Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.     

Induced colistin resistance as an identifying marker for Aeromonas phenospecies groups

AIMS: To investigate the taxonomic interest of colistin resistance as an identifying marker for Aeromonas phenospecies groups. METHODS AND RESULTS: Colistin resistance was investigated in 387 Aeromonas isolates identified at species level using a 14-test format protocol with miniaturized tests combined with determination of urocanic acid utilization whenever necessary. Colistin resistance, determined by the disc diffusion method, was unreliable when compared with minimum inhibitory concentration (MIC) determination. In some strains, the MIC values and resistance rates of colistin could be increased after overnight induction with a 50- microg colistin disc in 20 ml of Mueller-Hinton broth (2.5 mg l(-1)). Colistin-induced resistance level was raised to 85.8% in the Aeromonas hydrophila complex, 2.1% in the A. caviae complex and 2.5% in the A. veronii complex except for A. jandaei (100% colistin resistant). This new marker allowed the identification of 96.2 and 93.6% of Aeromonas isolates to phenospecies and species level, respectively. CONCLUSIONS: Colistin-induced colistin resistance is a new phenotypic marker for Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: With the present protocol, colistin resistance determination may improve the identification of Aeromonas isolates to phenogroup level, when results obtained by conventional biochemical methods are ambiguous.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Ribotyping of Burkholderia mallei isolates

In this study, the subspecies differentiation of 25 isolates of Burkholderia mallei was attempted based on their ribotype polymorphisms. The isolates were from human and equine infections that occurred at various times around the world. DNA samples from each isolate were digested separately with PstI and EcoRI enzymes and probed with an Escherichia coli-derived 18-mer rDNA sequence to identify diagnostic fragments. Seventeen distinct ribotypes were identified from the combined data obtained with the two restriction enzymes. The results demonstrate the general utility of ribotyping for the subspecies identification of B. mallei isolates.     

Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.     

Vibrio anguillarum serogroup O3 and V. anguillarum-like serogroup O3 cross-reactive species--comparison and characterization

Forty-five Vibrio anguillarum-like isolates reacting with V. anguillarum serogroup O3 antiserum were examined in 30 characters to clarify their phenotypical properties, while their genotype was examined by ribotyping. The strains were isolated from diseased and dead fish or from environmental sources such as water, sediment, plankton, and faeces and gills of healthy fish. Phenotypically, the similarity of all the strains was more than 90%. However, significant differences between the fish-associated and environmental strains were detected. Biochemically, deviations were found in the Voges-Proskauer test and lysine decarboxylase reaction. Clustering analysis of the ribotypes showed two distinct clusters with a similarity of only 32%. Two strains representing each of these groups were used in a LD50 study, which showed some difference also in the pathogenicity between environmental and fish strains. It is suggested that the environmental strains belong to another species than V. anguillarum, but serologically cross-reacting with the V. anguillarum serogroup O3. The ribotyping as well as biochemical results indicated that the environmental strains possibly belong to Vibrio aestuarianus. The bona fide V. anguillarum serogroup O3 strains proved to be very homogeneous both phenotypically and genotypically, and the similarity of ribotypes was more than 96%. The V. anguillarum-like, serogroup O3-reactive strains from the environment were more heterogeneous in their biochemical behaviour, and showed an approximately 70% similarity in ribotypes.     

Molecular,serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental,food, and clinical sources in North America and Asia

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.     

Antimicrobial Susceptibility and rRNA Gene Restriction Patterns among Staphylococcus intermedins from Healthy Dogs and from Dogs Suffering from Pyoderma or Otitis Externa

A total of 60 Staphylococcus intermedins strains from dogs were investigated by their sensitivity to various antibiotics (50 strains) and by their rRNA gene restriction patterns (ribotyping) (60 strains). Fifteen isolates were from healthy dogs, 9 with otitis externa, and 36 with pyoderma, including 10 strains from a previous study. Sixty per cent of the 50 strains tested for antibiotic susceptibility demonstrated resistance to penicillin, 24% to spiramycin, 20% to tetracycline, 16% to chloramphenicol, and 2% to fucidic acid. All isolates were susceptible to amoxycillin with clavulanic acid, enrofloxacin, and sulphonamides with trimethoprim. There were no significant differences in antimicrobial susceptibility patterns observed among isolates from pyoderma, otitis externa or healthy dogs. Among the 60 strains studied by ribotyping, 10 different ribotypes were identified: 6 different ribotypes among isolates from otitis externa, 8 among isolates from pyoderma, and 5 among isolates from healthy dogs. One ribotype (profile C) was dominant among the isolates from healthy dogs while another ribotype (profile A) was dominant among strains from dogs suffering from pyoderma. This profile was not demonstrated in any of the strains from healthy dogs. From 5 different dogs suffering from pyoderma, 2 different clones were demonstrated based on their plasmid profile and antibiogram. In these dogs 1 of the clones always belonged to ribotype A. The results concerning strains of S. intermedins isolated from furunculosis suggest the existence of distinct subpopulations with different pathogenicity to dogs.     

Traditional ribotyping shows a higher discrimination than the automated RiboPrinter system in typing Vibrio cholerae O1

Sixteen clinical Vibrio cholerae O1 strains from four different countries were selected for comparison by traditional ribotyping and an automated RiboPrinter system for identification and discrimination purposes. Automated ribotyping, which routinely uses the restriction enzyme EcoRI for typing all bacterial species, produced only five different ribotypes compared with 10 different EcoRI ribotypes obtained by the traditional method. Traditional and automated ribotyping using the restriction enzyme BglI, which is recommended for the ribotyping of V. cholerae, produced 10 and seven different ribotypes, respectively. The lower discrimination shown by the RiboPrinter system was caused mainly by an inability to differentiate closely located fragments due to a lower resolution and electrophoresis conditions, a parameter which cannot be changed in the automated system. The RiboPrinter system includes a database for bacterial identification. However, none of the V. cholerae O1 strains studied showed EcoRI ribotype patterns which matched any of the patterns included in the database. In conclusion, the existing RiboPrinter system is not adequate for taxonomic identification and classification of V. cholerae O1.     

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.     

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic

Aims:  Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods.
Methods and Results:  Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia , whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia . Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582^T and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same Eco RI ribotype profile and proved to be identical strains.
Conclusions:  Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification.
Significance and Impact of the Study:  To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.     

Molecular, Serological, and Virulence Characteristics of Vibrio parahaemolyticus Isolated from Environmental, Food, and Clinical Sources in North America and Asia

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.     

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus.

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

Ribotyping and biotyping of<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolated from hospital environment

The Staphylococcus strains acquired from scrapings from hospital environments were identified to the species level based on their biochemical properties. From the monitored sample the Staphylococcus epidermidis strains were selected for more accurate typing and tested on their virulence factor and ribotyped. The biotyping of S. epidermidis did not show any considerable intraspecific variation of these isolates and there were no atypical reactions, with the exception of three strains (out of 33). In contrast, the results of ribotyping showed greater heterogeneity of strains and unequivocally demonstrated the relation between the ribotype and the place of sample drawing. In addition to this fact, the found ribotypes repeat in the same environment in the long-term which suggests the occurrence and persistence of the same strains of conditionally pathogenic bacteria in hospital environment. We showed that ribotyping is a suitable method for precise and reliable detection of some coagulase-negative staphylococci.     

Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among Pseudomonas spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as P. fluorescens and P. putida by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.     

Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea

AIMS: To characterize 46 isolates of different Aeromonas spp. strains (26 Aeromonas hydrophila, 13 Aeromonas sobria and 7 Aeromonas salmonicida) isolated from coastal water and clinical sources in Sardinia, Italy. METHODS AND RESULTS: The isolates were analysed for the production of the following virulence properties: slime, haemolysin, gelatinase and protease production, and adhesion to eucaryotic epithelial cells. The presence of known virulence genes: A. hydrophila cytolytic enterotoxin gene AHCYTOEN; type IV pilus gene Tap; Bundle forming pilus genes BfpA and BfpG were investigated using polymerase chain reaction (PCR). In addition the enterobacterial repetitive intergenic consensus sequences (ERIC)-PCR fingerprinting was used to further differentiate the strains. CONCLUSIONS: This study confirms the presence of virulent Aeromonas strains in the Mediterranean sea. The study also found a greater prevalence of haemolysin, protease and gelatinase production, as well as a higher adhesion capacity, among strains isolated from patients with diarrhoea. SIGNIFICANCE AND IMAPCT OF THE STUDY: This is the first time that Aeromonads have been isolated and characterized from Sardinian waters and from patients with diarrhoea in Sardinia. This study adds to our knowledge of the ecology of this micro-organism and may in the future help prevent infections both in fish and in humans.     

Monitoring temporal and spatial variation in rhizosphere bacterial population diversity: A community approach for the improved selection of rhizosphere competent bacteria

The potential for developing a reliable strategy for selecting rhizosphere competent bacteria, based on an improved understanding of the community diversity and population dynamics of fluorescent pseudomonads, was investigated. Isolates from a collection of over 690 fluorescent pseudomonads, obtained from sugar beet and wheat plants grown in field soils in laboratory microcosms, were genotypically and phenotypically characterised. RFLP rRNA analysis (ribotyping) revealed that the sampled population was composed of 385 related but distinct ribotypes. Most ribotypes were isolated only once and represented a transient colonising population. However, representatives of 26 ribotypes were detected more often, of which five were isolated from rhizosphere soils sampled 7 months after the first sampling. Comparative phenotypic analysis of isolates (motility, antibiotic resistance and production, adherence, fatty acid composition, substrate utilisation patterns) demonstrated that the ability to utilise organic acids as carbon sources correlated with rhizosphere competence. Single inoculum and competitive colonisation studies in planted microcosms confirmed rhizosphere competence, but also demonstrated synergistic interactions. The colonisation ability and population densities of transient strains were significantly increased when co-inoculated with rhizosphere competent isolates. These data demonstrate potential cross-feeding and combined niche exploitation, rather than direct competition, confirming the multi-factorial nature of rhizosphere competence in diverse fluorescent pseudomonad communities. They also highlight the need to consider the use of mixed inocula for plant growth promotion and the systematic selection of strains for effective biotechnological exploitation.     

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.     

High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.     

Production of cytolethal distending toxin and other virulence characteristics of Escherichia coli strains of serogroup O86

Genetic and phenotypic virulence markers of different categories of diarrhoeagenic Escherichia coli were investigated in 106 strains of enteropathogenic E. coli (EPEC) serogroup O86. The most frequent serotype found was O86:H34 (86%). Strains of this serotype and the non motile ones behaved as EPEC i.e., carried eae, bfpA and EAF DNA sequences and presented localised adherence to HeLa cells. Serotypes O86:H2, O86:H6, O86:H10, O86:H18, O86:H27 and O86:H non determined, belonged to other categories. The majority of the strains of serotype O86:H34 and non motile strains produced cytolethal-distending toxin (CDT). The ribotyping analysis showed a correlation among ribotypes, virulence markers and serotypes, thus suggesting that CDT production might be a property associated with a universal clone represented by the O86:H34 serotype.