Arbitrarily Primed Polymerase Chain Reaction Research Articles

The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status.

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first-named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS-PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother-child pairs were analyzed by arbitrarily primed PCR (AP-PCR). Similar AP-PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra-familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP-PCR types were most obvious within periodontally healthy mother-child pairs.

Rapid differentiation of Staphylococcus aureus from staphylococcal species by arbitrarily primed-polymerase chain reaction.

An arbitrarily primed-polymerase chain reaction (AP-PCR) method was optimized to differentiate Staphylococcus aureus from other staphylococcal species, using DNA from crude cell extract. From the different assays carried out, the best resolution of the band patterns was obtained when the reaction mixture contained 200 micromol l(-1) dNTPs, 200 ng primer, 1 U Taq DNA polymerase and 3 mmol l(-1) MgCl2 and the amplification conditions were: initial denaturation of 94 degrees C for 1 min, primer annealing of 30 degrees C for 1.5 min, DNA extension at 55 degrees C for 5 min and final extension at 55 degrees C for 5 min. The results of the characterization of the staphylococcal isolates by AP-PCR are in accordance with those of the biochemical identification by the API Staph System, time of analysis of the AP-PCR being only 6-7 h. Thus, this technique could be a useful method for microbial quality assurance.

Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae.

It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (< or =88.8% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 88.8 and 100%. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (95.5% identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.

Interrelationship of apoptosis, mutation, and cell proliferation in N-methyl- N′-nitro- N-nitrosoguanidine (MNNG)-induced medaka carcinogenesis model

The present study examined the interrelationship of GSH depletion, apoptosis, mutation, and cell proliferation following carcinogen exposure. Medaka ( Oryzias latipes) were investigated following a 28 day, three times/week pulse exposure to N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Fish (5 weeks old) were exposed to MNNG at concentrations of 0, 0.5, and 1 mg l −1 and reared for 3, 5 and 7 more months after the last day of exposure. GSH levels were decreased in the higher concentration groups and longer-reared groups. Flow cytometric analysis revealed that fish from the groups reared 3 and 5 months showed active apoptotic changes in the dose- and time-dependent manner, but the group reared 7 months had fewer apoptotic, rather showed more necrotic and carcinogenic alterations. Mutational responses were detected by an arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting method using whole body DNA samples as templates and pBR primer. A mutational change was expressed by a loss or gain of a band. There was a time-dependent mutational change, but no distinctive concentration-dependent one. A band from normal fish sample that disappeared after treatment of MNNG was excised and sequenced. The band had an 869 base pair-long sequence, however, there was no putative protein-coding region based on an analysis by DNAsis. Spindle cell sarcomas invading muscle were detected on the whole body sections from three of ten fish examined, and immunohistochemical analysis with PCNA showed that tumor cells were actively proliferating. However, terminal deoxynucleotidyl transferase (TdT) assay showed that tumored fish still had active apoptotic cell changes in the tissues without tumor. This study shows not only the interrelationship of GSH depletion, apoptosis, mutation and cell proliferation, but also indicates that medaka is appropriate as a fish model for research on the passage of carcinogenesis.

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3[ratio ]K6 and O4[ratio ]K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Frequent Chromosome 8q Gains in Human Small Cell Lung Carcinoma Detected by Arbitrarily Primed-PCR Genomic Fingerprinting

The arbitrarily primed-polymerase chain reaction (AP-PCR) genomic fingerprinting method was applied to detect chromosomal numerical imbalances in 13 small cell lung carcinomas (SCLC). Increases and decreases in the intensity of the AP-PCR bands from several chromosomes, representing gains of chromosomes 1, 7, 16, and X, and losses of chromosomes 2, 10, and 22, were recurrent events in independent tumors. In addition, the most common alterations detected were increases in signal intensity of three AP-PCR bands derived from genomic sequences on chromosome 8q (eight of 13 tumors: 62%). To define whether the 8q gains observed in the AP-PCR analysis include the C-MYC gene at chromosome 8q24 or not, we performed targeted AP-PCR analysis of the C-MYC gene. The C-MYC gene was amplified in five of the eight tumors with gains of 8q, but in none of the remaining five tumors in which 8q gains were not detected. These results indicate that chromosome 8q gain occurs frequently in SCLC and gained regions often, but do not always, include the C-MYC gene. Moderate increases in copy number of the C-MYC gene and additional gene(s) in the 8q region appear to be under positive selection during SCLC progression.

Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis.

Actinobacillus actinomycetemcomitans leukotoxin is thought to be an important virulence factor in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. Some highly leukotoxic A. actinomycetemcomitans strains produce 10 to 20 times more leukotoxin than other minimally leukotoxic strains. The distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis. The polymerase chain reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by both AP-PCR and ribotyping. Highly leukotoxic A. actinomycetemcomitans was found only in subjects with localized juvenile and early-onset periodontitis. Fifty-five percent of the LJP subjects harbored highly leukotoxic A. actinomycetemcomitans isolates. Seventy-three percent of the A. actinomycetemcomitans isolates in these subjects were highly leukotoxic. Highly leukotoxic A. actinomycetemcomitans infected younger subjects (mean age 13.95 years, range 5 to 28 years) than minimally leukotoxic (mean age 35.47 years, range 6 to 65 years). Most subjects were infected with only one A. actinomycetemcomitans genotype. However, PCR of whole dental plaques and subsequent analysis of up to 130 individual oral isolates suggested a possible shift in A. actinomycetemcomitans over time in that a few subjects harbored both highly leukotoxic and minimally leukotoxic strains. AP-PCR analysis was consistent with intrafamilial A. actinomycetemcomitans transmission. Ribotyping and AP-PCR analysis confirmed a previous report that highly leukotoxic A. actinomycetemcomitans consists of a single clonal type. This study suggests that localized juvenile and other forms of Actinobacillus-associated periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.

Molecular genotyping of Candida species with special respect to Candida (Torulopsis) glabrata strains by arbitrarily primed PCR.

A set of 46 epidemiologically related or unrelated Candida (Torulopsis) glabrata isolates from four different medical centres in Germany and Hungary, and the type strain of this species, were genetically typed by arbitrarily primed PCR (AP-PCR). The resulting band patterns of C. glabrata strains were compared with those of other species of the genus Candida including C. albicans, C. guilliermondii, C. kefyr, C. parapsilosis, C. tropicalis and C. krusei. After preliminary trials of various reaction parameters and control experiments to test the reproducibility of this method, it was found that consistently reproducible amplification patterns were obtained only when rigorously optimised and standardised reaction conditions were employed. Discriminatory abilities were studied with 29 generated 10-mer oligonucleotides of different G+C content. Typing of clinical isolates with the optimised AP-PCR protocol was then performed with the primer 50-1, with a G+C content of 50%. Sufficiently discriminatory polymorphisms were observed among the band patterns of the Candida species included. The gel electrophoresis patterns of each species showed an adequate similarity. Variations in minor bands were characteristic for comparison at the isolate level. Only three AP-PCR genotypes were identified among the clinical isolates of C. glabrata tested. Two of these genotypes were closely related and appeared to be widespread within German and Hungarian isolates. The third genotype of C. glabrata showed a distinct band pattern. With optimised, validated and standardised assay conditions, the feasibility, sensitivity and rapidity of AP-PCR may offer a discriminatory method for genotyping of yeasts in epidemiological studies, as well as in the control of nosocomial infections.

Application of PCR to the identification of dermatophyte fungi.

Infection of the keratinised tissues (skin, hair and nails) in man and animals by keratinophilic fungi (dermatophytes) results in dermatophytosis (also known as tinea or ringworm). As conventional laboratory procedures for the identification of dermatophytes are either slow or lack specificity, improved diagnostic methods are required. The application of nucleic acid amplification technology has made rapid and precise identification of dermatophytes possible. Recent studies have shown that when one of the four random primers (OPAA11, OPD18, OPAA17 and OPU15) was used in arbitrarily primed PCR (AP-PCR), up to 20 of the 25 dermatophyte species or subspecies under investigation could be distinguished on the basis of characteristic band patterns detected in agarose gel electrophoresis. A combination of two random primers (OPD18 and OPAA17) used in separate reaction tubes identified 23 of the 25 dermatophyte species or subspecies examined. AP-PCR provides a rapid and practical tool for identification of dermatophyte isolates that is independent of morphological and biochemical characteristics and thus enhances laboratory diagnosis of dermatophytosis.

Arbitrarily Primed PCR for Differentiation of Salmonella enteritidis Strains

Bakshi, C.S., Singh, V.P., Sharma, B., Malik, M. and Singh, R.K. 2000. Arbitrarily primed PCR for differentiation of Salmonella enteritidis strains. J. Appl. Anim. Res., 17: 291–295. Strains of Salmonella enteritidis of avian origin isolated from Bangalore and Pune in India were analysed by Arbitrarily Primed PCR (AP-PCR), which generated eight different patterns and enabled the discrimination among them. It was concluded that the technique could be of use in the epidemiological studies of S. enteritidis strains.

Isolation and characterisation of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

Analysis of Leptospira isolates from mainland Portugal and the Azores islands

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) ( n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes ( n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting ( n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans ( n=3), Leptospira kirschneri ( n=8) and Leptospira borgpetersenii ( n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.

Analysis of Leptospira isolates from mainland Portugal and the Azores islands.

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.

Morphological and Genomic Diversity in the Genus Pteridium(Dennstaedtiaceae)

Morphometric comparisons of frond material grown under standardized garden conditions and DNA fingerprinting by arbitrarily-primed PCR were used to assess taxonomic groupings and relationships in the cosmopolitan bracken ferns of the genus Pteridium (Dennstaedtiaceae). The genus comprises a limited number of relatively stable, generally at least partially interfertile, morphotypes which were placed by R. M. Tryon (Rhodora43: 1–31, 37–67, 1941) in a single species containing 12 varieties. Both morphometric analysis and DNA fingerprinting of 72 accessions including 11 of these varieties (excluded is var. feei from Central America) resolves groupings corresponding to vars africanum, arachnoideum, esculentum, latiusculum and revolutum from each other. DNA fingerprinting further (a) distinguishes an additional grouping of Atlantic Island (Azores, Madeira) and European brackens as an ‘aquilinum complex’ including var. aquilinum and a number of morphotypes recognised by C. N. Page (The ferns of Britain and Ireland. 2nd edn. Cambridge: Cambridge University Press, 1997) and others at various taxonomic levels (near atlanticum, fulvum, pinetorum, osmundaceum); (b) confirms var. yarrabense as a tetraploid hybrid (4n = 208) of var. esculentum and var. revolutum; (c) establishes that at least those accessions of var. caudatum examined here are tetraploid hybrids involving var. arachnoideum as one progenitor; (d) indicates that the closest relatives of var. decompositum are var. latiusculum and var. revolutum; and (e) provides evidence of close genomic relationships between var. latiusculum, var. pseudocaudatum and var. pubescens in North America. These conclusions are consistent with the results of the morphometric analysis. The DNA evidence suggests that morphotypes in Pteridium are determined by specific qualitative and quantitative combinations of a limited number of highly conserved, additively assorted, genomic elements. A general model based on allopolyploidy followed by one or more rounds of autogamous allohomoploidy is proposed to account for the origin, maintenance and interrelationships of morphotypes in Pteridium. It is suggested that Tryon's varieties africanum, aquilinum, arachnoideum, decompositum, esculentum, latiusculum and revolutum might best be treated as species; pseudocaudatum and pubescens as varieties within latiusculum; yarrabense and caudatum (at least in part) as hybrids.

Inoculation, isolation and identification of Tuber melanosporum from old and new oak hosts in Israel

Five local oak species, Quercus boissieri, Q. calliprinus, Q. cerris, Q. ithaburensis and Q. libani were identified as hosts of Tuber melanosporum , the black truffle of Perigord, following inoculation of roots. Q. cerris and Q. libani developed abundant mycorrhizal roots, similar to the amount found in roots of Q. pubescens , the traditional host of this mycorrhizal fungus. Roots of Q. ilex, Q. hartwissiana and Q. pedunculiflora , introduced species in Israel, were also heavily colonized by the mycorrhizal fungus. Mycorrhized hazel ( Corylus avellana ) roots, were also obtained by the same inoculation procedure. Recovery of T. melanosporum from roots of inoculated oak was improved when chloramphenicol was added to the isolation medium. Recovery varied between 14 and 54% for C. avellana and Q. calliprinus , respectively. Recovery was highest for the two local species, Q. calliprinus (54%) and Q. boissieri (50%), followed by Q. pubescens (44%) and Q. ilex (41%). Identification of the isolated fungi was conducted using arbitrarily-primed PCR. Identical band patterns were observed among AP-PCR-amplified DNA extracted from an authentic culture of the fungus, ascocarps (truffles) and cultures isolated from roots. In addition, AP-PCR was reliable for differentiating between representative isolates of T. melanosporum, T. magnatum, T. borchii, T. maculatum, T. dryophilum, T. macrosporum and T. uncinatum.

Comparison of Arbitrarily Primed PCR, Antibiotic Resistance and Plasmid Profiling for Differentiating Salmonella enteritidis Isolated from Fish

A total of 28 isolates of Salmonella enteritidis isolated from Tilapia (Tilapia mossambica) were investigated through arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting using three primers, plasmid profiling and their antibiotic resistance patterns. All 28 isolates carried at least one plasmid ranging in size from 1.4 to 38 megadalton that enabled the S. enteritidis to be grouped into nine plasmid profiles. Isolates were resistant to nalidixic acid (21.1%), penicillin (39.3%) and streptomycin (71.4%), and were susceptible to carbenicillin, cephalothin, kanamycin, rifampicin and tetracycline. Seventeen isolates (60.7%) were resistant to a single antibiotic and eleven (39.3%) were resistant to two antibiotics; thus separating the isolates into six antibiotic resistance patterns. The AP-PCR results showed that the collection of isolates were genetically very heterogenous. AP-PCR enab led us to differentiate the isolates into 28 AP-PCR types. Analysis derived from these data in combination (AP-PCR, plasmid profiling and antibiotic resistance patterns) showed that all 28 isolates were distinct and were grouped into 28 individual groups. Our results demonstrate that AP-PCR fingerprinting method is more sensitive than plasmid profiling and antibiotic resistance patterns with respect to the individualization of the isolates used in this study.

Isolation and molecular characterization of Clostridium difficile strains from patients and the hospital environment in Belarus.

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2. 1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogeneous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile-associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.

Extensive gene flow within sibling species in the deep-sea fish Beryx splendens

Molecular markers allow insights into the population biology and ecology of deep-sea organisms, which are usually hardly accessible to direct observation and poorly known. Such a study was undertaken here for the deep-sea fish Beryx splendens, a species of growing interest to fisheries. B. splendens populations were sampled on seamounts and continental margins in the southwestern Pacific (New Caledonia, New Zealand, southeastern Australia) and in the northeastern Atlantic. Two hundred and fifty individuals were characterised by their single-strand DNA conformation (SSCP) of a ∼ 360-base-pair (bp) fragment of the mitochondrial cytochrome b gene, amplified by the polymerase chain reaction (PCR). Two major SSCP haplotypes were observed in New Caledonia, a and w, whose frequencies were negatively correlated along a north-to-south cline. All SSCP haplotypes in the total sample were sequenced on 273 bp. The phylogenetic tree of B. splendens haplotype sequences, rooted by two B. decadactylus sequences, showed that a and w belong to distinct mitochondrial clades, A and W, which are separated by ∼ 4–6 % nucleotide divergence. Thirty individuals from New Caledonia were characterised by their DNA fingerprint from arbitrary-primed PCR. The distribution of individual-pairwise similarity indices was strongly bimodal. The larger similarity values all corresponded to comparisons within a clade (A or W) while the lower values were all between clades. Therefore, there was a strict association between the mitochondrial type and the DNA (presumably, nuclear DNA) fingerprint of an individual. Altogether, these results point to the existence of two biological species (sp. A and sp. W) within the current taxon B. splendens. No within-species differentiation was detected at the regional scale (New Caledonia). A remarkable result is that the three cytochrome b haplotypes of northeastern Atlantic B. cf. splendens sp. A were also the three commonest in the southwestern Pacific populations of this species. Such a level of homogeneity in the distribution of haplotypes suggests there is, or recently has been, gene flow at the inter-oceanic scale.

Cluster analysis of AP-PCR generated DNA fingerprints of Vibrio vulnificus isolates from patients fatally infected after consumption of raw oysters

Arbitrarily-primed-polymerase chain reaction (AP-PCR) DNA fingerprints were generated for 10 Vibrio vulnificus strains isolated from patients who became infected and died between 1993 and 1996 as a result of consuming raw oysters. Analysis of the DNA fingerprints with gel imaging and cluster analysis software revealed significant genetic heterogeneity among these strains, suggesting that V. vulnificus has a high degree of variation in its genomic organization, and that multiple pathogenic strains with greatly diverse genomic arrangements, rather than a single type of infective strain or serogroup, caused these infections.

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction.

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.