Multilocus Variable Number Tandem Repeat Research Articles

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013

BackgroundBrucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking.MethodsA total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1).ResultsThe 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 – 1959, 1962 – 1969, 1971 – 1975, 1977 – 1983, 1985 – 1989, 1992 – 1997, 2000 – 2008 and 2010 – 2013 in China.ConclusionsBrucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA.Trial registrationIDOP-D-16-00101.

Single-nucleotide polymorphism typing analysis for molecular subtyping of Salmonella Tennessee isolates associated with the 2007 nationwide peanut butter outbreak in the United States

BackgroundIn 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak.Methods and resultsOne seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases.ConclusionsThe SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

Within-Flock Population Dynamics of Dichelobacter nodosus.

Footrot causes 70–90% of lameness in sheep in Great Britain. With approximately 5% of 18 million adult sheep lame at any one time, it costs the UK sheep industry £24–84 million per year. The Gram-negative anaerobe Dichelobacter nodosus is the causative agent, with disease severity influenced by bacterial load, virulence, and climate. The aim of the current study was to characterize strains of D. nodosus isolated by culture of swabs from healthy and diseased feet of 99 ewes kept as a closed flock over a 10-month period and investigate persistence and transmission of strains within feet, sheep, and the flock. Overall 268 isolates were characterized into strains by serogroup, proline–glycine repeat (pgr) status, and multi-locus variable number tandem repeat analysis (MLVA). The culture collection contained 87 unique MLVA profiles and two major MLVA complexes that persisted over time. A subset of 189 isolates tested for the virulence marker aprV2 were all positive. The two MLVA complexes (76 and 114) comprised 62 and 22 MLVA types and 237 and 28 isolates, respectively. Serogroups B, and I, and pgrB were associated with MLVA complex 76, whereas serogroups D and H were associated with MLVA complex 114. We conclude that within-flock D. nodosus evolution appeared to be driven by clonal diversification. There was no association (P > 0.05) between serogroup, pgr, or MLVA type and disease state of feet. Strains of D. nodosus clustered within sheep and were transmitted between ewes over time. D. nodosus was isolated at more than one time point from 21 feet, including 5 feet where the same strain was isolated on two occasions at an interval of 1–33 weeks. Collectively, our results indicate that D. nodosus strains persisted in the flock, spread between sheep, and possibly persisted on feet over time.

Genotyping Brucella canis isolates using a highly discriminatory multilocus variable-number tandem-repeat analysis (MLVA) assay

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

Imported human brucellosis in Belgium: Bio and molecular typing of bacterial isolates, 1996-2015.

ObjectivesThe aim of this study was to characterize by classical biotyping and Multi-Locus variable number tandem repeats (VNTR) Analysis (MLVA) all Brucella spp. derived from human cases in Belgium from 1996 to 2015. Final goals were to determine the species and biovar, to trace-back on genetic grounds the origin of each strain when patient history and risk factors were missing, and to survey for particular trends at the national level.MethodsA total of 37 Brucella strains, isolated from 37 patients in Belgium, were analyzed by both classical biotyping and MLVA, and the genetic patterns compared to those of human strains isolated worldwide.ResultsClassical biotyping revealed that isolates were mainly Brucella melitensis. Most of them belonged to biovar 3, the most abundant biovar in the Mediterranean region. MLVA confirmed that Brucella melitensis is too diverse in VNTRs to be able to make clusters associated to each biovar, but it allowed retrieving precious epidemiological information. The analysis highlighted the imported nature of the strains from all over the world with a dominant part from the Mediterranean countries. Findings of the MLVA11 testing were in line with the travel history of patients coming from Italy, Turkey, Lebanon and Peru. The analysis was particularly useful because it suggested the geographical origin of the infection for 12/16 patients for whom no case history was available.ConclusionClassical biotyping and MLVA analysis are not exclusive but remain complementary tools for Brucella melitensis strain surveillance. MLVA11 is sufficient for Brucella-free countries such as Belgium to trace the geographical origin of infection, but complete MLVA16 is needed to search for links with endemic areas.

Presence of Oenococcus oeni and other lactic acid bacteria in grapes and wines from Priorat (Catalonia, Spain)

Oenococcus oeni, the lactic acid bacterium (LAB) mainly responsible for malolactic fermentation, has been repeatedly isolated from wines, but hardly ever from grapes. In this study, a large survey of autochthonous LAB from the Catalan wine region of Priorat was made. A total of 761 LAB isolates, 254 from Grenache and Carignan grape berries and 507 from wines, were identified and typed. Around 70% of the isolates were O. oeni, mostly from wines, but remarkably, 53 of them were isolated from grapes. A minimum spanning tree of O. oeni strains constructed from the multilocus variable number tandem repeat analysis showed considerable phylogenetic diversity. Other non-Oenococcus species were also identified and typed, Lactobacillus plantarum being predominant in grapes. Other LAB isolates were Pediococcus pentosaceus, Fructobacillus tropaeoli, L. mali, L. lindneri and L. sanfranciscensis. High-throughput sequencing (HTS) analysis was also carried out with grape samples, and Oenococcus and Lactobacillus were detected in significant quantities, which corroborates the culturing results from the same samples.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

Severe pertussis infection in infants less than 6 months of age: Clinical manifestations and molecular characterization

ABSTRACTWe conducted a study to determine the main traits of pertussis among unimmunized infants less than 6 months of age. From August 2012 to March 2015, 141 nasopharyngeal aspirates (NPAs) were collected from infants with respiratory symptoms attending 2 major hospitals in Rome. Clinical data were recorded and analyzed. Lab-confirmation was performed by culture and realtime PCR. B. pertussis virulence-associated genes (ptxP, ptxA and prn), together with multilocus variable-number tandem repeat analysis (MLVA), were also investigated by the sequence-based analysis on the DNAs extracted from positive samples. Antibiotic susceptibility with Etest was defined on 18 viable B. pertussis isolates. Samples from 73 infants resulted positives for B. pertussis. The median age of the patients was 45 d (range 7–165); 21 infants were treated with macrolides before hospital admission. Cough was reported for a median of 10 d before admission and 18 d after hospital discharge among infected infants, 84% of whom showed paroxysmal cough. No resistance to macrolides was detected. Molecular analysis identified MT27 as the predominant MLVA profile, combined with ptxP3-ptxA1-prn2 associated virulence genes.Although our data may not be generalized to the whole country, they provide evidence of disease severity among infants not vaccinated against pertussis. Moreover, genetically related B. pertussis strains, comprising allelic variants of virulence associated genes, were identified.

Fatal community-acquired ribotype 002 Clostridium difficile bacteremia

Extra-colonic infections, and especially bacteremia, are infrequent manifestations of Clostridium difficile infection. C. difficile bacteremia is generally health-care associated and polymicrobial. We report the case of a patient on hunger strike that presented a C. difficile colitis and mono-microbial bacteremia on its admission to the hospital. Multilocus variable number tandem repeat analysis of stool and blood isolates indicated clonality. The strain was characterized as a ribotype 002, an emerging ribotype previously associated with high fatality rate. The patient received treatment by intra-venous amoxicillin-clavulanate and oral vancomycin but eventually died on the seventh day of admission with concomitant pneumonia and pulmonary embolism.

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013.

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector.

Multiple-locus variable-number tandem repeat analysis potentially reveals the existence of two groups of Anaplasma phagocytophilum circulating in cattle in France with different wild reservoirs.

Background Anaplasma phagocytophilum is the causative agent of tick-borne fever, a disease with high economic impact for domestic ruminants in Europe. Epidemiological cycles of this species are complex, and involve different ecotypes circulating in various host species. To date, these epidemiological cycles are poorly understood, especially in Europe, as European reservoir hosts (i.e. vertebrate hosts enabling long-term maintenance of the bacterium in the ecosystem), of the bacterium have not yet been clearly identified. In this study, our objective was to explore the presence, the prevalence, and the genetic diversity of A. phagocytophilum in wild animals, in order to better understand their implications as reservoir hosts of this pathogen.MethodsThe spleens of 101 wild animals were collected from central France and tested for the presence of A. phagocytophilum DNA by msp2 qPCR. Positive samples were then typed by multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), and compared to 179 previously typed A. phagocytophilum samples.Results Anaplasma phagocytophilum DNA was detected in 82/101 (81.2%) animals including 48/49 red deer (98%), 20/21 roe deer (95.2%), 13/29 wild boars (44.8%), and 1/1 red fox. MLVA enabled the discrimination of two A. phagocytophilum groups: group A contained the majority of A. phagocytophilum from red deer and two thirds of those from cattle, while group B included a human strain and variants from diverse animal species, i.e. sheep, dogs, a horse, the majority of variants from roe deer, and the remaining variants from cattle and red deer.ConclusionsOur results suggest that red deer and roe deer are promising A. phagocytophilum reservoir host candidates. Moreover, we also showed that A. phagocytophilum potentially circulates in at least two epidemiological cycles in French cattle. The first cycle may involve red deer as reservoir hosts and cattle as accidental hosts for Group A strains, whereas the second cycle could involve roe deer as reservoir hosts and at least domestic ruminants, dogs, horses, and humans as accidental hosts for Group B strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1888-4) contains supplementary material, which is available to authorized users.

Seroprevalence and risk factors of leptospirosis in wild boars ( Sus scrofa ) in northern Italy

Wild boar ( Sus scrofa ) is considered a potential source of several viral and bacterial pathogens that represent a risk to humans and other mammals. Among these the spirochete of the genus Leptospira causes Leptospirosis, a neglected zoonotic disease. This study investigates the presence of antibodies against pathogenic Leptospira spp. serovars in wild boar in different areas of the Lombardy region (northern Italy) and the risk factors associated with its presence in a specific population. Blood and tissue samples from wild boars were collected from 2008 to 2013 during a wildlife survey. A total of 2101 serum samples were analysed using a microscopic agglutination test (MAT) to detect antibodies against Leptospira interrogans sensu lato . Culture isolation and Leptospira DNA detection by PCR were carried out using 189 kidney and 159 urine samples, respectively. Antibodies against 5 serovars were detected in 321 serum samples (15.3 %). Bratislava was the most frequently identified serovar (14.6%; 95% C.I. 13.1–16.2%), followed by Copenhageni (1.48%; 95% C.I. 1.0–2.1%), Grippotyphosa and Pomona (0.48%; 95% C.I. 0.23–0.87%), and Canicola (0.05%; 95% C.I. 0–0.3%). Genotyping by multilocus sequence typing and multilocus variable number tandem repeat analysis of a single leptospire isolate confirmed the presence of L. interrogans serovar Bratislava with the same genetic profile as Jez Bratislava. The statistical analyses confirmed the wild boar’s age class as an important risk factor for the seroprevalence of leptospirosis, whereas no effect of wild boar abundance on seroprevalence was observed. In addition, a positive seroprevalence trend was observed, in particular that of Australis Bratislava showed a general increasing pattern over the years. Our results confirmed that wild boars are a potential source of pathogenic Leptospira spp., which can infect humans, domestic animals and other wild animal species in low-density regions, such as those on the Alps.

Molecular tracking investigation of melioidosis cases reveals regional endemicity in Hainan, China.

Sporadic cases of melioidosis have been reported in Hainan, China for decades; however, to the best of our knowledge, there are no accurate source-identification investigations confirming that melioidosis is endemic. Four indigenous melioidosis cases were identified, which prompted the performance of contact microbiologic and molecular techniques to evaluate endemicity. Environmental samples were collected from various locations surrounding each patient's residence. The samples were screened for Burkholderia pseudomallei (B. pseudomallei) using Ashdown culture medium, and confirmed by polymerase chain reaction and 16S ribosomal DNA sequencing. Clinical and environmental isolates of B. pseudomallei were evaluated by multilocus sequence typing (MLST) and 4-locus multilocus variable number tandem repeat analysis (MLVA-4) for evidence of homology between them. Analysis by MLST indicated that one environmental sample and one clinical colony were sequence type-46, as well as type (8, 3, 11, 9) by MLVA-4. The evidence indicates a likely geographical and epidemiological association. Taken together, B. pseudomallei from the environmental samples in addition to the high molecular homology between the clinical and environmental isolates indicates, at least, regional endemicity of melioidosis in Hainan, China.

Inter- and intra-strain variability of tandem repeats in Mycoplasma pneumoniae based on next-generation sequencing data.

To characterize inter- and intra-strain variability of variable-number tandem repeats (VNTRs) in Mycoplasma pneumoniae to determine the optimal multilocus VNTR analysis scheme for improved strain typing. Whole genome assemblies and next-generation sequencing data from diverse M. pneumoniae isolates were used to characterize VNTRs and their variability, and to compare the strain discriminability of new VNTR and existing markers. We identified 13 VNTRs including five reported previously. These VNTRs displayed different levels of inter- and intra-strain copy number variations. All new markers showed similar or higher discriminability compared with existing VNTR markers and the P1 typing system. Our study provides novel insights into VNTR variations and potential new multilocus VNTR analysis schemes for improved genotyping of M. pneumoniae.

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Whole genome sequencing of Salmonella Typhimurium illuminates distinct outbreaks caused by an endemic multi-locus variable number tandem repeat analysis type in Australia, 2014.

BackgroundSalmonella Typhimurium (STM) is an important cause of foodborne outbreaks worldwide. Subtyping of STM remains critical to outbreak investigation, yet current techniques (e.g. multilocus variable number tandem repeat analysis, MLVA) may provide insufficient discrimination. Whole genome sequencing (WGS) offers potentially greater discriminatory power to support infectious disease surveillance.MethodsWe performed WGS on 62 STM isolates of a single, endemic MLVA type associated with two epidemiologically independent, food-borne outbreaks along with sporadic cases in New South Wales, Australia, during 2014. Genomes of case and environmental isolates were sequenced using HiSeq (Illumina) and the genetic distance between them was assessed by single nucleotide polymorphism (SNP) analysis. SNP analysis was compared to the epidemiological context.ResultsThe WGS analysis supported epidemiological evidence and genomes of within-outbreak isolates were nearly identical. Sporadic cases differed from outbreak cases by a small number of SNPs, although their close relationship to outbreak cases may represent an unidentified common food source that may warrant further public health follow up. Previously unrecognised mini-clusters were detected.ConclusionsWGS of STM can discriminate foodborne community outbreaks within a single endemic MLVA clone. Our findings support the translation of WGS into public health laboratory surveillance of salmonellosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0831-3) contains supplementary material, which is available to authorized users.

Comparison of molecular typing methods for the analyses of Acinetobacter baumannii from ICU patients

Acinetobacter baumannii has emerged as an important cause of healthcare-associated infections causing great morbidity and mortality. Despite its clinical importance, it is still unknown which molecular typing method is the best to determine or confirm institutional outbreaks as well as to identify epidemiologically related isolates from different geographical areas. To determine the most discriminatory molecular typing method, we isolated A. baumannii from perianal swabs collected from intensive care unit (ICU) patients in a cohort study during 2002 and 2008. Strains from each year were analyzed by pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable-number tandem repeat analysis (MLVA). Genetic relatedness of the isolates was consistent between PFGE and MLST as well as between analyses of loci containing MLVA and MLST. Our data show that PFGE and MLVA are similar when discriminating between isolates and are both good methods to use when questioning whether two isolates are indistinguishable.

National outbreak of Yersinia enterocolitica infections in military and civilian populations associated with consumption of mixed salad, Norway, 2014.

In May 2014, a cluster of Yersinia enterocolitica (YE) O9 infections was reported from a military base in northern Norway. Concurrently, an increase in YE infections in civilians was observed in the Norwegian Surveillance System for Communicable Diseases. We investigated to ascertain the extent of the outbreak and identify the source in order to implement control measures. A case was defined as a person with laboratory-confirmed YE O9 infection with the outbreak multilocus variable-number tandem repeat analysis (MLVA)-profile (5-6-9-8-9-9). We conducted a case–control study in the military setting and calculated odds ratios (OR) using logistic regression. Traceback investigations were conducted to identify common suppliers and products in commercial kitchens frequented by cases. By 28 May, we identified 133 cases, of which 117 were linked to four military bases and 16 were civilians from geographically dispersed counties. Among foods consumed by cases, multivariable analysis pointed to mixed salad as a potential source of illness (OR 10.26; 95% confidence interval (CI): 0.85–123.57). The four military bases and cafeterias visited by 14/16 civilian cases received iceberg lettuce or radicchio rosso from the same supplier. Secondary transmission cannot be eliminated as a source of infection in the military camps. The most likely source of the outbreak was salad mix containing imported radicchio rosso, due to its long shelf life. This outbreak is a reminder that fresh produce should not be discounted as a vehicle in prolonged outbreaks and that improvements are still required in the production and processing of fresh salad products.

A genetic diversity study of antifungal Lactobacillus plantarum isolates

Lactobacillus plantarum is a lactic acid bacterium commonly found on fruits and vegetables and also used in a variety of food fermentations. Strains from this species are also regularly reported as having antifungal or probiotic activity. Genotyping methods can be used to differentiate strains of the same species thus determining if strains are related or not. However for L. plantarum, the currently used methods have limitations including DNA band profile interpretation difficulty and cost. In this study, a new genotyping method based on multi-locus variable number tandem repeat analysis (MLVA) was developed and compared to a previously reported randomly amplified polymorphic DNA-PCR (RAPD-PCR) method for L. plantarum. With a selection of 13 antifungal strains of L. plantarum isolated from heterogeneous sources (cheese, silage, sauerkraut, vegetables and a probiotic product), RAPD-PCR revealed 9 different profiles resulting in a Hunter-Gaston discrimination index (D-value) of 0.94. The new MLVA method which compares the lengths of 4 repetitive regions within LPXTG motif-containing surface protein genes differentiated the 13 L. plantarum strains into 10 different subtypes leading to a D-value of 0.95. Interestingly 11 additional L. plantarum isolates obtained in a previous study during a screen for antifungal activity against the common cheese spoilage mould Penicillium commune all possessed the same RAPD-PCR and MLVA profile as each other and the commercial probiotic strain L. plantarum 299v. This study demonstrates that the new MLVA method can be used to simply and inexpensively differentiate L. plantarum strains and provide information regarding strain relatedness and thus potential insight into strain properties.