MLVA Assay Research Articles

Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan.

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.

Genomic variability of Mycoplasma hyopneumoniae within pig lung lobes

BackgroundGenotypic variability in M. hyopneumoniae has been reported within and among herds. However, information regarding VNTR types within single lung lobes is lacking. The objective of his study was to analyse M. hyopneumoniae infections and their association with VNTR types and lung lesions at the lobe level. Lungs from 300 pigs from 10 farms experiencing an enzootic pneumonia outbreak were collected and scored. M. hyopneumoniae was detected by real-time PCR and genotyped by MLVA assay in all samples.ResultsThe results showed genotypic variability within single pigs and among lung lobes. At the lobe level, infection with one VNTR type (SN infection) was dominant. Lobes with lesion scores > 0 were associated with positive results for real-time PCR. At the lobe level, no relationship was observed between infections with more than one genotype (MX infections) and the proportion of Mycoplasma-like lesions. Lesion-free lobes presented a higher proportion of MX infections than lobes scored > 0. M. hyopneumoniae was detected more frequently in the right lobe of the lung (p < 0.05), with a similar distribution within lobes for SN and MX infections. The anatomic conformation of swine lungs led to a higher prevalence of infections in the right lobe. However, this study showed that this condition did not affect the distribution of infections with multiple VNTR types. Nevertheless, careful consideration of sample selection should be practised for M. hyopneumoniae genotype analyses, including lung lobes with no visible lesions.ConclusionThe results did not show a significant association between the number of detected genotypes and the severity of the lesions at the lung lobe level, but revealed the unexpected detection of M. hyopneumoniae genotypes in lesion-free lobes. These results imply that a representative sampling of all lobes may lead to an accurate identification of the VNTR-type distribution. Further studies including factors that can affect pathogenetic evolution of this bacterium could shed light on the complexity of the relationship between genotypes and the lung lesions magnitude.

A new inclusive MLVA assay to investigate genetic variability of Xylella fastidiosa with a specific focus on the Apulian outbreak in Italy

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains and from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

Genetic diversity and population structure of Pseudomonas savastanoi, an endemic pathogen of the Mediterranean area, revealed up to strain level by the MLVA assay

Pseudomonas savastanoi is a bacterial species included in the Pseudomonas syringae complex. It is further sub-typed in pathovars which are the causal agents of a group of diseases of woody plants, such as the “knot disease” on olive and oleander and the bacterial canker on ash. Given its long-established presence in the Mediterranean area, the pathogen causing the afore-mentioned diseases can be considered endemic. Here, an MLVA approach was developed to assess the genetic relationships among and within those pathovars, with a specific focus on P. savastanoi pv. savastanoi. By analyzing the genome of the P. savastanoi pv. savastanoi strain NCPPB 3335 (accession n° CP008742), 14 Tandem Repeat (TR) loci were identified and the corresponding primers were designed and used for the amplification of genomic DNAs from 84 strains belonging to Pseudomonas savastanoi pathovars. Data were analyzed using different approaches, such as hierarchical clustering, STRUCTURE, and k-means clustering with DAPC to evaluate the effectiveness of the assay in describing pathovars and population structure of the pathogen. Results reveal a very complex articulation of genetic relationships, as expected from a long-time evolving pathogen, while providing the possibility to discriminate the pathovars between each other. At intra-pathovar level, the MLVA assay clusters isolates mainly according to their hosts and geographic origin. This resulted particularly useful in the identification and tracking of P. savastanoi populations at local level.

Tandem repeat analysis for typing of Pseudomonas aeruginosa carrying resistance genes in southern Iran

Pseudomonas aeruginosa is known to be one of the most important causes of nosocomial infections, with high antimicrobial resistance. Thus, the current study was conducted to evaluate usefulness of multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA) plan for epidemiological studies and clustering of P. aeruginosa isolated in different wards of general hospital in Bandar Abbas, south of Iran, as well as assessment of presence of integrons and resistance genes in these strains. A total of 72 clinical isolates of P. aeruginosa were isolated from the patients hospitalized in the hospital from December 2017 to June 2018 in Bandar Abbas, southern Iran. MLVA of seven VNTR markers was performed for differentiating and genetic relatedness of isolates. PCR amplification was conducted for detection of bla HSV, bla VEB , int1 and int2 genes. MLVA assay with seven loci resolved 36 different genotypes. Nearly all the isolates were classified into 6 different clonal complexes and 15 singletons. High-level resistance rate was observed in ceftazidime (44; 61%). Nine (12.5%) of these isolates showed multiple drug resistance (MDR) phenotypes. Seven (9.7%), 5 (6.9%), 46 (63.8%) and 6 (8.3%) of isolates carried bla HSV, bla VEB , int1 and int2 genes, respectively. Seven VNTR markers used in this study provided a moderate discriminatory power and differentiated 36 genotypes among 72 isolates. In addition, presence of class 1 and class 2 integrons, bla HSV and bla VEB in different clones indicated a high risk of spreading of MDR isolates among these clones. Results obtained from specification of different tandem repeats in the current study can be helpful to be used for MLVA in epidemiological surveillance of P. aeruginosa isolates in different regions.

New insights about the complexity of Pseudomonas syringae pv. actinidiae across the world

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa), is an emblematic example of a catastrophic disease of fruit crops. In 2008, after decades of limited damages, a new, extremely virulent form emerged and devastated thousands of hectares of Actinidia spp. orchards all over the world with impressive economic losses. To elucidate the diffusion of different Psa types on world scale and their presumptive movements, it is necessary to recognize genetic diversities within its populations. We analyzed an exhaustive collection of 142 Psa strains, that included a conspicuous number isolated in the Asiatic area of origin of Actinidia spp. MLVA has been chosen as the investigative technique for its efficacy and reproducibility, as well as its simplicity and low cost. A panel of 13 VNTR loci was identified and used to study the Psa complex. Results indicate that this MLVA assay can reveal a much wider diversity among Psa populations throughout the world than previously assessed. The congruence of results with those of other molecular approaches was demonstrated and the VNTRs loci were divided in two different panels having an increasing discriminatory power. Ten main clonal complexes were depicted that correlate repeatedly with geographic origin of strains, but also with their virulence and isolation time. The recently originated hypervirulent type of Psa, in which, beside the known strains from Europe, New Zealand and Chile, only few strains from Shaanxi Province in China are also included, confirmed to be genetically very homogeneous. China proved to be the area where the broadest genetic variability resides but, interestingly, different Psa types are retrievable in both Japan and Korea. The low virulent strains group together and are deeply different from any other Psa, so that they can no longer be considered as a real Psa variant. The effectiveness of MLVA in resolving differences between Psa strains and related implications on pathogen spread are discussed.

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains.IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

Multiple-locus variable-number tandem repeat analysis (MLVA) for genotyping of Salmonella enterica subspecies enterica serotype Infantis isolated from human sources

Salmonella is an important cause of food-borne infection worldwide. Detection of outbreaks caused by Salmonella spp. relies on suitable and robust methods for genotyping. Little is known about the genetic diversity of the Salmonella enterica subspecies enterica serotype Infantis strains isolated from human sources in Iran. In this study, 40 isolates of S. Infantis, which were previously recovered from patients with gastroenteritis or diarrhea in Tehran between years 2007 and 2009, were subjected to multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and ERIC-PCR. Using MLVA method, 31 types were identified. The MLVA clustering of the isolates by the unweighted pair group method with arithmetic mean (UPGMA) revealed the presence of two major clusters. The discriminatory power of MLVA was superior to that of PFGE and ERIC-PCR. Overall, our data showed that MLVA assay could effectively differentiate closely related strains. It is technically simple and inexpensive to perform. Furthermore, MLVA can be used as a helpful method for epidemiological investigations.

Genotyping of Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis

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.

Comparison of 2 proposed MLVA protocols for subtyping non-O157:H7 verotoxigenic Escherichia coli

Multiple locus variable number tandem repeats (VNTRs) analysis (MLVA) has become a reliable tool, able to establish genetic relationships for epidemiological surveillance and molecular subtyping of pathogens such as verotoxigenic Escherichia coli (VTEC). This emerging pathogen whose primary reservoir is the cattle causes severe disease in humans, such as hemorrhagic colitis and hemolytic uremic syndrome. With the aim of comparing a recently proposed MLVA assay with that used routinely in our laboratory, we analyzed a set of VTEC isolates (n = 72) obtained from meat using both assays. All samples could be typed by the new MLVA assay, and an increase in the number of distinct profiles (31–43) was observed. However, intraserotype resolution was not significantly enhanced; thus, the incorporation of more VNTR loci is still needed to achieve a greater discrimination among non-O157:H7 serotypes.

Diversity of Mycoplasma hominis clinical isolates from Bordeaux, France, as assessed by multiple-locus variable-number tandem repeat analysis

BackgroundMycoplasma hominis is an opportunistic human mycoplasma species that can cause various urogenital infections and, less frequently, extragenital infections. The objective of this work was to study the genetic diversity of this species using a molecular typing method based on multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA).ResultsThe genome content of M. hominis PG21 was analysed for tandem repeats (TRs), and five of the 130 TRs identified were selected for use in an MLVA assay. The method was based on GeneScan analysis of VNTR loci using multiplex PCR with fluorescent dyes and resolution by capillary electrophoresis. This approach was used on a collection of 210 urogenital and extragenital French clinical isolates collected between 1987 and 2009. Forty MLVA types were found. The discriminatory index of our MLVA scheme was 0.924. Using this new typing tool, persistent infection was suggested for six patients and new infection for one patient. Furthermore, mother-to-child transmission was confirmed in the two cases studied. Application of MLVA to a wide range of M. hominis isolates revealed high genotypic diversity and no obvious link between the MLVA type and the isolate year of collection, the patient’s age or sex, the anatomical origin of the isolates or resistance to antibiotics was found.ConclusionsOur MLVA scheme highlights the high genetic heterogeneity of the M. hominis species. It seems too discriminatory to be used for large epidemiological studies but has proven its usefulness for molecular studies at the individual level.

Isolation of Central Asian Subspecies of Tularemia Agent in the Altai Territory

Within the frames of activities attributed to the Reference Center for tularemia monitoring at SRC AMB, genetically identified are 4 isolates of Francisella tularensis , isolated in 2011 in the Altai Territory. These bacteria prove to be virulent for BALB/c mice, DCL being lower than 10 CFU. Using single-primer PCR-typing and MLVA assay distinguished have been the subspecies of the isolates. Three of them refer to the Central Asian subspecies, one – to the Holarctic, the former being isolated in the territory of the Russian Federation for the first time ever.

A new highly discriminatory multiplex capillary-based MLVA assay as a tool for the epidemiological survey of Pseudomonas aeruginosa in cystic fibrosis patients

Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) has been shown to provide a high level of information for epidemiological investigations and the follow-up of Pseudomonas aeruginosa chronic infection. In the present study, an automatized MLVA assay has been developed for the analysis of 16 VNTRs in two multiplex polymerase chain reactions (PCRs), followed by capillary electrophoresis. The result in the form of a code is directly usable for clustering analyses. This MLVA-16(Orsay) scheme was applied to the genotyping of 83 isolates from eight cystic fibrosis patients, demonstrating that the same genotype persisted during eightyears of chronic infection in the majority of cases. Comparison with pulsed-field gel electrophoresis (PFGE) analysis showed that both methods were congruent, MLVA providing, in some cases, additional informativity. The evolution of strains during long-term infection was revealed by the presence of VNTR variants.

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri

BackgroundShigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri.ResultsThirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b.ConclusionsThe 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BackgroundThe genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.ResultsGenotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.ConclusionIn a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.