和调查取证提供了技术支撑。     

深圳市龙岗区鼠伤寒沙门菌耐药性和分子分型研究

目的研究深圳市龙岗区2010~2012年腹泻病人分离株鼠伤寒沙门菌的耐药性和分子分型。方法按国标GB 4789.4-2010对腹泻标本进行分离培养和生化及血清学鉴定,确定为鼠伤寒沙门菌的菌株采用纸片扩散法（K-B法）检测对22种抗生素的敏感性,通过脉冲场凝胶电泳（PFGE）对菌株进行分子分型,限制性内切酶XbaI为首选酶,AvrⅡ为次选酶,利用BioNumerics软件对酶切电泳图谱进行同源性分析。结果共分离出48株鼠伤寒沙门菌,50%的菌株来自7岁以下儿童患者。60.0%以上的鼠伤寒沙门菌对四环素和氨苄西林耐药。有34株菌株（占70.8%）耐3种及以上药物,耐8~10种药物有11株（占22.9%）,所有菌株对头孢西丁、美罗培南、亚胺培南和左氧氟沙星均敏感,多重耐药株在该区域普遍存在。XbaⅠ酶切分成40个带型,有6个型别,各含2~3株,AvrⅡ二次酶切将其重新分成12个型,其中XAP2、XAP3型2株。结论深圳市龙岗区鼠伤寒沙门菌分离株多重耐药较为严重,婴幼儿是重点防治人群。经PFGE分型鼠伤寒沙门菌菌型较多,说明本地区存在多个不同克隆株。     

鼠疫疫苗株脉冲场凝胶电泳操作规范的建立

目的建立鼠疫菌脉冲场凝胶电泳的操作规范,并对操作进行风险评估。方法应用疫苗株EV76进行实验,比较不同培养时间、两种酶切的电泳图形,以及胶块中细菌存活情况的检测。结果不同培养时间对电泳结果没有影响,FseI酶切图谱条带较容易分析,胶块内无细菌存活。结论严格执行标准操作程序,才能确保鼠疫菌脉冲场凝胶电泳(PFGE)操作的实验室生物安全。     

河南省20株布鲁氏菌鉴定与PFGE分子分型研究

目的检测与分析河南省2010-2012年20株布氏菌型别及PFGE脉冲场凝胶电泳指纹图谱特征。方法采集病人静脉血,分别以试管凝集试验(SAT)、双相血培养瓶分离培养、热裂解法制备DNA模板和AMOS-PCR鉴定4种布氏菌型别。采用脉冲场凝胶电泳技术(PFGE)对检出的布鲁氏菌进行分子分型鉴定。结果分离培养的20株布鲁氏菌经鉴定,19株为羊种,1株为牛种;羊种布鲁氏菌经XbaI酶切与脉冲场凝胶电泳后,共获得了8种不同带型,带型相似度在80%~100%之间,牛种与羊种菌株在带型相似度上差异较大,具有较好的分辨能力。结论河南省病人感染的布鲁氏菌以羊种为主要型别,AMOS-PCR与PFGE作为布鲁氏菌菌型鉴定与分子分型的技术手段,为布鲁氏菌病的病原学监测与应急检测提供依据。     

河南省20株布鲁氏菌鉴定与PFGE分子分型研究北大核心CSCD

目的 检测与分析河南省2010-2012年20株布氏菌型别及PFGE脉冲场凝胶电泳指纹图谱特征。方法 采集病人静脉血,分别以试管凝集试验（SAT）、双相血培养瓶分离培养、热裂解法制备DNA模板和AMOS-PCR鉴定4种布氏菌型别。采用脉冲场凝胶电泳技术（PFGE）对检出的布鲁氏菌进行分子分型鉴定。结果 分离培养的20株布鲁氏菌经鉴定,19株为羊种,1株为牛种;羊种布鲁氏菌经XbaI酶切与脉冲场凝胶电泳后,共获得了8种不同带型,带型相似度在80%~100%之间,牛种与羊种菌株在带型相似度上差异较大,具有较好的分辨能力。结论 河南省病人感染的布鲁氏菌以羊种为主要型别,AMOS-PCR与PFGE作为布鲁氏菌菌型鉴定与分子分型的技术手段,为布鲁氏菌病的病原学监测与应急检测提供依据。     

云南鼠疫菌AscI酶切分型及其流行病学意义

目的构建以AscI酶切的云南省鼠疫菌PFGE图谱库并探究其流行病学意义。方法采用限制性内切酶AscI对云南家、野鼠型及玉龙鼠疫菌进行酶切分析,并对电泳图谱进行聚类分析。结果30株被试菌株分为15种PFGE型别（相似性系数为88．9％～100．0％）、5个簇、4个群,除404号菌与EV76归为一群,其余3个群分别为家鼠型基因群、野鼠型基因群及玉龙基因群。结论云南鼠疫菌PFGE基因型与生态型相吻合,具有一定的区域聚集性;玉龙鼠疫菌是云南独立的一个基因群。     

致病性钩端螺旋体限制性内切酶酶切分析分型研究

为了建立一种简便、稳定的钩体分型方法,我们采用限制性内切酶ＥｃｏＲＩ对致病性钩端螺旋体八个血清群５４个血清型６４株国内外钩体参考株及２７株野生株进行限制性内切酶酶切分析（ＲＥＡ）。结果表明：９１株菌中共有５９种不同的限制性内切酶图谱（ＲＥＰｓ）,根据前５条高分子酶切片段可以区分不同的ＲＥＰｓ;除部分血清群中个别不同的血清型有相同的ＲＥＰ型外,大部份血清型都有其独特的ＲＥＰ型,同一血清群往往拥有共同的酶切片段;所研究的同一血清型的国内和国际参考株的ＲＥＰｓ不同;大多数野生株的ＲＥＡ型和参考株相同,差异仅表现为个别高分子带的缺少和增加,ＲＥＡ分型和血清分型吻合较好,通过ＲＥＡ分型基本可区分不同的血清型。     

O139群霍乱弧菌分子流行病学研究

目的分析2001-2006年广州地区霍乱暴发、散发事件及外环境监测中分离的O139群霍乱弧菌的致病相关基因型和PFGE型,追踪菌株的来源和变迁,探讨本地区O139群霍乱流行特点。方法采用多重PCR方法检测O139群菌株的4种致病相关基因,应用脉冲场凝胶电泳技术(PFGE)对菌株进行分子分型,采用软件Bio Numerics Version4.0对分型数据进行处理和分析。结果广州地区O139群菌株中存在2种致病相关基因型,即A型和C型,18株感染者相关菌株中,除1株外,均为致病相关基因A型;10株珠江水分离株均为致病相关基因C型。28株O139群霍乱弧菌分为20个不同的PFGE型,归为4个聚类群(Ⅰ、Ⅱ、Ⅲ、Ⅳ群)。珠江水中O139群菌株存在PFGE克隆型的多样性。O139群暴发中分离的菌株PFGE型相同或相近,O139群散发疫情中的病例分离株与部分暴发株也具有相同或相近的PFGE型。结论分子分型方法结合流行病学资料,可分析O139群霍乱菌株的流行特点,致病相关基因分型可代替噬菌体-生物分型来判断和区分O139群霍乱弧菌的流行株与非流行株,从而为霍乱的预防、控制和预警提供科学依据。     

An epidemiological study of outbreaks of Salmonella enterica serovar paratyphi A occurred in two Chinese families after traveled to different areas of China

We reported two familial clusters of paratyphoid fever after travel to China occurring in the same Yokohama ward from September to October 2002. Six Salmonella enterica serovar Paratyphi A (S. Paratyphi A) strains, 3 each from 2 clusters, were isolated and their characteristics analyzed using phage typing, susceptibility to antibiotics, and patterns of restriction endonuclease-digested DNA fragments in agarose gel following pulsed-field gel electrophoresis (PFGE). Mutations in genes for gyrA and parC, which determine sensitivity to fluoroquinolones, were also investigated. All isolates showed the same characteristics, i.e. "untypable", employing bacteriophages, resistant to antibiotics nalidixic acid and fosfomysin, and decreased susceptibility to fluoroquinolones. No difference was observed in PFGE patterns after digesting with 4 restriction enzymes, Xba I, Bin I, Spe I, and Xho I. We also found that the gyrA gene, which is one of the quinolone-resistance-determining regions (QRDR), was mutated at position 83 from serine to phenylalanine (from TTC to TCC) in all 6 strains. Other QRDR's, parC were not mutated commonly in them. Hearing from patients and family members, it was apparent that these 2 families had been contacted neither in Japan nor in China during ill or incubation period of paratyphoid fever, although a member of one cluster had a familial relationship with one of another family. It was also reported by them that typhoid fever is endemic in both of the areas of their visits. From these results, it was suggested that these 2 cluster cases were infected separately in China with the progeny of the same clone which is endemic in these regions.     

Urinary tract infection caused by Kluyvera ascorbata in an immunocompromised patient: case report and review

We describe an immunocompromised patient, aged 78 y, with urinary tract infection caused by Kluyvera ascorbata. Complete recovery was achieved on antibiotic therapy with ceftriaxone. Review of the literature clearly indicates that K. ascorbata is a potentially dangerous pathogen either in the immunocompetent or in the immunocompromised host. K. ascorbata, and in general Kluyvera species, deserve prompt identification in order to determine antimicrobial susceptibility pattern and a correct and aggressive antibiotic treatment.     

Rearrangements in the genome of the bacterium Salmonella typhi.

We have determined the genomic map of the bacterium Salmonella typhi Ty2, the causal organism of typhoid fever, by using pulsed-field gel electrophoresis. Digestion of the Ty2 genome with endonucleases Xba I, Bln I, and Ceu I yielded 33, 26, and 7 fragments, respectively, that were placed in order on a circular chromosome of 4780 kb. Transposon Tn10 was inserted in specific genes of Salmonella typhimurium and transduced into S. typhi, and thus, the positions of 37 S. typhi genes were located through the Xba I and Bln I sites of the Tn10. Gene order on chromosomes of Escherichia coli K-12 and S. typhimurium LT2 is remarkably conserved; however, the gene order in S. typhi Ty2 is different, suggesting it has undergone major genomic rearrangements during its evolution. These rearrangements include inversions and transpositions in the 7 DNA fragments between the seven rrn operons for rRNA (postulated to be due to homologous recombination in these rrn genes), another inversion that covers the replication terminus region (resembling inversions found in other enteric bacteria), and at least three insertions, one as large as 118 kb. Partial digestion of genomic DNA with the intron-encoded endonuclease I-Ceu I, which cuts only in rrn genes, shows chromosomal rearrangements, apparently due to homologous recombination in the rrn genes, that were detected in all wild-type strains of S. typhi tested. These rearrangements may have been selected to compensate for the insertions that otherwise would have altered the locations of genes with respect to the origin and terminus of replication. These observations are relevant to our view of the evolution of the bacterial genome and may be significant in the virulence of S. typhi.     

Studies on Vibrio vulnificus infection: molecular epidemiology of environment-derived strains and clinical isolates

To clarify the route and source of Vibrio vulnificus infection, we conducted molecular epidemiological investigation by DNA analysis of 355 environmental isolates (seawater-derived strain: 86, sea mud-derived strain:36, and oyster-derived strain: 233) and 65 human clinical isolates, for a total of 420 isolates, using pulse field gel electrophoresis (PFGE), with the following results. 1. When DNA was cleaved with 2 enzymes, Not I and Sfi I, and subjected to PFGE, Not I DNA interpretation was 76.9%, and Sfi I cleavage was 97.9%, showing that Sfi I was superior in cleaving DNA of this bacteria. 2. Sfi I-interpreted strains were subjected to PFGE and migration patterns were analyzed by UPGMA, but close classification was not possible because similarity was low, this infectious disease clearly originated from multiple rather than a single-clone. In this cluster, we concluded that this infectious disease was acquired through contact between the environment and human beings and viceversa. We identified an assortment of clinical isolates and environment-derived strains among more than 89% of strain groups tested, none of which could be expected to have the same origin. We conclued DNA analysis on these two types of restriction enzymes using PFGE, but were unable to classify test results in detail due to the proliferation of migration patterns and low degree of similarity.     

Kluyvera co‐infection in two solid organ transplant recipients: an emerging pathogen or a colonizer bystander?

Kluyvera species are opportunistic, gram-negative bacilli in the family Enterobacteriaceae. Ordinarily occurring as a commensal, Kluyvera have been reported to cause serious infections in immunosuppressed and immunocompetent hosts, causing diarrhea, urinary infections, peritonitis, and cholecystitis. We report Kluyvera infections in 2 solid organ transplant recipients. An 18-year-old female with alpha-1 antitrypsin deficiency underwent living donor liver transplantation and presented 6 months later with a liver abscess. The abscess aspirate grew mixed organisms including Kluyvera cryocrescens. A 22-year-old female with renal failure secondary to focal segmental glomerulosclerosis underwent a deceased donor kidney transplant and presented 3 months later with pyelonephritis; the urine culture grew Kluyvera ascorbata. Both patients improved only when their antibiotic coverage was broadened to include Kluyvera. The isolation of Kluyvera as a pathogen in transplant patients emphasizes that this commensal organism may be virulent in this patient population.     

Peritonitis due to Kluyvera ascorbata: case report and review

Kluyvera, a new genus in the family Enterobacteriaceae, was formerly known as enteric group 8 and as API group 1. Although Kluyvera species have been isolated from various clinical specimens such as sputum, urine, stool, and blood, the clinical significance of these isolates has not been established. Recently, we treated a child who developed peritonitis due to Kluyvera ascorbata. The repeated isolation of the organism in pure culture from the peritoneal fluid and its isolation from postmortem subdiaphragmatic microabscesses suggest that Kluyvera can be clinically significant. Review of the literature clearly indicates that Kluyvera strains are infrequent but potentially dangerous pathogens in humans. Further experience is needed to determine the therapeutic efficacy of the various antibiotics to which these bacteria are sensitive in vitro.     

Epidemiological characteristics and virulence factor of verotoxin-producing Eschericia coli O121:H19 isolated in Akita Prefecture in July 1997

Epidemiological characteristics and virulence factors of VTEC O121:H19 strains isolated in July 1997 from a 15 year old female and a 20 year old male patient suffering from bloody diarrhea and severe abdominal pain were examined. The 2 VTEC O121:H19 isolates showed identical antibiotic susceptibility patterns, biochemical characteristics and plasmid profile while slight differences were observed in their Xba I and Not I PFGE patterns, suggesting that closely related 2 VTEC O121:H19 strains evoked the sporadic infectious cases in July 1997. The 2 VTEC O121:H19 isolates, as well as VTEC O157:H7, possessed eaeA gene and a ca. 60 MDa plasmid which hybridised with CVD 419 probe and produced enterohemolysin. In addition, the VTEC O121:H19 isolates produced almost the same amount of VT-2 in vitro as VTEC O157:H7 did. These results suggested that VTEC O121:H19 possesed the virulence factor comparable to that of VTEC O157:H7. Incidence, molecular epidemiology and infectious source of VTEC O121:H19 in this country have not been sufficiently understood. Antiserum for E. coli serogroup O121 should be manufactured to clarify the epidemiology of the highly virulent VTEC strain.     

日本血吸虫单克隆抗独特型抗体NP30单链抗体基因的构建和表达

目的　构建日本血吸虫单克隆抗独特型抗体NP30单链抗体 (scFv)基因。　方法　通过PCR方法体外扩增并经测序验证的重链、轻链可变区 (VH、VL)基因先后重组入原核表达质粒 pTHA90相应的位点上 ,中间通过一连接肽 (Gly4 Ser) 3基因连接构建成单链抗体基因 (scFv) ,连接产物转化相应受体菌Top1 0 ,提取质粒 ,酶切鉴定重组克隆。表达产物经ELISA方法测定活性。　结果　重组克隆经酶切鉴定可见预期大小的片段 ,表明重组成功。表达产物经ELISA检测 ,OD4 92 值为 1 .0 6 ,高于阴性对照 3倍以上 ,证实具有与相应抗原结合的能力。　结论　成功地构建了scFv基因 ,且其表达产物保留了抗体的亲和性和特异性