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Mycoplasma spp. can cause diseases of the respiratory system as well as urogenital infections, infertility, and anemia. The members of this genus have a low G + C content compared to other bacteria. Because primers used in the random amplified polymorphic DNA (RAPD) technique are only 10 bp long and have high GC content, this method can be inadequate for genotyping Mycoplasma spp. isolates. The aim of this study was to develop and evaluate multiple-locus variable number tandem repeat analysis (MLVA) and two-primer RAPD (TP-RAPD) procedures for subtyping Mycoplasma cynos isolates. A total of 55 M. cynos isolates obtained from 162 bronchoalveolar lavage fluid samples from shelter and pet dogs were used in this study. Seventy-four tandem repeat regions were detected in the M. cynos genome, and two of these loci were determined to be suitable and used for development of the MLVA scheme. The results of variable number tandem repeat (VNTR) analysis and TP-RAPD-PCR were compared with RAPD-PCR. The discriminatory power of TP-RAPD-PCR (Hunter-Gaston diversity index [HGDI] = 0.84) was higher than those of RAPD-PCR (HGDI = 0.727), VNTR1 (HGDI = 0.8), and VNTR3 (HGDI = 0.757). We observed that the TP-RAPD-PCR and MLVA methods provide clearer data and are more successful in determining genetic diversity, in contrast to the RAPD-PCR method for this species. • TP-RAPD-PCR and MLVA were evaluated for genotyping of 55 Mycoplasma cynos isolates. • The discrimination power of TP-RAPD-PCR and MLVA are higher than RAPD-PCR. • RAPD-PCR may cause difficulty genotyping bacteria with low GC because of primers properties. [ABSTRACT FROM AUTHOR]

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation. • MLST had the lowest level of resolution for the Brucella abortus strains accessed. • MLVA16 showed a good discriminatory power and is a good strategy for endemic areas. • SNP techniques are the most indicated for B. abortus outbreak investigations. [ABSTRACT FROM AUTHOR]

A newly developed MLVA seven-loci scheme for Streptococcus pyogenes is described. The method can be successfully applied by using both agarose gel with visual inspections of bands and Lab on Chip technology. The potential of the present MLVA has been tested on a collection of 100 clinical GAS strains representing the most common emm types found in high-income countries plus 18 published gap-free genomes, in comparison to PFGE and MLST. The MLVA analysis defined 30 MLVA types with ten out of the considered 15 emm types exhibiting multiple and specific MLVA types. In only one occasion the same MLVA profile was shared between isolates belonging to two different emm types. A robust congruency between the methods was observed, with MLVA discriminating within clonal complexes as defined by PFGE or MLST. This new MLVA scheme can be adopted as a quick, low-cost and reliable typing method to track the short-term diffusion of GAS clones in inter-laboratory-based surveillance. [ABSTRACT FROM AUTHOR]

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups—O26, O111, O103, O121, O45, and O145—and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks. [ABSTRACT FROM AUTHOR]

Mycoplasma hyorhinis ( M. hyorhinis ) has re-emerged as an important swine pathogen in recent years causing significant economic losses in post weaning pigs. Genetic variability of M. hyorhinis has been described based on different molecular methods that have limited resolution and reproducibility. The present study was undertaken to develop a molecular epidemiological typing tool for M. hyorhinis based on multiple loci of variable number of tandem repeats in its genome, termed MLVA. The typing method was designed on the basis of the number of repeats in two hypothetical proteins, MHR_0152 and MHR_0298. A total of 205 samples were analyzed, including field isolates, clinical specimens, and a reference strain. Analysis of the combination of the 2 loci revealed 16 MLVA types in 165 of the 205 samples. In the remaining forty samples only one locus could be amplified. The most frequent types obtained from the set of samples were 8-4 (36.9%), 8-3 (11.5%), 7-4 (11.5%), 9-4 (10.9%) and 10-4 (9.3%). The Simpson's diversity index for the assay was D = 0.814 when the 165 samples were taken into account. No clustering was observed based on the geographical location, sample type, or year of isolation or sampling. The MLVA assay developed in this investigation showed to be a reproducible and portable assay which could be easily performed and transferred to other laboratories. The use of this technique will assist in epidemiological investigations and can be used to improve the understanding the molecular biology of M. hyorhinis variants. [ABSTRACT FROM AUTHOR]

Abstract: Xanthomonas arboricola is an important bacterial species, the pathovars of which are responsible for bacterial blight diseases on stone fruit, hazelnut, Persian walnut, poplar, strawberry, poinsettia and banana. In this study, we evaluated variable number tandem repeats (VNTR) as a molecular typing tool for assessing the genetic diversity within pathovars of X. arboricola. Screening of the X. arboricola pv. pruni genome sequence (CFBP5530 strain) predicted 51 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 51 loci were designed, and their discriminatory power was initially evaluated with a core collection of 8 X. arboricola strains representative of the different pathovars. Next, the 26 polymorphic VNTR loci present in all strains were used for genotyping a collection of 61 strains. MLVA is a typing method that clearly differentiates X. arboricola strains. The MLVA scheme described in this study is a rapid and reliable molecular typing tool that can be used for further epidemiological studies of bacterial diseases caused by X. arboricola pathovars. [Copyright &y& Elsevier]

Abstract: Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies. [Copyright &y& Elsevier]

Abstract: Yersinia pseudotuberculosis human infections caused by serotype O:1 and O:3 isolates have been common in Finland and have also caused outbreaks. Epidemiological studies on the outbreaks have been limited by the lack of accurate typing methods. During the recent years, multilocus variable-number tandem repeat analysis (MLVA) has been successfully applied for molecular typing of several bacterial pathogens. We designed an MLVA scheme based on seven loci for Y. pseudotuberculosis. The method was able to discriminate clinical isolates of serotypes O:1 and O:3 into several MLVA types. The MLVA profiles were based on the number of 6 to 9bp long tandem repeats in each locus. The number of repeats varied from 1 to 23 depending on the locus. The loci were all located in the bacterial chromosome for stability of the markers. The MLVA method developed was serotype-specific and will be a new additional tool for the epidemiological investigations of isolates associated with disease outbreaks and for comparison of sporadic isolates. [Copyright &y& Elsevier]

Abstract: Ralstonia solanacearum is an important soil borne bacterial plant pathogen causing bacterial wilt on many important crops. To better monitor epidemics, efficient tools that can identify and discriminate populations are needed. In this study, we assessed variable number of tandem repeats (VNTR) genotyping as a new tool for epidemiological surveillance of R. solanacearum phylotypes, and more specifically for the monitoring of the monomorphic ecotypes “Moko” (banana-pathogenic) and “brown rot” (potato-pathogenic under cool conditions). Screening of six R. solanacearum genome sequences lead to select 36 VNTR loci that were preliminarily amplified on 24 strains. From this step, 26 single-locus primer pairs were multiplexed, and applied to a worldwide collection of 337 strains encompassing the whole phylogenetic diversity, with revelation on a capillary-electrophoresis genotype. Four loci were monomorphic within all phylotypes and were not retained; the other loci were highly polymorphic but displayed a clear phylotype-specificity. Phylotype-specific MLVA schemes were thus defined, based on 13 loci for phylotype I, 12 loci for phylotype II, 11 loci for phylotype III and 6 for phylotype IV. MLVA typing was significantly more discriminative than egl-based sequevar typing, particularly on monomorphic “brown rot” ecotype (phylotype IIB/sequevar 1) and “Moko disease” clade 4 (Phylotype IIB/sequevar 4). Our results raise promising prospects for studies of population genetic structures and epidemiological monitoring. [Copyright &y& Elsevier]

Abstract: The Multi Locus Variable Number Tandem Repeat Analysis 16 loci panel (MLVA-16), involving singleplex PCRs and agarose gel electrophoresis, is the standard genotyping method for Brucella spp., used also for the Brucella international online database. We describe an alternative, reliable, high-throughput MLVA-16 protocol using multiplex PCRs and multicolor capillary electrophoresis. [Copyright &y& Elsevier]

Abstract: Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson''s index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology. [Copyright &y& Elsevier]

Abstract: Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis). Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains. The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies. [Copyright &y& Elsevier]

Abstract: Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains). [Copyright &y& Elsevier]

Abstract: We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson''s Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28. We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE. [Copyright &y& Elsevier]

Abstract: A PCR approach for multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae directly from respiratory samples was developed and evaluated on 54 specimens from M. pneumoniae-positive pneumonia patients. The method resulted in a clearly identifiable MLVA type in all samples tested and can be used for MLVA without laborious isolation of the pathogen. [Copyright &y& Elsevier]

Abstract: The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium, Escherichia coli (two assays), Listeria monocytogenes and Yersinia enterocolitica. The gram-negative invasive enteropathogenic bacterium Shigella is the most common cause of bacillary dysentery (shigellosis) worldwide, and is a global human health problem. It was of great interest to develop a rapid and robust MLVA-assay for this important pathogen. Though not common in Norway, we do receive isolates mostly associated with foreign travel and thus an outbreak may be possible. The resulting MLVA-assay is based on seven polymorphous VNTRs found by search in the published genomes of all Shigella species. The assay is fast (one multiplexed PCR reaction), robust and show high divergence among the Shigellae. A total of 235 Shigella spp. were typed with 194 distinct MLVA-genotypes. An outbreak cluster of Shigella sonnei was additionally identified during manuscript preparation. [Copyright &y& Elsevier]

Abstract: Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and the Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types. [Copyright &y& Elsevier]

Abstract: Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections. [Copyright &y& Elsevier]

Abstract: We report the development of a single-tube, multi-locus variable number tandem repeat analysis (MLVA) assay for simultaneous speciation and strain typing of Brucella, the etiologic agent of brucellosis. Our MLVA assay consists of eight loci, two of which are species-specific markers that allow for definitive identification of Brucella melitensis, B. abortus, and Brucella species while ruling out related pathogenic bacterial genera. The remaining six loci are moderately variable loci capable of discriminating between Brucella strains originating within our study area. We applied the assay to a collection of 110 B. melitensis isolates of primarily Mexican origin and to smaller sample sizes of four other Brucella species for a total of 161 isolates. Simpson''s index of diversity was 0.985 for B. melitensis and 0.938 for B. abortus. The assay accurately distinguished seven epidemiologically-linked clusters of B. melitensis infections and ascertained the source of infection in several laboratory-acquired cases. This assay is accessible to limited-resource settings due to its technological and economical feasibility. The timely and accurate information provided by this assay will potentially aid brucellosis control efforts, improve patient outcomes, and reduce the occurrence of laboratory-acquired infections. [Copyright &y& Elsevier]

Abstract: Multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) has been shown to be very promising for the typing of Burkholderia pseudomallei and mallei. The currently available set of loci requires high resolution allele size measurement due to short repeat units. The present work was aimed at expanding the available set of VNTR loci, and generating data from a collection of 102 B. pseudomallei strains isolated in Singapore between 1988 and 2004 including few additional strains of various origins as references. Ten new VNTRs with a longer array size have been identified compatible with standard agarose gel separation, and a reference database of 72 genotypes was created which can be queried on the Internet. [Copyright &y& Elsevier]

Abstract: The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles). [Copyright &y& Elsevier]

Abstract: The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson''s index of diversity of D =0.98. [Copyright &y& Elsevier]

Abstract: Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA–DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis. [Copyright &y& Elsevier]

The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned allele numbers, which were used for strain comparison. [Copyright &y& Elsevier]

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison. [Copyright &y& Elsevier]

Abstract: A multilocus variable number of tandem repeat analysis (MLVA) scheme was developed and 44 isolates of Bifidobacterium longum subsp. longum were typed, including 5 isolates recovered during a clinical trial. The MLVA scheme generated 19 profiles and proved to be a fast, reliable and relatively cheap typing method. [Copyright &y& Elsevier]