Multiple-locus Variable-number Tandem Repeat Analysis Assay Research Articles

Molecular epidemiology and antimicrobial resistance patterns of carbapenem-resistant Acinetobacter baumannii isolates from patients admitted at ICUs of a teaching hospital in Zunyi, China.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a predominant strain of healthcare-associated infections worldwide, particularly in intensive care units (ICUs). Therefore, it is imperative to study the molecular epidemiology of CRAB in the ICUs using multiple molecular typing methods to lay the foundation for the development of infection prevention and control strategies. This study aimed to determine the antimicrobial susceptibility profile, the molecular epidemiology and conduct homology analysis on CRAB strains isolated from ICUs. The sensitivity to various antimicrobials was determined using the minimum inhibitory concentration (MIC) method, Kirby-Bauer disk diffusion (KBDD), and E-test assays. Resistance genes were identified by polymerase chain reaction (PCR). Molecular typing was performed using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Among the 79 isolates collected, they exhibited high resistance tovarious antimicrobials but showed low resistance to levofloxacin, trimethoprim-sulfamethoxazole, and tetracyclines. Notably, all isolates of A. baumannii were identified as multidrug-resistant A. baumannii (MDR-AB). The bla OXA-51-like, adeJ, and adeG genes were all detected, while the detection rates of bla OXA-23-like (97.5%), adeB (93.67%), bla ADC (93.67%), qacEΔ1-sul1 (84.81%) were higher; most of the Ambler class A and class B genes were not detected. MLST analysis on the 79 isolates identified five sequence types (STs), which belonged to group 3 clonal complexes 369. ST1145Ox was the most frequently observed ST with a count of 56 out of 79 isolates (70.89%). MLST analysis for non-sensitive tigecycline isolates, which were revealed ST1145Ox and ST1417Ox as well. By using the MLVA assay, the 79 isolates could be grouped into a total of 64 distinct MTs with eleven clusters identified in them. Minimum spanning tree analysis defined seven different MLVA complexes (MCs) labeled MC1 to MC6 along with twenty singletons. The locus MLVA-AB_2396 demonstrated the highest Simpson's diversity index value at 0.829 among all loci tested in this study while also having one of the highest variety of tandem repeat species. The molecular diversity and clonal affinities within the genomes of the CRAB strains were clearly evident, with the identification of ST1144Ox, ST1658Ox, and ST1646Oxqaq representing novel findings.

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli

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.

Development and comparison of a generic multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis for typing of Salmonella enterica subsp. enterica.

Salmonella enterica subsp. enterica causes salmonellosis in humans and animals. Serovar-specific multiple-locus variable-number tandem repeat analysis (MLVA) is widely used for Salmonella surveillance; however, isolates have to be serotyped prior to MLVA typing and only the most common serovars can be typed. We developed a MLVA scheme for high-discriminatory typing of Salmonella. Sixty-six unique VNTRs were investigated and the polymorphisms of seven promising VNTRs were evaluated with a panel 163 diverse isolates of 14 serotypes of significance for human health. Five VNTRs were selected for MLVA analysis. The discriminatory power was evaluated within serovars by 163 isolates and MLVA yielded 79 genotypes (DI of 0·9790) and pulsed-field gel electrophoresis (PFGE) revealed 87 genotypes (DI of 0·9989). MLVA divided each serotype into 2-8 different profiles and identified six pairs of outbreak-related strains. The technique showed a high-discriminatory power within most serotypes comparable with or better than that of PFGE. This MLVA assay makes it possible to use a single typing method for Salmonella surveillance and outbreak investigations. This allows inexpensive and fast surveillance for laboratories without resources for both serotyping and molecular typing, e.g. PFGE or sequence-based methods, and thereby improve the effectiveness of epidemiological investigations of Salmonella infections globally.

Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA).

Vibrio parahaemolyticus is a leading cause of seafood-borne infections in the US. This organism has a high genetic diversity that complicates identification of strain relatedness and epidemiological investigations. However, sequence-based analysis methods are promising tools for these identifications. In this study, Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was performed on 58 V. parahaemolyticus isolates (28 of oyster and 30 of clinical origin), to identify differences in phylogeny. The results obtained by both methods were compared to Pulsed-Field Gel Electrophoresis (PFGE) patterns determined in a previous study. Forty-one unique sequence types (STs) were identified by MLST among the 58 isolates. Almost half of the isolates (22) belonged to a new ST and added to the MLST database. A ST could not be generated for 5 (8.6%) isolates, primarily due to an untypable recA locus. Analysis with eBURST did not identify any clonal complex among the strains analyzed and revealed 37 singeltons with 4 of them forming 2 groups (1 of them SLV, and the other a DLV). An established MLVA assay, targeting 12 total genes through three separate 4-plex PCRs, was successfully adapted to high resolution melt (HRM) analysis with faster and easier experimental setup; resulting in 58 unique melt curve patterns. HRM-MLVA was capable of differentiating isolates within the same PFGE cluster and having the same ST. Conclusively, combining the three methods PFGE, MLST, and HRM-MLVA, for the phylogenetic analysis of V. parahaemolyticus resulted in a high resolution subtyping scheme for V. parahaemolyticus. This scheme will be useful as a phylogenetic research tool and as an improved method for outbreak investigations for V. parahaemolyticus.

Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

ObjectiveTo evaluate antimicrobial resistance, invasion index and genetic profile in Listeria monocytogenes isolated from clinical samples. MethodsAt all, 170 clinical samples were collected from patients with spontaneous abortions hospitalized in Shariati hospital in Tehran during June 2010 to August 2013. Invasion index was determined using HeLa cells. The multiple-locus variable-number tandem-repeats analysis (MLVA) was used for evaluation of genetic relatedness. ResultsOut of 14 L. monocytogenes isolates, 4 (28.57%), 2 (14.28%), 0 (0%), 5 (35.71%) and 3 (21.42%) were isolated from placental tissue, urine, blood, vaginal and rectal swabs, respectively. High resistance to penicillin and multidrug resistant were found amongst isolates. The invasion index was in the range of 0.001–0.007. Seven different types were obtained by MLVA assay and type 2 and 3 with 4 strains were the most frequent type. Strains isolated from the vagina and the placenta of the same type were also more resistant to penicillin. ConclusionsSince MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method.

Development and evaluation of a multiple-locus variable-number tandem-repeats analysis assay for subtyping Salmonella typhi strains from Sub-Saharan Africa

Background: Typhoid fever a major public health problem in developing countries. Epidemiological investigations of Salmonella enterica serovar Typhi (Salmonella Typhi) infections using molecular sub-typing methods are challenging due to the highly homologous nature of Salmonella Typhi strains. In recent years, several approaches in using multiple-locus variable-number tandem-repeats analysis (MLVA) for molecular sub-typing of Salmonella Typhi have been made. To date, a standardized set of variable-number tandem-repeats (VNTR) loci for the typing of homologous Salmonella Typhi strains has not been established. The aim of our study was to develop and evaluate a MLVA assay consisting of 5 VNTR markers to analyse Salmonella Typhi strains from Sub-Saharan Africa (SSA). Methods & Materials: To develop and evaluate the MLVA assay, 50 Salmonella Typhi strains from humans were selected from the culture collection at the Centre for Enteric Diseases (CED) at the National Institute for Communicable Diseases (NICD) from a potential 1080 isolates. This strain panel represented the diverse Salmonella Typhi pulsed-field gel electrophoresis (PFGE) profiles in the CED database, specimen collection dates and geographic areas within the SSA region. The 50 Salmonella Typhi strains were used to evaluate 12 polymorphic VNTR loci that were previously published. These include TR1, TR2, TR3, TR4, TR5, Sal02, Sal06, Sal10, Sal16, Sal20, TR4500 and TR4600. The assay included PCR amplification of VNTR loci using fluorescently labelled primers and size determination of PCR products by capillary electrophoresis. Results: Of the 12 VNTR loci that were evaluated, 5 (TR3, TR4, TR5, Sal06, Sal10 and TR4500) were found unsuitable as they showed poor allele variation (between 1 and 3 alleles). The remaining 6 VNTR loci showed good allele variation and good diversity indices; with Sal20, Sal 16 and TR1 having 6, 9 and 11 alleles respectively; and Sal02, TR4699 and TR2 having 15, 20 and 23 alleles respectively. Conclusion: In summary, we have identified 6 polymorphic VNTR loci suitable for MLVA analysis of Salmonella Typhi strains. The five most diverse VNTR loci will be selected for the MLVA assay and will be used to analyse Salmonella Typhi strains from SSA. This work will assist in rapidly identifying strain relatedness and assist outbreak detection in typhoid fever outbreaks.

Molecular Strain Typing of Brucella abortus Isolates from Italy by Two VNTR Allele Sizing Technologies

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Antimicrobial susceptibility and molecular subtyping of 55 Turkish Bacillus anthracis strains using 25-loci multiple-locus VNTR analysis

Anthrax, which is caused by the bacterium Bacillus anthracis, is one of the oldest documented infectious diseases in both livestock and humans. The differentiation of B. anthracis strains is difficult because of their highly homogeneous genomes. We used multiple-locus variable-number tandem repeat analysis (MLVA) with 25 markers to genotype 55 B. anthracis isolates from 16 distinct regions of Turkey. The antimicrobial susceptibility of the isolates was investigated using the agar dilution method. An eight-loci MLVA assay revealed six unique genotypes (G(K)13, G(K)27, G(K)35, G(K)43, G(K)44, and G(K)61). However, the 25-loci MLVA was more discriminatory, revealing the presence of ten genotypes instead of six. The additional genotypes resulted from the split of four subtypes: G(K)35 (b and c), G(K)43 (a and f), G(K)44 (d and e), and G(K)61 (i and j). All of the Turkish B. anthracis isolates were susceptible to ciprofloxacin, levofloxacin, tigecycline, linezolid, and vancomycin. One isolate was resistant to penicillin and to doxycycline. A total of 34 isolates were susceptible, 20 isolates were partially susceptible, and one isolate was resistant to erythromycin. None of the isolates exhibited susceptibility to cefotaxime. A total of 53 isolates were susceptible to gentamicin, and two were resistant. The genotypes G(K)35 (n=24), G(K)44 (n=13), and G(K)43 (n=10) were the most prevalent in 10, 6, and 5 regions, respectively, of the total 16 provinces. The B. anthracis isolates collected from these regions implied that the movement of B. anthracis is a result of the increased transportation of livestock and the resultant cross contamination.

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

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.

Method comparison for molecular typing of French and Tunisian Mycoplasma genitalium-positive specimens

In this study, 76 French and Tunisian urogenital specimens were subjected to molecular typing by using the two main Mycoplasma genitalium molecular typing methods, the mgpB single nucleotide polymorphism (SNP) typing method and the combination analysis of a variable-number tandem-repeat (VNTR) marker in MG309 and mgpB SNP. Furthermore, we tried to develop a multiple-locus VNTR analysis (MLVA) method. The genome of M. genitalium G37(T) was analysed for VNTRs and four VNTRs were used for an MLVA. The method, applied directly on clinical specimens, was based on a genescan analysis of VNTR loci labelled with fluorescent dyes by using multiplex PCR and capillary electrophoresis. This method had a 1.00 diversity index (DI) while the mgpB SNP typing and the combination of MG309 and mgpB SNPs had DIs of 0.853 and 0.989, respectively. However, among the sets of two concurrent specimens, taken at the same time from the urogenital tracts of 12 patients, only nine had matching MLVA profiles, while the two other methods gave identical profiles for all specimens amplified, except for one set. Moreover, eight new sequence types were described with the mgpB SNP typing method. The three molecular typing methods revealed a genetic heterogeneity, suggesting that M. genitalium was endemic in France and Tunisia and that the infections were not due to the clonal dissemination of one strain. Comparison of the typing results obtained with the three methods showed that the MLVA assay seemed too discriminatory to be used in future studies of sexual networks of M. genitalium infection. According to the discriminatory power and the feasibility of each mgpB-based method, we recommend that the mgpB analysis be used for general epidemiological studies and that the combination of MG309-STR and mgpB SNP methods should be used for sexual-network studies of M. genitalium infection.

Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Human Brucella Isolates from Turkey

A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16(Orsay)) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16(Orsay) yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA)

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additonal research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.

Multiple-locus variable-number tandem repeat analysis for subtyping of Salmonella enterica subsp. enterica serovar Enteritidis

Salmonella enterica subsp. enterica serovar Enteritidis is a major food-borne pathogen that caused most of Salmonella infections worldwide. S. Enteritidis phage type 4 (PT4) especially presents a real challenge for the classical typing methods. We developed a simple multiple-locus variable-number tandem repeat analysis (MLVA) assay based on three hypervariable variable-number tandem repeat (VNTR) loci for subtyping of Salmonella Enteritidis. Testing an arbitrary chosen strain collection of 110 S. Enteritidis isolates, comprising PTs 4, 8, and 21, the MLVA assay yielded a higher discriminatory power, corresponding to a Simpson's index of diversity (ID) of 0.91, when compared to pulsed-field gel electrophoresis (PFGE) which had a Simpson's ID of 0.41. To simplify interpretation of results, we developed a VNTR allele code based on the repeat unit number. This code can easily be exchanged. In conclusion, MLVA is a promising new tool to investigate outbreaks of S. Enteritidis and constitutes a useful addition to the current phage typing scheme.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing

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).

Harmonization of the multiple-locus variable-number tandem repeat analysis method between Denmark and Norway for typing Salmonella Typhimurium isolates and closer examination of the VNTR loci

Harmonization and evaluation of the multiple-locus variable-number tandem repeat analysis (MLVA) method for sub-typing Salmonella enterica ssp. enterica serovar Typhimurium (Salm. Typhimurium) in Denmark and Norway, and analysis of the typing data. The Salm. Typhimurium MLVA (STMLVA) method, which uses length polymorphisms in five tandem-repeated DNA loci to differentiate isolates, was harmonized between Denmark and Norway, using a common set of 14 isolates. The MLVA assay that is routinely used at the Norwegian Institute of Public Health was set up at the Statens Serums Institute. Both the institutes used an ABI-310 Genetic Analyzer for capillary separation of PCR products, and the same internal size standard. Running the same set of 14 test isolates in both countries and comparing the results showed an excellent typing match at all loci in all isolates. Subsequently, 461 isolates were genotyped in Norway and 454 isolates were genotyped in Denmark. The STMLVA assay displayed a large number of allelic profiles that were distinct for each country as well as shared profiles. Differences in variable number of tandem repeats allele frequencies and absence of amplification products were observed between Denmark and Norway. The MLVA method was set up in two different laboratories and produced completely matching typing data that could be shared rapidly by e-mail for comparison. Notably, differences in allele frequencies and absence of amplification were noted between the countries. The STMLVA method was shown to be easily implemented and to produce typing data, which were shared over the Internet. This enables increased speed of typing and comparison of data between countries, when compared with earlier typing methods. Information embedded in the allele frequencies might give clues to the origin and source of isolates.

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis

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.

DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA)

BackgroundThe ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen.MethodsIn all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification.ResultsThe 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated.ConclusionThe findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.