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Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and >200-fold coverage. Based on the whole-genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used for characterizing chipmunk genotype I in 25 human specimens from four U.S. states and Sweden, one specimen each from an eastern gray squirrel, a chipmunk, and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among the animals, water, and humans, the 15 subtypes identified differed mostly in the numbers of trinucleotide repeats (TCA, TCG, or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the nonrepeat region. Some geographic differences were found in the subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the numbers of a 30-bp minisatellite repeat. Thus, Cryptosporidium chipmunk genotype I isolates from humans and wildlife are genetically similar, and zoonotic transmission might play a potential role in human infections.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Sporadic and epidemiologically linked Yersinia enterocolitica strains ( n = 379) isolated from fecal samples from human patients, tonsil or fecal samples from pigs collected at slaughterhouses, and pork samples collected at meat stores were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA) with six loci, i.e., V2A, V4, V5, V6, V7, and V9. In total, 312 different MLVA types were found. Similar types were detected (i) in fecal samples collected from human patients over 2 to 3 consecutive years, (ii) in samples from humans and pigs, and (iii) in samples from pigs that originated from the same farms. Among porcine strains, we found farm-specific MLVA profiles. Variations in the numbers of tandem repeats from one to four for variable-number tandem-repeat (VNTR) loci V2A, V5, V6, and V7 were observed within a farm. MLVA was applicable for serotypes O:3, O:5,27, and O:9 and appeared to be a highly discriminating tool for distinguishing sporadic and outbreak-related strains. With long-term use, interpretation of the results became more challenging due to variations in more-discriminating loci, as was observed for strains originating from pig farms. Additionally, we encountered unexpectedly short V2A VNTR fragments and sequenced them. According to the sequencing results, updated guidelines for interpreting V2A VNTR results were prepared.

In order to switch from IS 6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% ( n = 3,123). Of the remaining 855 cases, 12% ( n = 479) of the cases were clustered only by VNTR, 7.7% ( n = 305) only by RFLP typing, and 1.8% ( n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% ( n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS 6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems.

Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain.

This study describes the attempt to trace the first Mycobacterium bovis outbreak in alpacas ( Lama pacos ) in Spain by spoligotyping and variable-number tandem-repeat (VNTR) analysis. Due to high genotype diversity, no matching source was identified, but local expansion of a clonal group was found and its significance for molecular tracing is discussed.

The p1 genes of 60 Mycoplasma pneumoniae clinical isolates were sequenced and compared to previously reported p1 gene sequences. An AGT trinucleotide variable-number tandem repeat was identified that ranged in copy number from 5 to 14 among the isolates. In addition, a novel p1 gene variant named 2c was identified in 6 of the isolates.

The prevalence of Neisseria gonorrhoeae in the Netherlands has increased in recent years. A multiple-locus variable-number tandem repeat analysis (MLVA) was developed to assess the molecular epidemiology of N . gonorrhoeae and to elucidate transmission networks in high-risk groups in Amsterdam. The MLVA was evaluated using 5 variable-number tandem repeat (VNTR) loci with various degrees of polymorphism that were amplified in 2 multiplex PCRs and were then separated and sized on an automated sequencer. The assessed number of repeats was used to create MLVA profiles that consisted of strings of 5 integers. The stability of the VNTR loci was assessed using isolates obtained from multiple anatomical locations from the same patient ( n = 118) and from patients and their sexual partners ( n = 55). When isolates with a single locus variant were considered to belong to the same MLVA type, 87% of samples from multiple anatomical locations and 88% of samples from sexual partners shared an MLVA type. MLVA was ultimately performed on 880 isolates that were previously genotyped by restriction fragment length polymorphism (RFLP) analysis of the por-opa genes. Hierarchical cluster analysis of the MLVA profiles from 716 patient visits (one anatomical location per visit) classified 430 patient visits into 14 larger clusters (≥10 patient visits). In 7 clusters, 81% to 100% of isolates came from men who have sex with men (MSM); in 5 clusters, 79% to 100% of isolates came from heterosexuals; and 2 clusters contained isolates from fully mixed populations. Clusters also differed in characteristics such as ethnic background and coinfections. MLVA provided accurate identification of genetically related N. gonorrhoeae strains and revealed clusters of MSM and heterosexuals reflecting distinct transmission networks.

The multiple-locus variable-number repeat analysis of 90 human Brucella melitensis isolates from a large urban area in central Peru revealed variations at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) out of 16 loci investigated, of which 1 (Bruce42) also is used for species identification. Ten genotypes were identified, separated by the number of Bruce42 repeats into two groups that may have distinct phenotypic characteristics. Whereas genotypes with five or six Bruce42 repeats were cultured mainly from adult patients, genotypes with three Bruce42 repeats were isolated from children and young adolescents as well as from adults. In addition, the isolates with three Bruce42 repeats were obtained more often from patients with splenomegaly ( P = 0.02) or hepatomegaly ( P = 0.006). An annual variation in the diversity of genotypes was observed, possibly reflecting changes in sources of fresh dairy products, supply routes to city shops and markets, and the movement of infected dairy goat herds.

A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS 6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.

Standardized mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing based on 15 and 24 loci recently has been proposed for Mycobacterium tuberculosis genotyping. So far, this optimized system has been assessed in a single, 1-year population-based study performed in Germany (M. C. Oelemann, R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann, and P. Supply, J. Clin. Microbiol. 45: 691-697, 2007). Here, we evaluated these optimized formats in a much larger population-based study conducted during 39 months in the Brussels capital region of Belgium. Isolates from 807 patients were genotyped. The resolution power, cluster, and lineage identification by the standardized MIRU-VNTR sets were compared to those obtained using standardized IS 6110 -restriction fragment length polymorphism (RFLP), spoligotyping, and a previous 12-MIRU-VNTR-locus set. On a subset representing 77% of the cases during a 16-month period, a high concordance was observed between unique isolates or strain clusters as defined by standardized MIRU-VNTR and IS 6110 -RFLP (i.e., more than five IS 6110 bands). When extended to the entire population-based collection, the discriminatory subset of 15 loci decreased the strain-clustering rate by almost twofold compared to that of the old 12-locus set. The addition of the nine ancillary MIRU-VNTR loci and/or spoligotyping only slightly further decreased this strain-clustering rate. Familial, social, and/or geographic proximity links were found in 48% of the clusters identified, and well-known risk factors for tuberculosis transmission were identified. Finally, an excellent correspondence was determined between our MIRU-VNTR-spoligotyping strain identifications and external reference strain lineages included in the MIRU-VNTR plus database and identified by, e.g., large sequence polymorphisms. Our results reinforce the proposal of standardized MIRU-VNTR typing as a new reference genotyping method for the epidemiological and phylogenetic screening of M. tuberculosis strains.

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae . In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

The genome of Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 was analyzed for direct repeats, and 54 sequences containing variable-number tandem repeat loci were identified. Ten primer pairs that anneal upstream and downstream of each selected locus were designed and used to amplify PCR targets in isolates of S. enterica serovars Typhimurium and Newport. Four of the 10 loci did not show polymorphism in the length of products. Six loci were selected for analysis. Isolates of S. enterica serovars Typhimurium and Newport that were related to specific outbreaks and showed identical pulsed-field gel electrophoresis patterns were indistinguishable by the length of the six variable-number tandem repeats. Isolates that differed in their pulsed-field gel electrophoresis patterns showed polymorphism in variable-number tandem repeat profiles. Length of the products was confirmed by DNA sequence analysis. Only 2 of the 10 loci contained exact integers of the direct repeat. Eight loci contained partial copies. The partial copies were maintained at the ends of the variable-number tandem repeat loci in all isolates. In spite of having partial copies that were maintained in all isolates, the number of direct repeats at a locus was polymorphic. Six variable-number tandem repeat loci were useful in distinguishing isolates of S. enterica serovars Typhimurium and Newport that had different pulsed-field gel electrophoresis patterns and in identifying outbreak-associated cases that shared a common pulsed-field gel pattern.

In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS 6110 -based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.

PCR-based characterization techniques have been adopted in most laboratories for Mycobacterium bovis typing. We report a molecular characterization of human multidrug-resistant M. bovis isolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.

Single-nucleotide repeats (SNRs) are variable-number tandem repeats that display very high mutation rates. In an outbreak situation, the use of a marker system that exploits regions with very high mutation rates, such as SNRs, allows the differentiation of isolates with extremely low levels of genetic diversity. This report describes the identification and analysis of SNR loci of Bacillus anthracis . SNR loci were selected in silico, and the loci with the highest diversity were used to design and test locus-specific primers against a number of B. anthracis strains with the same multilocus variable-number tandem repeat analysis (MLVA) genotype. SNR markers that allowed strains with the same MLVA genotype to be differentiated from each other were identified. The resulting SNR marker system can be used as a molecular epidemiological tool in a natural outbreak or bioterrorism event, offering the best chance of distinguishing very closely related isolates.

Multilocus variable-number tandem repeat analysis (MLVA) is a validated molecular subtyping method for detecting and evaluating Escherichia coli O157:H7 outbreaks. In a previous study, five outbreaks with a total of 21 isolates were examined by MLVA. Nearly 20% of the epidemiologically linked strains were single-locus variants (SLV) of their respective predominant outbreak clone. This result prompted an investigation into the mutation rates of the seven MLVA loci (TR1 to TR7). With an outbreak strain that was an SLV at the TR1 locus of the predominant clone, parallel and serial batch culture experiments were performed. In a parallel experiment, none (0/384) of the strains analyzed had mutations at the seven MLVA loci. In contrast, in the two 5-day serial experiments, 4.3% (41/960) of the strains analyzed had a significant variation in at least one of these loci ( P < 0.001). The TR2 locus accounted for 85.3% (35/41) of the mutations, with an average mutation rate of 3.5 × 10 −3 ; the mutations rates for TR1 and TR5 were 10-fold lower. Single additions accounted for 77.1% (27/35) of the mutation events in TR2 and all (6/6) of the additions in TR1 and TR5. The remaining four loci had no slippage events detected. The mutation rates were locus specific and may impact the interpretation of MLVA data for epidemiologic investigations.

We applied a high-resolution PCR-based typing method, multiple-locus variable-number tandem repeat analysis (MLVA), for discrimination of 30 multidrug-resistant clinical isolates of Staphylococcus epidermidis . The results of MLVA were congruent with results obtained by pulsed-field gel electrophoresis (PFGE). MLVA generated discrete character data, and its discriminatory capacity was comparable to that of PFGE.

We report ulceroglandular tularemia affecting an 8-year-old boy and the first recovery of Francisella tularensis in Denmark. A novel real-time PCR assay was used to identify the strain as F. tularensis subsp. holarctica (type B). Multiple-locus variable-number tandem repeat analysis demonstrated a close genetic relationship to strains from Norway.

We analyzed a set of variable-number tandem-repeat (VNTR) loci to assess their nucleotide sequence diversity in isolates of three Mycobacterium ulcerans genotypes. Sequence variants in two loci resulted in intraspecies resolution of Southeast Asian and Asian genotypes in contrast to a homogenous sequence composition among African isolates. Nucleotide sequence polymorphism in repeat units can enhance discrimination of VNTR loci.

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae . To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multicase households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis . Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices ≥ 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS 6110 -based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 × 10 9 allele combinations, obviously showed discriminating capacity over the IS 6110 RFLP method for M. tuberculosis isolates with fewer than six IS 6110 -hybridized bands, whereas it had a slightly better resolution than IS 6110 RFLP for the isolates having more than five IS 6110 -hybridized bands. Allelic diversity of many VNTR loci varied in each IS 6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS 6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis . The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus . We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus . Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.

Sixty-eight ancestral Mycobacterium tuberculosis isolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol. 42: 1986-1993, 2004). These TbD1 + ancestral isolates were further characterized and typed in this study by IS 6110 restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS 6110 RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS 6110 RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS 6110 RFLP is proposed to achieve maximal discrimination for unrelated TbD1 + strains. This typing strategy may be useful in areas where TbD1 + ancestral strains are prevalent.

We have analyzed, using complementary molecular methods, the diversity of 43 strains of “ Mycobacterium canettii ” originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest “ M. canettii ” strains, this diversity within “ M. canettii ” subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC.

The need for molecular tools for the differentiation of isolates of Mycobacterium leprae , the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.

We report on the development of a scheme for the typing of Pseudomonas aeruginosa , multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme Spe I distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosa based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa . A free, online strain identification service based on the genotyping data produced herein has been developed.

An evaluation of the utility of IS 6110 -based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken. A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined. Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method. The number of copies of tandem repeats of the five loci ETR A to ETR E and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433). The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing. The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A. All of these isolates had identical MIRU and VNTR types. A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns. The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in “real time,” influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals.

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis . To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS 6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS 6110 RFLP profiles. In a single case, serial isolates with an unchanged IS 6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS 6110 RFLP to define ongoing transmission clusters.

Various genetic markers have been exploited for fingerprinting the Mycobacterium tuberculosis complex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47 Mycobacterium bovis field isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology 144: 1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47 M. bovis isolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47 M. bovis isolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing of M. bovis , with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.

Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli strains. Although MLVA—like MLST—was highly discriminatory (index of diversity, 0.988 versus 0.984), a low level of concordance demonstrated the limited ability of MLVA to reflect long-term evolutionary events.

In this study, we developed a single-tube multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay to type Mycoplasma pneumoniae directly from respiratory samples collected from children with respiratory infections. The multiplex PCR included four fluorescently primed VNTRs (Mpn13, Mpn14, Mpn15, and Mpn16) and was carried out in a single tube. A total of 137 M. pneumoniae -positive specimens, collected in 2013 from Beijing, China, were divided among four types (M4-5-7-2, M4-5-6-2, M3-5-6-2, and M5-5-7-2) using the amended MLVA system. The most prevalent genotype was M4-5-7-2. No correlation was found between macrolide resistance in the M. pneumoniae samples and the MLVA types. To our knowledge, this is the first study to type and analyze M. pneumoniae clinical specimens using multiplex PCR-capillary electrophoresis in a single tube. This novel low-cost method can be used to rapidly type M. pneumoniae clinical specimens directly and shows great potential for monitoring outbreaks of M. pneumoniae .

Yersinia pestis , the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the Y. pestis genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the Y. pestis genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity ( R = 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.

The present study was designed to evaluate the use of variable number tandem repeat (VNTR) and IS 6110 -restriction fragment length polymorphism (RFLP) analyses in combination as a two-step strategy for discrimination (as measured by the Hunter-Gaston Discrimination Index [HGDI]) of both high- and low-copy-number IS 6110 Mycobacterium tuberculosis isolates compared to IS 6110 -RFLP alone with an unselected collection of isolates. Individually, IS 6110 -RFLP fingerprinting produced six clusters that accounted for 69% of the low-copy-number IS 6110 isolates (five clusters) and 5% of the high-copy-number IS 6110 isolates (one cluster). A total of 39% of all the isolates were clustered (HGDI = 0.97). VNTR analysis generated a total of 35 different VNTR allele profile sets from 93 isolates (HGDI = 0.938). Combining IS 6110 -RFLP analysis with VNTR analysis reduced the overall percentage of clustered isolates to 29% (HGDI = 0.988) and discriminated a further 27% of low-copy-number isolates that would have been clustered by IS 6110 -RFLP alone. The use of VNTR analysis as an initial typing strategy facilitates further analysis by IS 6110 -RFLP, and more importantly, VNTR analysis subdivides some IS 6110 -RFLP-defined clusters containing low- and single-copy IS 6110 isolates.

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS 6110 -restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of Mycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS 6110 -RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS 6110 -RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS 6110 -RFLP clustering (ca. 15%). All but one IS 6110 -defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS 6110 -RFLP. Reinterpretation of previous IS 6110 -RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS 6110 -RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.

A panel of seven variable-number tandem-repeat (VNTR) markers was selected for Acinetobacter baumannii typing analysis (MLVA-7). Compared with pulsed-field gel electrophoresis (PFGE), MLVA-7 provided greater discrimination. We modified the criteria for MLVA complex assignments proposed previously, and a remarkable congruence between MLVA-7- and PFGE-based strain clustering was observed.

In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae . The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited ( n = 4) number of M. pneumoniae- positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations.

The performance characteristics of Neisseria gonorrhoeae multilocus variable-number tandem-repeat analysis were evaluated, by comparison with N. gonorrhoeae multiantigen sequence typing and full-length porB sequence typing. Assessment of the relatedness of intra- and interpatient isolates showed that all three genotyping techniques display a high resolution and typeability.

Variable-number tandem repeat (VNTR) and spoligotyping analyses were used to assess transmission of Mycobacterium bovis between humans. VNTR was more discriminatory than spoligotyping. Low case numbers, despite a substantial animal reservoir, and resolution of all isolates provided no evidence of recent human-to-human transmission or recent significant infection from animals.

A modified multiple-locus variable-number tandem-repeat analysis (MMLVA) method was validated on Clostridium difficile -infected stool specimens from institutional outbreaks. The method allows simultaneous detection of toxin genes, deletions, and tandem repeats from cultured isolates or stool specimens. Results were used to aid institutional outbreak investigation by identifying clusters of NAP1/027.

An assay based on multiple-locus variable-number tandem-repeat analysis allowed differentiating and studying diversity and persistence of Mycoplasma hyopneumoniae strains in pig herds without prior cultivation. The test had a discriminatory index of >0.99 and was applied reliably to porcine bronchoalveolar lavage fluid and tracheal swabs.

A novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates. The assay accuracy was confirmed by a multiple-target sequence analysis. The duplex PCR assay is a one-step, reliable, and accurate assay for discriminating M. abscessus species.

The present study evaluates the performance of the staphylococcal interspersed repeat unit (SIRU) method applied to a diverse collection of 104 Staphylococcus aureus isolates previously characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec typing for methicillin-resistant S. aureus . The SIRU method distributed the 104 strains into 81 SIRU profiles that could be clustered into 12 groups and 29 singletons. The discriminatory power of the method at the profile level, translated by Simpson's index of diversity (SID), was similar to that of PFGE subtyping (SID = 99.23% versus 99.85%) and slightly higher than that of spa typing (SID = 97.61%). At the group level, the SIRU SID (93.24%) was lower than that of PFGE typing (95.41%) but higher than that of MLST (SID = 91.77%). The adjusted Rand (AR) coefficient showed that SIRU typing at the group level had the highest congruence with MLST (AR = 0.5736) and with clonal complex (CC) (AR = 0.4963) but the lowest congruence with PFGE subtype (AR = 0.0242). The Wallace coefficient indicated that in the present collection, two strains with the same SIRU profile have a 100% probability of belonging to the same CC, a 90% probability of sharing the same spa type, and an 83% probability of being classified in the same sequence type. The high discriminatory power of the SIRU method, along with its apparent concordance with MLST results, makes it potentially valuable for S. aureus short-term epidemiological investigations and population dynamics as well.

The present study evaluated new markers for molecular typing of Mycobacterium tuberculosis with a collection of strains circulating in Bulgaria. A study sample included 133 strains from epidemiologically unlinked patients from different regions of the country. Spoligotyping was used as a primary typing tool; it subdivided these strains into 37 types, including 15 clusters and 22 singletons. Traditional IS 6110 -restriction fragment length polymorphism (RFLP) typing and novel 24-locus variable number tandem-repeat (VNTR) typing methods were applied to the selection of 73 strains. Discriminatory power (Hunter-Gaston index [HGI]) of these methods was found to be 0.983 and 0.997, respectively. The 73 strains were subdivided into 66 types by a 24-locus mycobacterial interspersed repetitive unit (MIRU)-VNTR scheme, 62 types by a classical 12-locus MIRU-VNTR scheme, 51 types by IS 6110 -RFLP typing, and 31 types by spoligotyping. A combination of the five most polymorphic loci (MIRU40, Mtub04, Mtub21, QUB-11b, and QUB-26) was shown to achieve a high discrimination (HGI = 0.984). To conclude, a complete 24-locus scheme excellently differentiated strains in our study, whereas a reduced 5-locus set provided a sufficiently high differentiation and may be preliminarily suggested for the first-line typing of M. tuberculosis isolates in Bulgaria.

For species with low genetic diversity, typing using the differences in PCR fragment length resulting from variations in numbers of short tandem repeats has been shown to provide a high level of discrimination. This technique has been called multilocus microsatellite typing (MLMT) or multiple-locus variable-number tandem repeat analysis, and studies usually employ genetic or sequence analyzers to size PCR fragments to a high degree of precision. We set out to validate one such system that has been developed for Aspergillus fumigatus (H. A. de Valk, J. F. G. M. Meis, I. M. Curfs, K. Muehlethaler, J. W. Mouton, and C. H. W. Klaassen, J. Clin. Microbiol. 43:4112-4120, 2005). The sizes of the alleles were compared both by sequencing and from two genotyping laboratories, where they used capillary electrophoresis (CE) for sizing. Size differences of up to 6 bases were found between the actual sizes reported by sequencing and the sizes reported by CE. In addition, because the two genotyping laboratories used different machines and running conditions, differences of up to 3 bases were identified between them. As the microsatellite markers used differ by repeat units of 3 or 4 bases, it was not possible to assign PCR fragments to the correct alleles without confirming the sizes of a range of alleles by direct sequencing. Lines of best fit were plotted for each CE machine against actual sizes and will therefore enable unsequenced PCR fragments to be assigned to the correct alleles. This study highlights the care required to ensure that an MLMT system undergoes a suitable correction procedure before data can be merged between different laboratories involved in the typing of individual species.

We identified many variable-number tandem repeat (VNTR) loci in the genomes of Neisseria meningitidis serogroups A, B, and C and utilized a number of these loci to develop a multiple-locus variable-number tandem repeat analysis (MLVA). Eighty-five N. meningitidis serogroup B and C isolates obtained from Dutch patients with invasive meningococcal disease and seven reference strains were analyzed using MLVA and multilocus sequence typing (MLST). MLVA, based on eight VNTR loci with limited variability in the number of repeats, yielded clustering of the strains similar to that obtained by MLST, with congruence between both methods amounting to 69%. The ability to recognize clonal complexes makes MLVA a valuable high-throughput method to serve as a tool complementary to MLST. Four highly variable VNTR loci were used in a second assay to analyze N. meningitidis serogroup C strains collected during an outbreak of meningococcal disease in The Netherlands. Typing based on the latter VNTR loci enabled differentiation of isolates with identical MLST sequence types and grouped epidemiologically related strains.

Thirty-one meningococcal isolates from carriers and disease cases belonging to the sequence type (ST) 162 complex, isolated in Greece in 1999 and 2000, were studied by the use of variable-number tandem repeat analysis. Our study demonstrated that the isolates belonging to the ST-162 clonal complex were a heterogeneous group. Based on this heterogeneity, it is unlikely that the disease-associated isolates represent an outbreak.

We used multiple-locus variable-number tandem repeat analysis (MLVA) to type 64 Bacillus anthracis isolates from outbreaks that have occurred during the past 40 years in Italy. MLVA of the 64 isolates revealed 10 unique genotypes; 9 of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes (G1 and G3), which differ by a single mutation in the pX01 locus, account for the majority of isolates in the country (53/63). The low diversity of B. anthracis genotypes in Italy suggests a single, dominant historical introduction, followed by limited localized differentiation.

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum.

Multilocus variable-number tandem-repeat analysis (MLVA) for seven genomic loci was developed for Enterococcus faecalis . MLVA and pulsed-field gel electrophoresis (PFGE) resulted in 37 and 31 genotypes among 83 strains, respectively. Both typing schemes were highly concordant (90.4%). MLVA is an excellent alternative to PFGE.