Comparison of Pulsed-Field Gel Electrophoresis, Ribotyping, and Plasmid Profiling for Typing of Vibrio anguillarum Serovar O1

A total of 75 Vibrio anguillarum serogroup O1 strains were studied with respect to their plasmid contents, ribotypes, and pulsed-field gel electrophoresis (PFGE) patterns. Eight plasmid profiles and six ribotypes were demonstrated, and one profile was dominant by both typing methods. In contrast, PFGE had very high discriminatory power, demonstrating 35 profiles. On the basis of PFGE patterns, a similarity matrix and a dendrogram were constructed. The results indicated that Scandinavian strains and southern European isolates (with some exceptions) belong to two different clonal lineages. A few strains from the United States and United Kingdom deviated considerably from each other and from Scandinavian and southern European strains.     

大连市两种来源的副溶血性弧菌分子分型

目的 分析食品来源、患者来源及2种来源的副溶血性弧菌之间的PFGE图谱的关系,从分子流行病学角度探讨2种来源的副溶血性弧菌的关联.方法 收集患者和食品2种来源的副溶血性弧菌178株,经限制性内切酶SfiI酶切,用脉冲场凝胶电泳方法进行电泳,凝胶成像仪获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析.结果 ...     

Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed-field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two-replicon structure is common throughout Vibrio species.     

Pulsed-field gel electrophoresis analysis of Vibrio vulnificus strains isolated from Taiwan and the United States

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.     

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.     

Pulsed-Field Gel Electrophoresis Analysis of Vibrio vulnificus Strains Isolated from Taiwan and the United States

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.     

Comparison of direct genome restriction enzyme analysis and pulsed-field gel electrophoresis for typing of Vibrio vulnificus and their correspondence with multilocus sequence typing data

We compared the potential of direct genome restriction enzyme analysis (DGREA) and pulsed-field gel electrophoresis (PFGE) for discriminating Vibrio vulnificus isolates from clinical (23) and environmental (17) sources. The genotypes generated by both methodologies were compared to previous multilocus sequence typing (MLST) data. DGREA established clearer relationships among V. vulnificus strains and was more consistent with MLST than with PFGE. DGREA is a very promising tool for epidemiological and ecological studies of V. vulnificus.     

Comparison of ribotyping,randomly amplified polymorphic DNA,and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis

Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams. In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD). Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested. The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters. Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6%. Cluster 2 included again only the isolate 0202RD. RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R. decussatus from those isolated from other clam species. Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests. On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint. In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.     

Stenotrophomonas maltophilia as a nosocomial pathogen

The aim of this study was to investigate the molecular epidemiology of Stenotrophomonas maltophilia in a university hospital in Turkey. Thirty nine clinical isolates were collected from 37 patients and one from an environmental source between 1998 and 2001. Susceptibility to 11 antimicrobials was studied. The isolates were categorised into six groups: A through F. Trimethoprim sulfamethoxazole was the most active agent against the tested isolates. Genotypic analysis by pulsed-field gel electrophoresis (PFGE) of clinical isolates identified 21 different PFGE patterns. Three most common clusters were composed of 11, seven and four strains. Antimicrobial susceptibility identified multi-resistant phenotype in all S. maltophilia PFGE clones. All the remaining 18 isolates (45%) revealed unique PFGE patterns. Resistance was not lower in unique strains. The clones mainly with two unique macrorestriction profiles strongly suggests nosocomial transmission of these strains from either a common source and/or between patients.     

Characteristics of Vibrio parahaemolyticus O3:K6 from Asia

A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of V. parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains.     

Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains

Abstract Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis. Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks. We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with Sma I or Nru I, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C. difficile belonging to 8 serotypes. The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping. Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains. PFGE was more time-consuming than RAPD but its patterns were easier to analyze.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Genomic heterogeneity of environmental and clinical aeromonads

Thirty-three isolates of Aeromonas from environmental sources and clinical samples were tested and the results, obtained using the pulsed field gel electrophoresis (PFGE) technique, were compared with those obtained by biochemical typing. On the basis of their biochemical characteristics 31 strains was assigned to one of the recognised groups or species within the Aeromonas genus and 2 strains to the species Vibrio fluvialis. These latter were nevertheless found to belong to the Aeromonas genus on the basis of the chromosomal DNA analysis. Among the clinical isolates the biochemical analysis showed greater uniformity. A low correlation between molecular and traditional typing methods was observed with a wider heterogeneity at the genomic level. The results showed the difficulty of discriminating Aeromonas isolates by conventional biochemical methods. The genomic analysis performed by PFGE can be a more effectual technique, which can be used for epidemiological and ecological studies of the microorganisms belonging to the Aeromonas group.     

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.     

一起沙门菌引起的食源性疾病爆发的溯源分析

目的:对一起沙门菌引起的食源性疾病爆发进行溯源分析。方法:采用GB4789法对采集的样品进行分离及鉴定,采用16S r RNA基因分型方法及PFGE分型方法对分离的菌株进行分子生物学分析,并对爆发进行溯源分析。结果:生化及血清学结果表明,该起爆发分离的菌型为伦敦沙门氏菌。16S r RNA基因分型表明爆发所分离的菌株均为肠道沙门菌肠道亚种,菌株12 sam与其他4个菌株分子发育距离较远,均为16S r RNA基因分型的TYPE1-11型;PFGE分型结果表明菌株10 sam、16 sam、27 sam及29sam的PFGE带型相似度为100%,菌株12sam跟其他菌株相似率为96%。结论:GB4789法结果表明该起爆发是由伦敦沙门氏菌引起的,16S r RNA基因分型及PFGE分型方法的结果均表明该起食源性疾病来源一致。     

Phenotypic and Genotypic Characterization Vibrio cholerae O139 of Clinical and Aquatic Isolates in China

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.     

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Comparison of Streptococcus thermophilus strains by pulse field gel electrophoresis of genomic DNA

Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII. PFGE profiles could be used for strain identification and were also useful in predicting relatedness of certain strains. Genetic variations between specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study. Statistical analysis of SmaI restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics.     

Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.