Insertion sequence IS200 fingerprinting of Salmonella typhi: an assessment of epidemiological applicability.

When Pst I-generated digests of genomic DNA from each of the type strains of 49 of the Vi phage types of Salmonella typhi were probed with a PCR-amplified IS200 gene probe, all strains were found to possess at least 11 IS200 elements carried on fragments in the range 24.2-1.2 kb. Fourteen fingerprints were identified but two patterns designated IS200Sty1 and IS200Sty2 predominated. In one strain, a plasmid-mediated IS200 element was identified. When IS200 fingerprinting was applied to epidemiologically-unrelated strains of S. typhi isolated in Ecuador, 3 patterns were identified in 10 strains belonging to 9 different phage types. It is concluded that Vi phage typing remains the method of choice for the primary differentiation of S. typhi but that IS200 fingerprinting may be of limited use in laboratories which do not have access to phage typing.     

rDNA fingerprinting as a tool in epidemiological analysis of Salmonella typhi infections.

Characterization of 169 strains of Salmonella typhi of phage types C1, C4, D1 and D9 isolated in 1975-88 was carried out by rDNA gene restriction pattern analysis. Twenty-four isolates had been recovered during four large waterbone outbreaks in the last 20 years in Sicily; 145 strains, isolated from apparently sporadic cases of infection in Southern Italy in the same period of time, were also examined. Application of rRNA-DNA hybridization technique after digestion of chromosomal DNA with Cla I showed the identity of patterns of the epidemic strains of phage types C1 and D1, confirming attribution of the outbreaks to single bacterial clones. Patterns of the two available strains of lysotype D9 were slightly different, whilst the 12 epidemic strains of phage type C4 could be assigned to two distinct patterns scarcely related to each other and, consequently, to two different clones. A considerable heterogeneity was detected among all apparently sporadic isolates of the four phage types under study. This fingerprinting method appears a reliable tool to complement phage typing in characterizing isolates of S. typhi. In particular, epidemiological features of spread of this salmonella serovar in areas, where simultaneous circulation of indigenous and imported strains occurs, can be elucidated.     

Use of AFLP and PFGE to discriminate between Salmonella enterica serovar Typhimurium DT126 isolates from separate food-related outbreaks in Australia

In 2001 Salmonella enterica serovar Typhimurium definitive phage-type (DT) 126 was isolated at higher frequency in Australia compared to other S. Typhimurium phage types and in comparison to previous years. Associated with this increase was the implication of this phage type in a number of food-related outbreaks. We compared fluorescent amplified fragment length polymorphism (FAFLP) to pulsed-field gel electrophoresis (PFGE), the current 'gold standard' for molecular typing of Salmonella for the discrimination between outbreak-associated isolates and epidemiologically unrelated DT126 strains. FAFLP showed a greater ability to discriminate between isolates than PFGE, with 16 groups of clusters or individual isolates with < 90% similarity to each other compared to three groups as determined by PFGE. Both methods were able to discriminate between isolates from two separate outbreaks in South Australia and isolates associated with an outbreak at a restaurant in New South Wales. The resolving power of both methods was not sufficient to separate all epidemiologically unrelated DT126 isolates from the outbreak isolates. We conclude that amplified fragment length polymorphism is a useful tool to assist in the discrimination of S. Typhimurium DT126 isolates.     

Tandem repeat analysis for surveillance of human Salmonella Typhimurium infections

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all Salmonella enterica serotype Typhimurium isolates are currently subtyped by using phage typing, antimicrobial resistance profiles, and pulsed-field gel electrophoresis (PFGE). We evaluated the value of real-time typing that uses multiple-locus variable-number tandem-repeats analysis (MLVA) of S. Typhimurium to detect possible outbreaks. Because only a few subtypes identified by PFGE and phage typing account for most infections, we included MLVA typing in the routine surveillance in a 2-year period beginning December 2003. The 1,019 typed isolates were separated into 148 PFGE types and 373 MLVA types. Several possible outbreaks were detected and confirmed. MLVA was particularly valuable for discriminating within the most common phage types. MLVA was superior to PFGE for both surveillance and outbreak investigations of S. Typhimurium.     

乳酸杆菌及嗜热链球菌脉冲场凝胶电泳分子分型方法建立及应用

目的 建立乳酸杆菌及嗜热链球菌的PFGE分子分型方法,并对北京市售酸奶中分离的乳酸杆菌及嗜热链球菌进行分子分型.方法 选取ApaⅠ、NotⅠ、sfiⅠ、XbaⅠ和SmaⅠ共5种PFGE分析中常用的限制性内切酶,对从北京市售酸奶中分离到的52株乳酸杆菌、嗜热链球菌以及相应的标准菌株进行酶切,优化PFGE限制性内切酶种类及电泳条件,并用优化出的实验条件对菌株进行分子分型,同时进行聚类分析,与生化鉴定及16s rRNA基因鉴定结果进行对比分析.结果 限制性内切酶NotⅠ对保加利亚乳酸杆菌、发酵乳酸杆菌和德氏乳酸杆菌的酶切效果较好,而限制性内切酶Apa Ⅰ对嗜热链球菌、嗜酸乳酸杆菌及干酪乳酸杆菌的酶切效果较好.24株保加利亚乳酸杆菌被分为8个PFGE型,15株嗜热链球菌被分为8个PFGE型,7株嗜酸乳酸杆菌被分为3个PFGE型,2株德氏乳酸杆菌分属于2个不同的PFGE型.结论 建立的PFGE方法分析结果与生化鉴定及16s rRNA基因鉴定结果高度符合,所建方法适用于乳酸杆菌及嗜热链球菌的分子分型.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseed-fennel-caraway infusion

During 2002-2003 increased numbers of notified salmonellosis due to S. enterica serovar Agona were observed in Germany. In order to understand the recent spread of this serovar and to trace the route of infection to its source, a new phage-typing scheme and pulsed field gel electrophoresis (PFGE) were used to analyse these isolates. By using 14 bacteriophages, 52 phage types were distinguished among the S. Agona strains. PFGE also differentiated 52 different patterns. A combination of both methods generated 94 clonal types among 165 S. Agona strains originating from Germany and other countries including the United States, United Arab Emirates, Turkey, India, Austria and Finland, indicating a great biological diversity within this serovar. However, 36 recent S. Agona isolates from infantile gastroenteritis in Germany, from an untreated batch of aniseed imported from Turkey and from fennel-aniseed-caraway infusion (packed in tea bags) revealed clonal identity indicating their epidemiological relatedness as a new source of infection. It is suggested that strains of S. Agona will continue to be of public health concern, and that phage typing together with PFGE typing should be applied as reliable and rapid tools for epidemiological subtyping and future monitoring.     

Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina

Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed.     

Molecular typing methods to investigate transmission of Escherichia coli O157:H7 from cattle to humans.

The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157:H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0.85, 0.69 and 0.93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157:H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157:H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157:H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157:H7 from cattle to humans.     

2008年-2010年上海市部分伤寒副伤寒沙门菌耐药分析及分子分型

目的:了解上海市2008年-2010年伤寒、副伤寒沙门菌的耐药及分子分型特点,初步掌握伤寒、副伤寒沙门菌的分子流行特点,为今后的防制工作提供科学依据。方法:采用WHO推荐的改良K-B纸片法,对18株伤寒、副伤寒沙门菌进行13种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对18株伤寒、副伤寒沙门菌进行分子分型及聚类分析。结果:18株伤寒副伤寒沙门菌对利福平100%耐药,对奈啶酸的耐药率为44.4%,其中伤寒沙门菌对奈啶酸的耐药率为37.5%,5株甲型副伤寒沙门菌对奈啶酸100%耐药,乙型副伤寒对奈啶酸无耐药株。所有菌株对其他11种抗生素均敏感;18株伤寒、副伤寒沙门菌共产生16种PGFE带型,有3株甲型副伤寒沙门菌表现为同一PFGE型别。结论:2008年-2010年伤寒、副伤寒沙门菌对抗生素的敏感性较高;甲型副伤寒沙门菌之间有较高的同源性,伤寒沙门菌的PFGE带型比较分散。     

PCR-RFLP analysis of the coagulase gene of Staphylococcus aureus: application to the differentiation of epidemic and sporadic methicillin-resistant strains.

Preventing cross-infection with epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) requires effective control measures. These call for simple, rapid, discriminatory and reproducible methods for typing this pathogen. In this study 140 isolates/strains from 105 hospitals in England and Wales, representing 72 diverse phage types, were analysed by bacteriophage typing and PCR coagulase (coa) gene restriction fragment length polymorphism (RFLP). Isolates gave a coa gene PCR product that was either 660 base pairs (bp), 603 bp or 547 pb in size. The PCR products were digested with Alu I and Cfo I, and the fragments separated by gel electrophoresis. Eight coa gene RFLP patterns, numbered 1 to 8, were observed. Pattern 3 was most common (N = 25 isolates), followed by patterns 2 and 5 (18 isolates each), pattern 1 (14 isolates), pattern 4 (11 isolates), pattern 7 (10 isolates), pattern 8 (eight isolates) and pattern 6 (six isolates). Isolates of the same phage type often gave different coa gene RFLP patterns, and the patterns within the epidemic types EMRSA-03, EMRSA-15 and EMRSA-16 were heterogeneous. Thus, representatives of EMRSA-03 were subtyped to coa RFLP patterns 1 and 2, those of EMRSA-05 to coa RFLP patterns 1, 2, 7 and 8, and those for EMRSA-16 to coa RFLP patterns 2, 3, 4, 5 and 6. The range of patterns within single phage types of S. aureus could help to discriminate between isolates/strains, and in a hierarchical approach coa gene RFLP could occupy an intermediate position between phage typing and pulsed-field gel electrophoresis (PFGE).     

Salmonellosis in Indonesia: phage type distribution of Salmonella typhi.

The distribution of phage types was studied among 577 strains of Salmonella typhi from Indonesia. Chemotype, colicinogeny, and tetrathionate reductase activity were also studied for most of these strains. The current phage type formula for Java was determined to be: A, D2, D6, E1a, E2, M1, and 46, but two other large groups of strains were also found, I + IV and degraded Vi+ strains. Significant differences in S. typhi strain distributions were noted between two localities on Java with respect to phage type and tetrathionate reductase activity. Comparisons were made with past phage typing studies in Jakarta as well as with more recent studies in other parts of south-east Asia. Phage types A, D1, D2, and E1 persisted at a rather steady level in Jakarta for 28 years. Evidence was found for epidemiological links to European and Asian areas. Antibiotic resistance among these Indonesian S. typhi strains was rare.     

Molecular subtyping of Staphylococcus aureus from an outbreak associated with a food handler

On 6 May 2000, a staphylococcal food poisoning outbreak occurred at a high school, affecting 10 of the 356 students who attended the breakfast. Twenty-seven Staphylococcus aureus isolates, producing enterotoxin A (SEA), SEB-, or non-SEA-E, were recovered from 7 patients, 2 food handlers and left-overs. To investigate the outbreak, we genotyped the isolates by using pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: inter-IS256 PCR typing, protein A gene (spa) typing, and coagulase gene restriction profile (CRP) analysis. Our results show that PFGE was the most discriminatory technique, whereas the three PCR-based techniques were insufficient in the discriminatory power to distinguish the S. aureus isolates from the outbreak. Based on the enterotoxin-producing types and the results of genotyping, three distinct types of strains (A1111, B2221 and N3221) were designated. Both the A1111 and B2221 strains were found in the specimens from the patients and a hand lesion of a food handler, suggesting that the source of contamination for the outbreak was most likely originated from a food handler.     

食品及环境阪崎肠杆菌分离株的MLST和PFGE分型方法比较

目的:对PFGE和MLST方法在阪崎肠杆菌分型研究中的分辨力和潜在价值进行了比较研究和论述。方法:采用PFGE和MLST方法对本实验室分离的19株阪崎肠杆菌和一株标准菌株ATCC51329进行分型研究。结果:PFGE方法可将20株阪崎肠杆菌分为6大类群,共16个PFGE型,分辨力为0.9474。而MLST方法可将20株菌株分为12个ST型,分辨力为0.9263。结论:PFGE法较MLST法具有较高的分辨力,可用于阪崎肠杆菌的溯源研究,而MLST分型方法能通过全球比对数据库得到更多的关于进化和亲缘关系的分析资料,在致病研究、流行病学调查、进化研究方面优于PFGE法。     

2005年河南省局部地区伤寒副伤寒沙门菌株脉冲场凝胶电泳分型研究

目的 了解河南省伤寒、副伤寒沙门菌主要流行菌型.方法 用脉冲场凝胶电泳(PFGE)进行分型,NTSYSpc 2.1软件聚类分析.结果 来自河南省两个监测点的伤寒、副伤寒沙门菌6株分离菌,根据XbaI酶切PFGE基因指纹图谱分析,6株菌出现5个PFGE基因型别:甲、乙副伤寒沙门菌,伤寒沙门菌3个PFGE型别.结论明确了2...     

Molecular epidemiology ofSalmonella enteritidis

Sixteen strains ofSalmonella enteritidis isolated in 1991 from 13 unrelated poultry-associated sources, 7 strains from 2 community outbreaks, and 18 human sporadic isolates were investigated by phage typing, analysis of rRNA gene restriction patterns (ribotyping) and plasmid profiles. Four different phage types and 10SphI patterns were found, whereas plasmids were identical in all but 4 isolates. Only one ribotype (RT A) occurred among both human and avian strains. This particular ribotype was also responsible for the two outbreaks investigated, suggesting that such strains may be of special significance for the increase ofS. enteritidis infections.     

Two consecutive outbreaks of Stenotrophomonas maltophilia (Xanthomonas maltophilia) in an intensive-care unit defined by restriction fragment-length polymorphism typing.

OBJECTIVE: To investigate and control consecutive outbreaks of Stenotrophomonas maltophilia infections in intensive-care-unit (ICU) patients. DESIGN: Epidemiological investigation; restriction fragment-length polymorphism typing by pulsed-field gel electrophoresis (PFGE) of genomic DNA of outbreak strains; institution of infection control measures to limit spread. SETTING: The medical-surgical ICU in an 800-bed tertiary-care center in Calgary, Alberta, Canada. RESULTS: S. maltophilia was recovered from 14 ICU patients (12 infected, 2 colonized) between February 1993 and February 1994. Ten of the 14 patient isolates and 1 environmental isolate were available for PFGE typing. Patient isolates from 6 of the first 10 patients were identical. Isolates from the next 3 of 4 patients and an isolate recovered from a ventilator being used by a patient not infected with S. maltophilia also were identical, but different from the first 6. The ventilator isolate was temporally associated with the latter 4 patients. CONCLUSION: Molecular typing allowed us to determine that there were two separate consecutive S maltophilia outbreaks rather than a single protracted outbreak. Recovery of S. maltophilia from patient ventilators and an in-line suction catheter suggests that the organism may have been spread by cross-contamination from contaminated equipment or from an environmental source.     

可变数目串联重复序列技术用于中国C群脑膜炎奈瑟菌基因分型的研究

目的利用脑膜炎奈瑟菌基因组中可变数目串联重复序列（VNTR）特征,对中国C群脑膜炎奈瑟菌菌株进行基因分型.方法中国C群脑膜炎奈瑟菌菌株109株,选择脑膜炎奈瑟菌DNA中4个VNTR位点,PCR扩增含有串联重复序列的DNA片段,选择每一个VNTR位点有差别的PCR产物进行测序,序列比对,测算串联重复序列的拷贝数.Bio-Rad Gel DocTM XR凝胶成像分析系统计算PCR产物DNA片段的碱基含量,换算成串联重复数;对109株菌株4个位点的串联重复序列拷贝数进行聚类分析,依据聚类分析结果进行基因分型,并将VNTR基因分型结果与脉冲场凝胶电泳基因分型（PFGE）结果进行比较.结果109株C群脑膜炎奈瑟菌菌株分为22个VNTR基因型,同一暴发来源的菌株具有相同VNTR特征;VNTR基因分型方法与PFGE基因分型具有相关关系.结论应用VNTR技术可以对中国C群脑膜炎奈瑟菌进行基因分型和分子流行病学方面的研究,VNTR基因分型可较好地应用于追溯流行性脑脊髓膜炎暴发传染源.     

广东伤寒菌株的Vi Ⅱ噬菌体型别与药物敏感性监测

对从广东省收集的72株伤寒杆菌进行Vi Ⅱ噬菌体分型及药物敏感性测定,结果表明能明确定型的为62株(86.1%)。噬菌体分型以M₁(41.9%)、E₁(30.6%)和A(19.4%)为常见菌型。72株地方株与国家参考标准株一样,对氯霉素、庆大霉素、新霉素和多粘菌素B 100%敏感,而对另14种常用抗菌药物,则有不同程度的敏感或耐药。今昔菌株药敏特性无大变化,氯霉素等仍可作为治疗常选药物。