MLVA

Subtyping of Listeria monocytogenes strains is an essential epidemiological tool to trace contamination and determine evolutionary relationships among different strains. Pulsed field gel electrophoresis (PFGE) is the current gold standard method for Listeria characterization. Multiple‑Locus Variable‑number tandem repeat Analysis (MLVA) is a rapid subtyping method based on Polymerase Chain Reaction (PCR) amplification that has been successfully developed for subtyping bacterial genera. The purpose of this study was to evaluate MLVA for subtyping L. monocytogenes strains isolated from Italian cheese and to compare it with PFGE. The type ability and discriminatory power of MLVA was determined on a collection of 90 isolates corresponding to 5 serotypes and 29 pulsotypes with enzymes AscI and ApaI. A panel of 5 variable‑number tandem repeat loci was used. MLVA and PFGE showed very similar discriminatory power (Simpson's Index of diversity 0.840 with 95% Confidence Interval [CI] 0.780/0.899 and 0.837 with 95% CI 0.768/0.906, respectively). MLVA is an easy test to perform. It is relatively fast, reproducible and could be implemented in any molecular laboratory but, according to the performed protocol, it is not sufficient for discriminating the L. monocytogenes strains isolated from cheese. This method could be combined with PFGE to increase the discrimination in molecular subtyping of these strains.

The genetic diversity of Brucella melitensis and Brucella abortus strains isolated in 199 cattle and sheep from 156 brucellosis outbreaks which occurred in 8 regions of Southern Italy in 2011, was determined using a Multiple-Locus Variable Number of Tandem Repeats Analysis (MLVA‑16) approach. The existence of possible genetic clusters was verified through a hierarchical cluster analysis based on 'single link', which is closely related to the minimum spanning tree. The Hamming weighted distance matrix was adopted in the analysis. All calculations were performed using R and the additional libraries phangorn and Cluster. For a number of clusters, ranging from 2 to 15, the average silhouette width was calculated. The number of clusters adopted was identified according to the maximum average silhouette width. For B. abortus and B. melitensis, 6 and 11 genetic clusters were identified, respectively. Three out of 6 B. abortus clusters included the 96.7% of all B. abortus isolates. Clusters were clearly geographically separated, and this highlighted the known epidemiological links among them. Brucella melitensis genotypes resulted more heterogeneous; the 3 more representative genetic clusters included 79.7% of all B. melitensis isolates. A clear geographical clusterization of genotypes is recognizable only for 1 cluster, whereas the others are more widespread across Southern Italy. The genetic characterization of Brucella strains isolated from animals may be a useful tool to better understand the epidemiology and dissemination patterns of this pathogen through host populations.

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven mi-crosatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more dis-criminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC phylotype III.

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.

Estimated has been efficacy of MLVA25 method, used for differentiation and clusterization of Yersinia pestis strains, in reference
to Yersinia pseudotuberculosis strains, as well as the search of Y. pseudotuberculosis strains mostly closely related to Y. pestis. Applied
was the reduced version of the technique deploying 14 out of 25 VNTR loci. Comparative study of 71 Y. pseudotuberculosis isolates
and five Y. pestis strains by means of MLVA25 and MLVA14 variants revealed 75 and 54 genotypes, respectively. Y. pseudotuberculo�sis strains of certain serovars corresponded to certain clusters of MLVA types. According to MLVA14 typing of 221 Y. pseudotubercu�losis isolates the clusters including the strains belonging to MLST types ST19 (serovar O:3) and ST43 (serovar O:1b) were the most
closely related to Y. pestis.

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNase I and was followed by DNase inactivation at 94°C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase, Deep-Vent Exo-®polymerase (New England Biolabs, Beverly, MA), and super-Taq® polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq® polymerase (Perkin-Elmer Cetus, Norwalk, CT), Ampli-Taq LD® polymerase (Perkin-Elmer Cetus) or Deep-vent® polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.

A female wild boar, aged approximately two years, was found dead by local veterinary services in Pianola di Roio in L'Aquila Province situated in the Abruzzo Region of central Italy. The carcass was submitted to the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' in Teramo for necropsy. Brucella suis biovar 2 was isolated from submandibular lymph nodes. This is the first report of isolation of B. suis in the Abruzzo Region. Several authors agreed in the past on the hypothesis that B. suis biovar 2 had been introduced into Italy through the importation of hares from European countries in which the infection is endemic in wild populations. This lead the Italian authorities to reinforce existing controls for hares imported for restocking purposes. However, no provisions for brucellosis control are currently in place (or have been in place in the past) for wild boar movements either at the national or the European level. The isolation of B. ...

Background.Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of theRalstonia solanacearumspecies complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III.Methods.We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Differ...

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. D...