Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD

Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction- (PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future.     

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.     

Proteomics and multilocus sequence analysis confirm intraspecific variability of Vibrio tapetis

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams. Phenotypic, antigenic and genetic variability have been demonstrated, with three groups being established associated with host origin. In this work we analyze the variability of representative strains of these three groups, CECT 4600(T) and GR0202RD, isolated from Manila clam and carpet-shell clam, respectively, and HH6087, isolated from halibut, on the basis of the whole proteome analysis by 2D-PAGE and multilocus sequence analysis (MLSA). A quantitative analysis of the proteome match coefficient showed a similarity of 79% between the clam isolates, whereas fish isolate showed similarities lower than 70%. A preliminary mass spectrometry (MS) assay allowed the identification of 27 proteins including 50S ribosomal protein L9, riboflavin synthase β subunit, ribose-phosphate pyrophosphokinase and succinyl-CoA synthase α subunit. The MLSA approach gave similar results, showing a 99.4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship between the two clam isolates.     

Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities

AIMS: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south-western Spain, was performed. METHODS AND RESULTS: Characterization of 123 presumptive Vibrio spp. was carried out using 94 phenotypic tests. Simple matching and Jaccard similarity coefficients were used for numerical analysis. Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0.81 similarity. Large phena corresponded to Vibrio tubiashii, V. splendidus biotype I and V. harveyi (phena 1, 5 and 9, respectively). The species V.splendidus biotype II, V. natriegens, V. mediterranei and V. alginolyticus were also represented. The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V.tapetis, the aetiological agent of BRD, was also investigated. Only five V. tubiashii isolates inhibited the growth of V. tapetis strains. The antimicrobial effect was inhibited by heating and depended on the culture medium. CONCLUSIONS: The main Vibrio species associated with manila clams were V. tubiashii, V.spendidus and V. harveyi. The antagonistic relationship established between V. tapetis and the Vibrio spp. clam microbiota may explain the failure of isolation in plating medium of V.tapetis from BRD-affected clams on the south Atlantic coast of Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

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.     

Comparative evaluation of three molecular typing methods in their applicability to differentiate Lactobacillus strains with human origin

We isolated a total of 49 strains of lactic acid bacteria from the faeces of healthy donors. The species in that group were determined as L. plantarum (11 strains), L. casei (11 strains), L. rhamnosus (seven strains), L. fermentum (seven strains), L. gasseri (six strains), L. delbrueckii ssp. lactis (four strains), L. salivarius (two strains), and L. acidophilus (one strain). Genotyping at strain level was performed using random amplification of polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE) with endonucleases ApaI and XhoI and amplified fragment length polymorphism (AFLP) with enzymes XhoI and TaqI. The main objective was the comparison of three molecular typing techniques: AFLP, PFGE and RAPD in their applicability to determine the genetic diversity among the isolates. RAPD was the easiest, comparatively rapid and fairly strain discriminative tool. PFGE was the most laborious method but producing the most stable profiles with satisfactory discriminatory power. AFLP proved to be the most discriminative approach for typing of the strains. AFLP could differentiate strains with the same PFGE profiles. Therefore, AFLP successfully could replace the labor consuming PFGE. The specially developed AFLP and PFGE proved very high potential to evaluate the strain diversity of Lactobacillus spp. with human origin.     

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge

This work compared the effect of challenge with Vibrio tapetis, the etiologic agent of brown ring disease (BRD) in clams, and other bacterial strains on defence-related factors in four bivalve species: Ruditapes philippinarum (highly susceptible to BRD), R. decussatus (slightly susceptible to BRD), Mercenaria mercenaria and Crassostrea virginica (both non-susceptible to BRD). Results show that bacterial challenge modulated defence-related factors, namely total and differential haemocyte counts, percentage of viable haemocytes, and lysozyme activity, both in haemolymph and extrapallial fluid. Injection with bacteria induced a response that was dependent upon the bacterial and bivalve species investigated, and upon the site of inoculation: external (pallial cavity), pseudo-internal (extrapallial space), or internal compartment (adductor muscle). The most conspicuous changes were systematically measured in R. philippinarum injected with V. tapetis, indicating a bacterial pathogenicity particular to the host in which it causes a specific disease syndrome. Alterations of defence-related factors were maximal in haemolymph of clams injected with V. tapetis in the muscle, and in the extrapallial fluid when the bacteria were injected into the pallial or the extrapallial cavity. Resistance to the development of the BRD symptom was not related to the extent of the haemocyte reaction measured following in vivo challenge.     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Genetic (RAPD-PCR) and technological diversities among wild Lactobacillus helveticus strains

Diversity in 25 Lactobacillus helveticus strains isolated from natural whey cultures for Argentinian hard cheese production was studied by means of RAPD-PCR patterns and technological parameters (acidifying and proteolytic activities, salt tolerance, diacetyl, H₂O₂ and slime production, phage sensitivity). In the RAPD diversity study, 10 Lact. helveticus strains from the CNRZ collection were also included.
The clustering of RAPD patterns from the Argentinian strains revealed the existence of two Lact. helveticus biotypes. Cluster 1 contained 22 strains (15 wild and seven CNRZ collection strains), Cluster 2 grouped 10 wild strains and Cluster 3 contained only three CNRZ collection strains. RAPD groups could be related to specific cheese-making characteristics (cheese plants). Analysis of technological characteristics in the Argentinian strains showed the occurrence of different natural variants. According to their capacity for growing in milk, they were classified as 'fast', 'intermediate' and 'slow' variants. Among the strains, low salt tolerance and widespread phage resistance were demonstrated. The genetic diversity (RAPD-PCR clustering) did not show any clear relationship with phenotypical diversity. Study of genetic and technological diversity allows a better characterization of wild strains belonging to Lact. helveticus .     

Comparison of repetitive PCR and pulsed-field gel electrophoresis for the genotyping of Mannheimia haemolytica

Mannheimia haemolytica is an opportunistic pathogen that can cause fibrinonecrotic pneumonia in cattle and is the main bacterial agent implicated in bovine respiratory disease-complex (BRD). Despite its economic importance to the cattle industry, few studies have characterized the genetic nature of M. haemolytica and none have genotyped isolates from feedlots. Identifying and monitoring genetic variants of M. haemolytica is important to understanding the etiology of BRD in cattle. We investigated the capacity of three genotyping techniques (BOX-PCR, (GTG)₅-PCR and PFGE analysis of SalI-restricted DNA) to discriminate among 24 reference strains from the family Pasteurellaceae and 40 M. haemolytica isolates collected from feedlot cattle. From cluster analysis of the M. haemolytica isolates, PFGE was revealed as most discriminating, followed by BOX-PCR and then (GTG)₅-PCR (Simpson's diversity index > 0.98, 0.82, and 0.72, respectively). Of these methods, PFGE also had the greatest mean repeatability (0.96). The PFGE and BOX-PCR assays grouped all M. haemolytica in a single cluster but only BOX-PCR and (GTG)₅-PCR grouped the Mannheimia glucosida and Mannheimia ruminalis strains together. Refinement of genotyping procedures for M. haemolytica could offer new insight into the etiology of this pathogen in BRD.     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.
Methods and Results:  Random amplification of polymorphic DNA (RAPD) was used to discriminate between 58 Tunisian strains and 21 strains from various other countries of P. savastanoi pv. savastanoi , the causal agent of olive knot disease. Isolates were separated into three groups by cluster analysis and principal coordinate analysis of RAPD fingerprint data obtained with three primers (OPR-12, OPX-7 and OPX-14). Group 1 contained isolates from the southeast of Tunisia and European strains. Group 2 comprised strains isolated from the north of Tunisia exclusively while group 3 encompassed the majority of isolates obtained from five orchards located in the centre of Tunisia.
Conclusions:  The results indicated that isolates of P. savastanoi pv. savastanoi were genetically distinct according to geographic regions. RAPD grouped isolates derived from the same orchard as identical.
Significance and Impact of the Study:  This is the first application of RAPD in the delineation of P. savastanoi pv. savastanoi strains.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

Multi-virulence-locus sequence typing of Listeria monocytogenes

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.