Comparison of ribotyping,randomly amplified polymorphic DNA,and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis

Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams. In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD). Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested. The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters. Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6%. Cluster 2 included again only the isolate 0202RD. RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R. decussatus from those isolated from other clam species. Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests. On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint. In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.     

Use of randomly amplified polymorphic DNA analysis to differentiate isolates of Vibrio parahaemolyticus from cockles (Anadara granosa)

A total of 35 Kanagawa-negative strains of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) were investigated by randomly amplified polymorphic DNA fingerprinting with three primers and their plasmid profiles. Eighteen strains carried small plasmid(s) of 2.4 to 7.3kb that enabled the V. parahaemolyticus to be grouped into eight plasmid patterns. The three primers generated polymorphisms in all 35 strains of V. parahaemolyticus tested, producing bands ranging from 0.25 to 3.9kb. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested, and that cockles in the study area are populated by genetically polymorphic strains of V. parahaemolyticus.     

Versatile low-copy-number plasmid vectors for cloning in Escherichia coli

Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques. pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin. pLG338 (7.3 kb) has unique restriction endonuclease sites for BamHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene. pLG339 (6.2 kb) lacks the KpnI site, but has unique SphI and PvuII sites. These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids.     

Stability of related human and chicken Campylobacter jejuni genotypes after passage through chick intestine studied by pulsed-field gel electrophoresis

The genomic stability of 12 Campylobacter jejuni strains consisting of two groups of human and chicken isolates was studied by analysis of their PFGE (pulsed-field gel electrophoresis) patterns after passage through newly hatched chicks' intestines. The patterns of SmaI, SalI, and SacII digests remained stable after intestinal passage, except for those of two strains. One originally human strain, FB 6371, changed its genotype from II/A (SmaI/SacII) to I/B. Another strain, BTI, originally isolated from a chicken, changed its genotype from I/B to a new genotype. The genomic instability of the strains was further confirmed by SalI digestion and ribotyping of the HaeIII digests. In addition, heat-stable serotype 57 of strain FB 6371 changed to serotype 27 in all isolates with new genotypes but remained unchanged in an isolate with the original genotype. Serotype 27 of strain BTI remained stable. Our study suggests that during intestinal colonization, genomic rearrangement, as demonstrated by changed PFGE and ribopatterns, may occur.     

The adaptation and resistance of Clostridium aminophilum F to the butyrivibriocin-like substance of Butyrivibrio fibrisolvens JL5 and monensin

A collection of 45 epidemiologically unrelated Streptococcus agalactiae strains (group B Streptococcus, GBS), belonging to different serotypes, isolated from pregnant women in China and Russia was studied. Strains were characterized by pulsed-field gel electrophoresis (PFGE) employing hybridization with nine genes potentially involved in virulence. Molecular sizes of GBS genomes varied from 2030 to 2290 kb. Location of the genes under study bac, bca, glnA, scpB, cyl, hylB, lmb, scaA and cfb on the GBS genomes was found to be conserved irrelevant to the serotype. Potential virulence genes scpB, hylB, lmb were located on a 91-kb SmaI fragment that is equal to 4.5% of total genome. Ribotyping of the strains under study revealed three different HindIII, nine EcoRI and 12 PvuII ribotypes among 45 strains. A strong correlation between the PvuII ribotype and the presence of the bac gene was observed, with 21 of 22 bac-positive strains belonging to the same PvuII ribotype P1. PFGE patterns of bac-positive strains were also similar. The possibility of close genetic relatedness of all bac-positive strains is discussed.     

Isolation and characteristics of a recombinant pOV13 plasmid as a vector for DNA cloning in a broad range of bacterial hosts

The hybrid plasmid pOV13 proposed as a potential vector for DNA cloning in a broad bacterial host range has been constructed on the basis of the broad host range plasmid RSF1010 and a shortened derivative of RP4, the plasmid pVZ115 serving a marker DNA fragment. The plasmid pOV13 contains the genes for streptomycin, kanamycin and tetracycline resistance and single cleavage sites for restriction endonucleases BamHI, BgIII, SalI, SmaI, PvuII, XhoI, as well as double cleavage sites for restriction endonucleases PstI and HindIII permitting one to clone DNA with insertional inactivation of genes. The physicogenetical map of the birepliconed plasmid pOV13 is presented.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

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.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas.

In the genus Aeromonas there are at least 13 DNA hybridization groups, which are difficult to differentiate biochemically. We investigated the usefulness of rRNA gene restriction patterns for characterization and identification of the various groups. Genomic DNA was digested with restriction endonuclease SmaI, transferred to a nylon membrane, and hybridized with biotinylated plasmid pKK3535 containing the rrnB operon of Escherichia coli. The SmaI bands at 0.8 to 4 kb but not those at positions corresponding to sizes larger than 4 kb showed a good correlation with hybridization groups, allowing identification of strains to the level of genetic species. We demonstrated that the 567-bp fragment localized between positions 80 and 647 of the 16S ribosomal gene of E. coli was essential for hybridization to the low-molecular-weight fragments, whereas the remainder of the operon did not hybridize to these fragments. On the basis of these results, we concluded that the Aeromonas chromosome contains multiple rRNA operons which may be used for species identification.     

Intraspecific characterization of Vibrio alginolyticus isolates recovered from cultured fish in Spain

AIMS: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain. MATERIALS AND RESULTS: Thirty-four Vibrio alginolyticus strains isolated from cultured fish were intraspecifically characterized on the basis of biochemical and exoenzymatic patterns, outer membrane protein (OMP) profiles, ribotyping and plasmid analyses. The typing methods used did not allow to group V. alginolyticus isolates on the basis of their sources of collection. A higher homogeneity was observed in OMP profiles. A high percentage of isolates were plasmidless. Ribotyping was the highest discriminatory typing method, as all the isolates tested presented 23 profiles using the HindIII restriction enzyme. On the basis of the ribotyping pattern, a similarity matrix and a dendrogram were constructed. CONCLUSIONS: The results obtained indicate that V. alginolyticus strains isolated from southwestern Spain belong to different clonal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown differences with other similar studies carried out in other areas of Europe with strains of V. alginolyticus with respect to the clonal lineages of the strains isolated in southwestern Spain.     

Evidence for the role of horizontal transfer in generating pVT1, a large mosaic conjugative plasmid from the clam pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600(T) reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.     

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.     

Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis)

The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no. of strains/no. of ribotypes between 1.1 and 1.5. Cluster analysis of V. splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.     

Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated.     

Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus

Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine.     

Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar.

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.     

Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping, and serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.