A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern different coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, 'V. anguillarum biovar II', V. diazotrophicus, and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis, and Aeromonas spp.     

A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern difference coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, ' V. anguillarum biovar II', V. diazotrophicus , and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis , and Aeromonas spp.     

Vibrio navarrensis sp. nov., a species from sewage.

A group of 11 strains, mostly isolated from sewage water in the Province of Navarra, Spain, were found to constitute a DNA relatedness group which is 2 to 39% related to 23 species of the genus Vibrio and 2 to 3% related to two Aeromonas species. Phenotypically, these strains have all of the properties that define the genus Vibrio. However, they differ from the previously described species by three or more properties. The strains are negative for arginine, ornithine, and lysine decarboxylase activities and the Voges-Proskauer test and are unable to utilize putrescine, gluconate, glucuronate, and histidine. They utilize and produce acid from sucrose and grow at 40 degrees C. All strains grow in the presence of 0.5% (wt/vol) NaCl, and seven strains grow weakly in peptone water lacking NaCl. The group of strains which we studied can also be differentiated from other Vibrio species by fatty acid content. The G+C ratio of the DNA is 45 to 47 mol%. The name Vibrio navarrensis sp. nov. is proposed for these strains; strain 1397-6 (= CIP 103381) is the type strain.     

Biochemical characteristics and virulence of environmental group F bacteria isolated in the United States.

Bacteria phenotypically resembling Aeromonas hydrophila, but requiring NaCl for growth, have been isolated form the New York Bight. The bacteria proved to be identical to group F organisms isolated from cases of human diarrhea in Indonesia and Bangladesh. Anaerogenic strains initiated responses in Y-1 tissue culture and rabbit ileal loop, consistent with those associated with cytotoxin- and enterotoxin-producing Aeromonas spp. strains. Separation on the basis of production of gas from glucose by group F strains was correlated with differences in mean guanine-plus-cytosine deoxyribonucleic acid base composition and in deoxyribonucleic acid relative reassociation. Both aerogenic and anaerogenic strains reassociated to a significantly greater extent with Vibrio spp. than with Aeromonas spp. and indeed should be considered a new species of the genus Vibrio.     

Numerical analysis of electrophoretic protein patterns of 'Achromobacter' group B, E and F strains from human blood

Thirty-two clinical strains representing 'Achromobacter' groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins. All the strains were isolated from blood samples from hospital patients in the United Kingdom. The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands. The 32 'Achromobacter' strains formed two clusters at the 77% S level. The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F. The strains in each phenon were characterized by a clearly distinct pattern of protein bands. Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD. Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria. Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies.     

A strain of Aeromonas veronii isolated from a patient suspected of cholera]

Investigations were carried on a strain isolated from patient hospitalized in Poland. The patient returned from Odessa, a region with cholera epidemic. The strain was suspected to be Vibrio cholerae, but it did not agglutinate in V. cholerae 01 serum. The aim of this study was establishment of taxonomical classification of this strain, in order to demonstrate whether it is a representative of the non-cholera vibrio group (not agglutinating) or another representative of the family Vibrionaceae. The strain was resistant to bile salts in TCBS medium and demonstrated several properties from a borderline of two Vibrio and Aeromonas species. Its properties were similar to the group of recently described pathogenic vibrio, but it did not require higher concentrations of NaCl in the medium and was resistant to the vibriostatic factor 0/129. These properties were considered as decisive in differentiation of Vibrio and Aeromonas. The strain was finally classified as Aeromonas veronii. It is a first case in Poland of isolation of A. veronii from the feces of patient with cholera-like symptoms. As newly described pathogenic species of genus Vibrio and Aeromonas were not isolated in Poland, we present schemes which may serve for beginning of this type of diagnostic investigations.     

Numerical analysis of electrophoretic protein patterns of 'Achromobacter'group B, E and F strains from human blood

Thirty-two clinical strains representing ' Achromobacter 'groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins. All the strains were isolated from blood samples from hospital patients in the United Kingdom. The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands. The 32 ' Achromobacter ' strains formed two clusters at the 77% S level. The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F. The strains in each phenon were characterized by a clearly distinct pattern of protein bands. Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD. Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria. Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies.     

A chemotaxonomic study of some moderately halophilic Gram-positive isolates

A chemotaxonomic study was carried out on some moderately halophilic Gram-positive motile cocci, previously isolated from the Salar de Atacama (Chile) and grouped in two phenons (A and B) by numerical taxonomic analysis. Strains included in phenon A had a DNA base composition ranging between 42.0 and 44.0 mol%, while that of phenon B ranged from 48.0 to 48.8 mol%. The results of DNA-DNA hybridization studies on representative strains from phenons A and B, indicated that the strains assigned to phenon A comprise a genomically homogeneous group, with a high degree of homology (80%) to the type strain of Marinococcus albus. Similarly, phenon B constituted a homogeneous group and the representative strain studied showed a DNA-DNA homology of 70% with the type strain of Marinococcus halophilus. Representative strains studied from each phenon had meso-diaminopimelic acid in the cell wall and menaquinone systems with seven isoprene units (MK-7) as a major component. All these results, together with those previously reported, indicate that strains included in phenons A and B constitute additional strains of the species Marinococcus albus and Marinococcus halophilus , respectively.     

Numerical analysis of electrophoretic protein patterns of Campylobacter laridis and allied thermophilic campylobacters from the natural environment

Twenty-one strains comprising Campylobacter laridis (nine), nalidixic acid sensitive campylobacters (NASC) (four), and urease-positive thermophilic campylobacters (UPTC) (eight) were characterized by one-dimensional SDS-PAGE of cellular proteins. The UPTC and NASC strains included six from river water, two from mussels and four from sea water. The type strains of three other Campylobacter species were included for reference. The protein patterns, which contained 45–50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 21 strains formed nine clusters at the 80% similarity (S) level. The typical C. laridis strains were restricted to two phenons (2 and 5); the atypical strains being distributed among the remaining phenons. In the second analysis, which excluded the principal protein bands (40–48.5 kD range), the 21 strains formed five clusters at the 80% S level. The typical C. laridis strains were relatively homogeneous and fell into a single phenon (2) within which two subgroups were discernable. The atypical strains were more heterogeneous with respect to background protein pattern, with representatives appearing in all five phenons. An electropherotyping scheme comprising six electropherotypes, and based on both analyses is proposed. The high within-group S level and separation from reference strains of Campylobacter in the second analysis, suggested that UPTC and NASC strains belonged within C. laridis possibly as biovars.     

Monoclonal antibodies for a comparative serological study of strains of Vibrio anguillarum

Murine monoclonal antibodies against characteristic determinants of heat stable somatic antigens of typical serovar I (820/15/8 Kop) and serovar II (134/82/1 Kiel) of European Vibrio anguillarum strains were produced. Three stable hybridoma cell lines, called aVaI/1F4, aVaI/6F4 and aVaI/2H5, produced monoclonal antibodies each against a typical serovar I determinant of strain 820/15/8 Kop. One hybridoma cell line (aVaII/5A4) producing monoclonal antibodies against Vibrio anguillarum strain 134/82/1 Kiel (serovar. II) was established. Fourteen Vibrio strains and Aeromonas salmoniáda strains were serologically compared by Enzyme-Linked Immunosorbent Assay (Elisa ).     

Numerical analysis of electrophoretic protein patterns of Campylobacter laridis and allied thermophilic campylobacters from the natural environment

Twenty-one strains comprising Campylobacter laridis (nine), nalidixic acid sensitive campylobacters (NASC) (four), and urease-positive thermophilic campylobacters (UPTC) (eight) were characterized by one-dimensional SDS-PAGE of cellular proteins. The UPTC and NASC strains included six from river water, two from mussels and four from sea water. The type strains of three other Campylobacter species were included for reference. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 21 strains formed nine clusters at the 80% similarity (S) level. The typical C. laridis strains were restricted to two phenons (2 and 5); the atypical strains being distributed among the remaining phenons. In the second analysis, which excluded the principal protein bands (40-48.5 kD range), the 21 strains formed five clusters at the 80% S level. The typical C. laridis strains were relatively homogeneous and fell into a single phenon (2) within which two subgroups were discernable. The atypical strains were more heterogeneous with respect to background protein pattern, with representatives appearing in all five phenons. An electropherotyping scheme comprising six electropherotypes, and based on both analyses is proposed. The high within-group S level and separation from reference strains of Campylobacter in the second analysis, suggested that UPTC and NASC strains belonged within C. laridis possibly as biovars.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and IB comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and 1B comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.     

Characterization of plasmid-borne and chromosome-encoded traits of Agrobacterium biovar 1, 2, and 3 strains from France

We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general. Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine.     

An Electrophoretic Analysis of Variation in the Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio fluvialis

A total of 59 isolates of Vibrio cholerae, V. parahaemolyticus and V. fluvialis were studied using the techniques of enzyme electrophoresis. The enzymes used were malate dehydrogenase (E.C.I. 1.1.37) and glucose-6-phospate dehydrogenase (E.C.I. 1.1.49). The results in general confirmed classical methods of vibrio taxonomy. The three species could be separated from each other and identified by their enzyme variant types.
The term zymovar, a group of strains having similar enzyme variants, is introduced. In the V. cholerae isolates six zymovars were found. All V. cholerae serovar 01 strains were classified in the same zymovar except for some strains of environmental origin, which occurred in another zymovar. In V. fluvialis two zymovars were detected corresponding with biovars 1 and 2 of this organism. All isolates of V. parahaemolyticus gave the same enzyme type.
The technique of enzyme electrophoresis appears to be a useful tool in vibrio taxonomy and used in conjunction with other methods may aid in the elucidation of the systematics of the genus.     

Typing of Aeromonas strains from patients with diarrhoea and from drinking water.

Aeromonas strains (187) from human diarrhoeal stools and from drinking water (263) in The Netherlands were typed by three different methods. Biotyping alone was found to be of little value for epidemiological studies because 84% of all strains belonged to only 10 biotypes. Common biotypes could be further differentiated by serotyping. Gas-liquid chromatography of cell wall fatty acid methyl esters (FAME) was useful for species identification as well as for typing: 86% of all strains could be identified to the species level, and within this group 92% of all identifications corresponded with the biotype. Cluster analysis and principal component analysis of FAME profiles could be used for comparison of strains from different sources and gave the same general conclusions as bio- and serotyping. There was little overall similarity between Aeromonas strains from human (diarrhoeal) faeces and from drinking water, differences being most pronounced for Aeromonas caviae and least for A. sobria.     

Typing of Aeromonas strains from patients with diarrhoea and from drinking water

A.H. HAVELAAR, F.M. SCHETS, A. VAN SILFHOUT, W.H. JANSEN, G. WIETEN & D. VANDER KOOIJ. 1992. Aeromonas strains (187) from human diarrhoeal stools and from drinking water (263) in The Netherlands were typed by three different methods. Biotyping alone was found to be of little value for epidemiological studies because 84% of all strains belonged to only 10 biotypes. Common biotypes could be further differentiated by serotyping. Gas-liquid chromatography of cell wall fatty acid methyl esters (FAME) was useful for species identification as well as for typing: 86% of all strains could be identified to the species level, and within this group 92% of all identifications corresponded with the biotype. Cluster analysis and principal component analysis of FAME profiles could be used for comparison of strains from different sources and gave the same general conclusions as bio- and serotyping. There was little overall similarity between Aeromonas strains from human (diarrhoeal) faeces and from drinking water, differences being most pronounced for Aeromonas caviae and least for A. sobria.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.