Linda Verdonck

Phylogenetic position of phytopathogens within the Enterobacteriaceae

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

TRANSFER OF ERWINIA-ANANAS (SYNONYM, ERWINIA-UREDOVORA) AND ERWINIA-STEWARTII TO THE GENUS PANTOEA EMEND AS PANTOEA-ANANAS (SERRANO 1928) COMB-NOV AND PANTOEA-STEWARTII (SMITH 1898) COMB-NOV, RESPECTIVELY, AND DESCRIPTION OF PANTOEA-STEWARTII SUBSP INDOLOGENES SUBSP NOV.

Eight Erwinia stewartii strains, 11 Erwinia ananas strains, and 7 Erwinia uredovora strains, as well as 9 phenotypically similar Erwinia herbicola strains and Enterobacter agglomerans LMG 5342, were compared by examining electropherograms prepared from their soluble proteins and were grouped into nine protein electrophoretic groups. The levels of DNA relatedness among these electrophoretic groups were determined spectrophotometrically from the renaturation rates at 74°C of the DNAs of 13 selected strains. The representatives from five protein electrophoretic groups, including E. ananas LMG 2665T (T = type strain) and E. uredovora LMG 2667T, exhibited 76 to 100% DNA binding to each other and constituted DNA hybridization group 2665. All E. stewartii strains (including LMG 2715T) were electrophoretically very similar; representatives of this species exhibited 93 to 99% DNA binding to each other and constituted DNA hybridization subgroup 2715. The strains belonging to the remaining three protein electrophoretic groups exhibited 94 to 96% DNA binding to each other and formed DNA hybridization subgroup 2632. The latter two subgroups were 60 to 83% (average, 73%) interrelated and exhibited 30 to 39% DNA binding to group 2665. It is proposed that E. ananas and E. uredovora should be united in a single species, which should be classified in the genus Pantoea as Pantoea ananas (Serrano 1928) comb. nov.; its type strain is strain LMG 2665 (= NCPPB 1846). This species also includes DNA hybridization group VI of Brenner et al. (D. J. Brenner, G. R. Fanning, J. K. Leete Knutson, A. G. Steigerwalt, and M. I. Krichevsky, Int. J. Syst. Bacteriol. 34:45-55, 1984). The transfer of E. stewartii to the genus Pantoea and the creation of two separate subspecies within Pantoea stewartii (Smith 1898) comb. nov. are also proposed. Pantoea stewartii subsp. stewartii (Smith 1898) comb. nov. (synonym, Erwinia stewartii) contains the strains belonging to subgroup 2715, and its type strain is strain LMG 2715 (= NCPPB 2295); and Pantoea stewartii subsp. indologenes subsp. nov. contains the strains belonging to subgroup 2632, and its type strain is strain LMG 2632 (= NCPPB 2280). As determined by principal-component analysis of the cellular fatty acid compositions, P. ananas, P. stewartii subsp. stewartii, and P. stewartii subsp. indologenes are separated from each other mainly by differences in the relative contents of cis-9-hexadecenoic acid (C16:1 cis 9), cyclo-heptadecanoic acid (C17:0 cyclo), and straight-chain octodecenoic acids (C18:1). P. stewartii subsp. stewartii can also be differentiated from P. ananas and P. stewartii subsp. indologenes by its inability to produce indole, to utilize citrate, to grow on cis-aconitate, and to form acid from seven carbohydrates. P. stewartii subsp. indologenes can also be separated from P. ananas by its inability to form acid from sorbitol and α-methyl-D-mannoside. Descriptions of P. ananas and P. stewartii and its two subspecies are given, and the description of the genus Pantoea is emended.

Identification and typing of Vibrio anguillarum - a comparison of different methods.

Summary The majority (91%) of 260 isolates initially identified as Vibrio anguillarum , that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.

Taxonomic evidence that Vibrio carchariae Grimes et al. 1985 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al, 1981.

A collection of 94 Vibrio isolates closely related to Vibrio harveyi, together with named reference and type strains, were investigated for phenotypic and genotypic properties. Using amplified fragment length polymorphism (AFLP), nine clusters were recognized. The largest cluster (n = 36), considered to be the bona fide V. harveyi group, contained the type strains of V. harveyi and Vibrio carchariae and most of the strains isolated from fish. The type strains of all other species, including Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio campbellii and Vibrio natriegens, clustered outside this group. By ribotyping, V. harveyi and V. carchariae patterns were very similar, insofar as they shared most bands. The V. campbellii type strain had several bands in common with the type strains of both V. harveyi and V. carchariae, whereas the other species were clearly distinct from these three species. DNA-DNA hybridization experiments showed 88% DNA binding between the type strains of V. harveyi and V. carchariae, whereas the DNA binding between V. harveyi and V. campbellii was 40%. Although the delineation of the species V. harveyi is still uncertain, the authors propose, on the basis of a number of tests, to delineate a core of V. harveyi strains which contained the type strains of both V. harveyi and V. carchariae. It is concluded that V. carchariae is the junior synonym of V. harveyi.

A comparison of methods for the typing of fish-pathogenic Vibrio spp.

Summary The validity and distinctiveness of Vibrio anguillarum (= Listonella anguillara), V. (= Photobacterium) damsela, V. ordalii and V. salmonicida was confirmed. However, strains received as V. cholerae and V. splendidus were heterogeneous. Ribotyping, phenotypic (BIOLOG-GN fingerprints and API 20E profiles), chemotaxonomic (lipopolysaccharide [LPS] and outer membrane proteins [OMP]), serogrouping and plasmid profiling data were not always congruent. V. anguillarum isolates were recovered in a single ribotype cluster, but many serogroups. There was little variation in OMP profiles, but not so for LPS profiles and plasmid composition. Heterogeneity was recorded in the phenotypic characters, particularly with the API 20E rapid identification system. V. damsela displayed heterogeneity by ribotyping, but homogeneity by BIOLOG-GN fingerprints and API 20E profiles. Four serogroups were defined, but only one LPS profile was recognised. V. ordalii was homogeneous by ribotyping, serogrouping and plasmid profiling, was accommodated in two LPS groups, but was more heterogeneous by BIOLOG-GN and API 20E. Despite its more exacting cultural requirements, V. salmonicida autoagglutinated and could not be serogrouped, but was accommodated in a single LPS group showing a profile associated with rough strains, and contained plasmids of 4.7 and 42 kb. Heterogeneity was recorded with the API 20E rapid identification system.

A Polyphasic Approach to Study the Intraspecif ic Diversity Amongst Vibrio vulnificus Isolates

Summary A polyphasic taxonomic approach using phenotypic and molecular genetic techniques, was carried out on the species Vibrio vulnificus in order to study its intraspecific diversity. Seven techniques, including phenotypic (API 20E, BIOLOG, total protein profiles, serotyping, ELISA), and genotypic methods (ribotyping and AFLP), were employed on 80 V. vulnificus strains of biotypes 1 and 2, including 9 reference cultures. The isolates came from different geographic origins (USA, Spain, Belgium, Sweden, Norway, Japan, Thailand) and types of samples (clinical, health/diseased fish, seafood, water). Diversity indexes calculated for strains of both biotypes revealed a higher phenotypic and genetic diversity of biotype 1 strains in contrast with the homogeneity shown by biotype 2 strains. A species profile was established for the species V. vulnificus by BIOLOG and SDS-PAGE. Serotyping allowed a clear separation within species level, and grouped typical and atypical biotype 2 strains in one serovar. Genetic fingerprinting techniques, especially AFLP, were able to differentiate within strains belonging to the same serovar, allowing the tracking of specific clones in epidemiologic or ecological studies. The techniques employed do not support the maintenance of the two biotypes established for the species.