Efficient tracing of global isolates of Yersinia pestis by restriction fragment length polymorphism analysis using three insertion sequences as probes

Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from rodent to rodent and from rodent to humans by fleabites. Multiple copies of three insertion sequences (IS100, IS285, and IS1541) are scattered over the Y. pestis genome. The genomic instability generated by these insertion sequences (IS) creates a polymorphism of the hybridizing restriction fragments (restriction fragment length polymorphism [RFLP]) which can be used to subtype this relatively clonal species. The aim of this work was to evaluate and compare the potential of the three IS-RFLP techniques, individually or in combination, to define clusters of strains according to their focus of origin. The analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each IS-RFLP used individually. In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination (D = 0.999) and a robust clustering of the isolates according to their biovar and geographical origin. This geographical clustering was observed even within the Orientalis group, although these strains had only a short period of time (one century) to diverge from the original clone that spread globally. Therefore, 3IS-RFLP is a technique that may be useful for addressing epidemiological problems and forensic issues. When plague reemerges after several decades of silence in a quiescent focus, it may help in determining whether the disease was reimported or reactivated. It may also be of value to identify the origin of a strain when plague cases appear in a previously plague-free region. Finally, this technique could be useful for the tracing of a Y. pestis isolate that has been used as a biological terrorism threat.     

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.     

Analysis of different molecular methods for typing methicillin-resistant Staphylococcus aureus isolates belonging to the Brazilian epidemic clone

The extensive geographic spread of MRSA isolates belonging to the Brazilian epidemic clone (BEC) limited the value of pulsed-field gel electrophoresis (PFGE) in epidemiological studies of outbreaks caused by these strains. Thus, the discriminatory power of eight different molecular methods was evaluated in an attempt to establish a methodology for genotyping BEC isolates involved in intra-hospital outbreaks. BEC isolates from five hospitals in Teresina City, Piaui State were genotyped by conventional electrophoresis or PFGE of Cla I- or Sma I-digested genomic DNA hybridised with specific labelled mecA, Tn554, IS257 and IS256 probes. The combination of PFGE with Cla I/mecA, Cla I/Tn554, Cla I/IS257, Sma I/mecA and Sma I/IS257 probe-fingerprinting techniques provided a very poor discriminatory power for BEC strains. Although Cla I/IS256 fingerprinting discriminated 17 different polymorphisms among the isolates displaying PFGE A1 pattern, this strategy was not reproducible. In contrast, the combination of PFGE and Sma I/IS256 polymorphisms differentiated BEC isolates into nine stable polymorphisms. Thus combination of PFGE and hybridisation with IS256 probe may be recommended as a useful means of typing BEC strains involved in intra-hospital infections.     

Genotypic characterization of five subspecies of Mycobacterium kansasii.

Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.     

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.     

Comparison of an automated ribotyping system to restriction endonuclease analysis and pulsed-field gel electrophoresis for differentiating vancomycin-resistant Enterococcus faecium isolates

The RiboPrinter Microbial Characterization System was compared with pulsed-field gel electrophoresis (PFGE), restriction endonuclease analysis (REA), and epidemiological data for typing 45 vancomycin-resistant Enterococcus faecium (VRE) isolates. In 21 clinically related isolates, 90 to 100% were similar by PFGE and REA, but only 57% were similar by the RiboPrinter. In another eight clinically related isolates, three isolates similar by PFGE and REA were all unique by the RiboPrinter. In contrast, in 16 clinically unrelated isolates, the predominant RiboPrinter ribotype represented 50% of the strains, while the largest PFGE and REA clones represented less than 19% of the strains. These data suggest that the RiboPrinter is not reliable for VRE investigation.     

Molecular analysis of Clostridium difficile at a university teaching hospital in Japan: a shift in the predominant type over a five-year period

Clostridium difficile isolates recovered from patients admitted to a teaching hospital in Japan over a 5-year period were analyzed. Two molecular typing systems, PCR ribotyping and pulsed-field gel electrophoresis (PFGE) analysis, were used. Twenty-six PCR ribotypes were found among the 148 isolates. The predominant type at our hospital appeared to shift during the study period, from PCR ribotype a in 2000 (15/33, 45%) to PCR ribotype f in 2004 (18/28, 64%). By using PFGE with thiourea added to both the gel and running buffer, all 148 Clostridium difficile isolates were successfully classified into 37 types and 61 subtypes. The PCR ribotype f isolates were further classified into four types and 11 subtypes by PFGE. The PFGE patterns of the 11 subtypes differed from each other by only 1 to 4 bands, suggesting that these differences might reflect genetic changes during patient-to-patient transmission over the 5-year period analyzed, and that PCR ribotype f isolates might be outbreak-related. In addition, the PCR ribotype f was identical to the PCR ribotype designated smz, which is reported to have caused multiple outbreaks in Japan. These results confirmed that PCR ribotype f (type smz) has specific virulence or survival factors that make it more likely to cause nosocomial outbreaks at Japanese hospitals. PCR ribotype 027, which has been reported to have caused recent outbreaks in North America and Europe, was recovered from one patient in this study; however, this strain was community-acquired. Our findings emphasize the importance of monitoring specific strains to control and prevent nosocomial infection.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Practical evaluation of molecular subtyping and phage typing in outbreaks of infection due to Salmonella enterica serotype typhimurium

Identification and control of food-poisoning outbreaks due to salmonellosis depend on prompt microbiological diagnosis and subtyping to identify the causative strain. In Australia, Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) is responsible for 40-70% of cases of human salmonellosis. Phage typing is the usual method of subtyping S. typhimurium, but on its own, has limitations. We compared it with three molecular subtyping methods using 100 isolates of S. typhimurium, representing four different phage types (PT 1, 9, 126 and 135) and comprising 74 isolates from three presumed outbreaks, 25 isolates from sporadic cases of salmonellosis and S. typhimurium ATCC 10428 (phage type 126). The isolates were divided into 11 subtypes by IS200 restriction fragment length polymorphism (RFLP) typing, four each by ribotyping and pulsed-field gel electrophoresis (PFGE) and 17 distinct strains using a combination of phage and molecular typing. Isolates from two presumed outbreaks were resolved into multiple strains, possibly explaining the failure to identify a common source for either during the original investigations. IS200 RFLP analysis was the most discriminatory and reproducible typing method. Several strains were identifiable within and shared between phage types 1, 9 and 126. Phage and IS200 RFLP typing together, would provide improved definition of S. typhimurium outbreaks.     

Evaluation of the Mycobacterium bovis Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of Mycobacterium tuberculosis Strains in Ireland

A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of Mycobacterium bovis. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of Mycobacterium tuberculosis from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS6110 strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS6110 (41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS6110 and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS6110 isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.     

Molecular typing of Mycobacterium bovis isolates from Cameroon

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.     

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.     

Genetic diversity of mycobacterium avium recovered from AIDS patients in the caribbean as studied by a consensus IS1245-RFLP method and pulsed-field gel electrophoresis

A total of 45 strains of Mycobacterium avium from 31 human immunodeficiency virus (HIV)-positive patients in the French Caribbean islands and Guiana were subjected to DNA fingerprinting using a recently described consensus IS1245 restriction fragment length polymorphism (RFLP) method, and pulsed-field gel electrophoresis (PFGE). The IS1245-RFLP resulted in three distinct clusters composed of three 27-banded isolates from two patients (cluster A), nine two-banded isolates from six patients (cluster B), and three 20-banded isolates from three patients (cluster C). PFGE results obtained after XbaI and DraI digestions gave similar clustering results irrespective of the enzyme used, and confirmed the molecular clonality for high IS1245 copy number isolates (clusters A and C). However, PFGE further discriminated the low IS1245 copy number cluster B into two distinct subclusters: subcluster I containing six isolates from four patients during the same time period from a single hospital in Guadeloupe, and subcluster IV composed of four isolates from two patients, out of which three isolates were from a single patient (patient 19). Interestingly, in the latter case, PFGE grouped together all three isolates from patient 19 despite the fact that IS1245 fingerprinting permitted grouping only two of the three isolates (the remaining unclustered isolate contained two additional bands of 3.5 and 5 kbp, and was initially considered as evidence of polyclonal infection). A combined numerical analysis of the IS1245-RFLP and PFGE results corroborated the existence of four instead of three clusters. A comparison of IS1245-RFLP versus PFGE results suggested that the standardized RFLP procedure is compatible with PFGE only for M. avium isolates containing > or = 5 copies of IS1245. Consequently, the typing results for low IS1245 copy number isolates (31% of isolates in this study) should be reconsidered for secondary typing using PFGE. Lastly, the absence of a predominant genotype of M. avium infecting HIV-positive patients over a 5-year period in this tropical region argues in favor of a lack of a privileged ecological niche for M. avium, and instead suggests that microepidemics of M. avium may prevail during limited periods of time.     

Evaluation of Mycobacterium tuberculosis typing methods in a 4-year study in Schleswig-Holstein, Northern Germany

In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci.     

Rapid diagnostic test that uses isocitrate lyase activity for identification of Yersinia pestis.

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.     

Identifying sources of human exposure to plague

Yersinia pestis, the etiologic agent of plague, has shaped the course of human history, killing millions of people in three major pandemics. This bacterium is still endemic in parts of Asia, Africa, and the Americas, where it poses a natural disease threat to human populations. Y. pestis has also recently received attention as a possible bioterrorism agent. Thus, rapid methods to distinguish between bioterrorism and naturally occurring plague infections are of major importance. Our study is the first to demonstrate that variable-number tandem repeats (VNTRs) in the Y. pestis genome can link human case isolates to those obtained from suspected environmental sources of infection. We demonstrate the valuable utility of VNTR markers in epidemiological investigations of naturally occurring plague and the forensic analysis of possible bioterrorism events.     

Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes). Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements. In general, strains which were different by MEE or ribotyping also had different PGFE patterns. PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping. All V. cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns. PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia. Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive. PFGE also distinguished nontoxigenic isolates endemic to the U.S. Gulf Coast from unrelated nontoxigenic isolates. In the present study PFGE was more discriminating than other previously described subtyping assays for V. cholerae O1 and appears to be a useful epidemiologic tool.     

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.     

Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates from Japan.

The nucleotide sequences of the outer surface protein C gene (ospC) from Borrelia burgdorferi sensu lato isolates representing six different restriction fragment length polymorphism (RFLP) ribotype groups were determined, and the deduced amino acid sequences were aligned in comparison with the previously published OspC protein sequences. The sequence similarity analysis revealed the high sequence variability of OspC protein, and the degree of amino acid similarity ranged from 53.8 to 100% among 25 isolates. It has been reported that the representatives belonging to the three species of B. burgdorferi sensu lato showed a species-specific amino acid sequence motif at positions 23 to 35 (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rössler, E. Soutschek, and R.C. Johnson, J. Clin. Microbiol. 33:103-109, 1995). Alignment with the OspC sequences of RFLP ribotype group IV, V, and VI isolates revealed that a sequence motif of all the isolates was quite similar to that of Borrelia garinii. A phylogenetic analysis based on OspC protein sequences also showed that most of the Japanese isolates were closely related to the species B. garinii. THe RFLP ribotype group IV species is predominant among clinical isolates of Lyme disease patients, reservoir rodents, and adult ticks in Japan. Although the isolates differed from type strains of the three delineated genospecies in genetic and immunological characteristics, it is likely that the spirochetes diverged within the species level.(ABSTRACT TRUNCATED AT 250 WORDS)