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Gram-negative bacteria were isolated from knots induced by Pseudomonas savastanoi in olive trees (Olea europaea L.). A total of nine endophytic bacterial strains were isolated, each from inside a different tree knot. Biochemical characterization indicated that all the strains belong to the family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA genes of these novel isolates revealed that they formed a homogeneous cluster within Erwinia species. DNA signatures of these isolates were identical to those described for the genus Erwinia. The strains formed a homogeneous group as shown by DNA–DNA hybridization analysis and numerical analysis of phenotypic data, clearly differentiated from all species of Erwinia with validly published names. The data provide strong evidence of the differentiation of these strains from the most closely related species. Therefore, these isolates represent a novel species, for which the name Erwinia toletana sp. nov. is proposed. The isolates are available at CFBP, CECT and ATCC. The G+C content is 52±0·5 mol%. The type strain is CFBP 6631T (=A37T=ATCC 700880T=CECT 5263T)., This work was supported in part by a grant from the Universidad San Pablo CEU (Facultad de Ciencias Experimentales y de la Salud), Spain and NIH grant AI34829.

Bacterial blight is an emerging disease that affects primarily onion, but also garlic and Welsh onion. The present study was undertaken to characterize the causative xanthomonad(s) by a polyphasic approach using a worldwide collection of 33 bacterial strains. Analysis of 16S rRNA gene sequence similarities indicated that the causal agent belongs to the campestris core in the genus Xanthomonas, which is in agreement with results of phenotypic characterization (analyses of carbon source utilization and fatty acid methyl esters). However, DNA–DNA hybridization, thermal stability of DNA reassociation and fluorescent amplified fragment length polymorphism analysis allowed the causal agent to be identified as a pathovar of Xanthomonas axonopodis.

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.

Ramlibacter gen. nov. is proposed for two aerobic, chemo-organotrophic, cyst-producing soil bacterial strains. These bacteria are Gram-negative, non-flagellated rods or cysts, isolated from subdesert soil in Tataouine, Tunisia. Phylogenetic analyses of the rrs sequences of the two strains showed that they do not constitute a robust clade at the genus level with any previously described bacteria and that they are a deep branch of a clade also grouping the genera Acidovorax and Hydrogenophaga within the beta-Proteobacteria. They belong to two different species, as verified by DNA-DNA hybridization (23.5% reassociation). The type species of the genus is Ramlibacter tataouinensis sp. nov., with the type strain TTB310T (=DSM 14655T =ATCC BAA-407T =LMG 21543T). The second species is Ramlibacter henchirensis sp. nov., with the type strain TMB834T (=DSM 14656T =ATCC BAA-408T =LMG 21542T). The G + C contents of R. tataouinensis and R. henchirensis are 69.6 and 66.6 mol%, respectively.

A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described. This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process. Preliminary results using a Cellulomonas strain cultivated in a liquid medium containing FWA, indicated that this component is non-toxic at a final concentration of 0.01 per thousand (v/v) and that the fluorescence decreased during the first 24 h of incubation, i.e. during exponential growth phase, suggesting an adsorption of FWA on bacterial cells. Consequently, all experiments have been performed with a liquid medium containing FWA (0.01 per thousand v/v) and white paper (8.0 g/l) as cellulose source. Mixed bacterial populations (MBPs) were prepared from refuse samples. These MBPs, which mainly consisted of bacterial rod cells, were used as inocula and fluorescence was measured after 30 h of incubation, i.e. after the stationary phase was reached. A high linear correlation (R(2) = 0.979) was found between the percentages of degraded paper (%P) deduced from residual paper weight and the fluorescence values (F) of the culture medium and the following equation between %P and F was determined: %P = 8.71x10(-5) x F. An additional experiment using a second MBP showed a strong correlation (R(2) = 0.990) between the measured %P and the %P estimated from F values, confirming the reproducibility of the method. Moreover, the time course of paper degradation by five replicate flasks from a unique MBP was set up. Paper degradation was detected 3 to 5 days after the beginning of the stationary phase. The average degradation rate between the 7th and the 11th day of incubation was 11.4% per day. Rates of paper degradation ranged from 31 to 60% after 10 days and from 77 to 88% after 3 weeks of incubation, depending on the inoculum.

Six dairy cows were immunized subcutaneously with purified type 5 capsular polysaccharide (CP5) of Staphylococcus aureus or CP5-ovalbumin conjugate in Freund's incomplete adjuvant. The CP5 antibodies elicited were measured in sera and analysed with regard to isotypes by enzyme-linked immunosorbent assay. At the doses tested, the purified CP5 did not induce a humoral response in the cows. Immunization of two cows with the CP5-ovalbumin conjugate elicited a CP5 antibody response mainly of the IgG2 isotype, which culminated 4 weeks later. A second injection of conjugate, 3 months after the first one, resulted in a rapid and durable anti-CP5 response without exceeding the first antibody peak value. Intramammary infusion of purified CP5 failed to provoke an inflammatory response in the milk of the immunized cows. In contrast, a marked recruitment of cells was recorded in the milk of the sensitized cows after intramammary infusion of ovalbumin. These results demonstrate that injection of CP5-protein carrier conjugate in cows entails both antibody response against CP5 and carrier-specific recruitment of cells in milk of immunized animals.

Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl(+) Plt(+) strains isolated from tobacco, cotton or wheat on different continents. Phl(+) Plt(+) strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the 'P. syringae', 'P. fluorescens' and 'P. chlororaphis' species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl(+) Plt(+) strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl(+) strains, the Phl(+) Plt(+) strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl(+) Plt(+) fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0(T) (=CFBP 6595(T), =DSM 19095(T)) as the type strain.

Given the high level of background flora in sludge, methods for detecting Listeria monocytogenes are not well established. In this study, two critical parameters for the detection of L. monocytogenes were evaluated: the concentration of Listeria sp. in a modified Fraser broth (first stage of the method) and the proportion of L. monocytogenes on Palcam agar (second stage of the method). Concentrations of Listeria sp. estimated in 118 modified Fraser enrichment broths inoculated with four types of sludge, reached 10(4) bacteria per mL for 83% of the positive enrichment broths. Proportion of L. monocytogenes on Palcam agar, which was estimated by transferring all characteristic colonies of Listeria sp. onto Rapid'L Mono agar, was highly variable regardless of the type of sludge. According to these results, we proposed a protocol that consisted of an enrichment in modified Fraser broth for 48 h at 37 degrees C, followed by plating 0.1 mL of appropriate dilutions of broth onto Palcam agar. After an incubation of 48 h at 37 degrees C, a systematic identification of characteristic colonies of Listeria sp. on Rapid'L Mono agar allowed to enhance the detection of Listeria monocytogenes.

In this work, we focused on the molecular characterisation of Spanish Pseudomonas syringae pv. phaseolicola isolates. PCR amplification profiles of repetitive DNA were similar among the Spanish P. s. pv. phaseolicola isolates and the reference P. s. pv. phaseolicola strains, but partially different from the patterns shown by P. s. pv. glycinea. Taking these profiles into consideration, two P s. pv. phaseolicola genomic groups have been distinguished. Additionally, using a DNA heteroduplex mobility assay (HMA) performed on the internal transcribed spacer (ITS) region between 16S and 23S rRNA genes, it was possible to differentiate Group 1 isolates, which clearly present a unique HMA band, from Group 2 isolates, which show multiple HMA bands.

International audience; To re-examine the taxonomic status of Pseudomonas corrugata, 27 strains of this species were studied using a polyphasic approach. Numerical analysis of phenotypic data revealed two phena, A (including the P. corrugata type strain) and B, which could be clearly differentiated by the assimilation of mesotartrate, 2-ketogluconate and histamine. The mean DNA reassociation values with labelled DNA of P. corrugata type strain CFBP 2431T (phenon A) and strain CFBP 5447T (phenon B) were high for strains belonging to the same phenon (96.9 and 98.5%, respectively), whereas the DNA relatedness between the two phena was assessed as being close to 70%, which represents the value that is accepted for the definition of a bacterial species. Phena A and B were also differentiated by means of DNA profiles generated by heteroduplex mobility assay of PCR products of 16S rDNA hypervariable region 2, HaeIII restriction of the amplified internal transcribed spacer, REP- and BOX-PCR profiles, and by PCR with two pairs of specific primers. A comparison of the 16S rRNA sequences of strains CFBP 5447T and CFBP 5458 from phenon B with the available sequences of Pseudomonas species showed that these strains formed a cluster distinct from the P. corrugata type strain. Thus, a new species, Pseudomonas mediterranea, is proposed for strains of phenon B. The type strain is strain CFBP 5447T (= ICMP 14184T); its G+C content is 60.2 mol%

Bacterial strains isolated from diseased erythrina (Erythrina sp.) trees in Martinique (French West Indies) were studied using phenotypic tests, 16S rDNA sequence analysis and DNA-DNA hybridization. Numerical analysis of phenotypic characteristics showed that these strains formed an homogeneous phenon among plant-pathogenic Enterobacteriaceae, and gave useful and updated information for the identification of these bacteria. Results of DNA-DNA hybridization indicated that strains from erythrina belonged to a discrete genomospecies (89-100% hybridization) and had low levels of DNA relatedness (2-33% hybridization) with reference strains of phytopathogenic Erwinia, Brenneria, Pectobacterium, Pantoea and Enterobacter species. 16S rDNA sequence analysis using three different methods revealed that the position of strain CFBP 5236T isolated from erythrina was variable in the different trees, so that strains from erythrina could not be assigned to any recognized genus. It is proposed that these strains are included in a new genus, Samsonia. The name Samsonia erythrinae is proposed for the new species. The G+C content of the DNA of the type strain, CFBP 5236T (= ICMP 13937T), is 57.0 mol%.

International audience; Enumeration and phenotypic characterization of aerobic cellulolytic bacteria were performed on fresh, 1 year old and 5 years old refuse samples of a French landfill site. Numbers of cellulolytic bacteria ranged from 1.1×106 to 2.3×108 c.f.u. (g dry wt.)−1 and were lower in 5 years old refuse samples. A numerical analysis of phenotypic data based on 80 biochemical tests and performed on 321 Gram-positive isolates from refuse, revealed a high phenotypic diversity of cellulolytic bacteria which were distributed into 21 clusters. Based on the phenotypic analysis and the sequencing of 16S rDNA of five representative strains of major clusters, the predominant cellulolytic groups could be assigned to the family of Bacillaceae and to the genera Cellulomonas, Microbacterium and Lactobacillus. Furthermore, chemical parameters such as pH, carbohydrates and volatile solid contents influenced the composition of the cellulolytic bacterial groups which were reduced essentially to the family of Bacillaceae in the oldest refuse samples.

Fluorescent oxidase negative pseudomonads (FONP) include Pseudomonas syringae pathovars and related species (P. amygdali, P. avellanae, P. ficuserectae, P. meliae, P. savastanoi and P. viridiflava). Using DNA-DNA hybridization, 9 genomospecies have been delineated among FONP, two of them being elevated to species level, P. tremae and P. cannabina (2). Analyses of 16S rDNA sequences have shown that Pseudomonas sensu stricto included two intrageneric clusters, designated P. aeruginosa and P. fluorescens. Five lineages were defined in P. fluorescens cluster : P. fluorescens, P. syringae, P. cichorii, P. putida and P. agarici lineages (3). Comparisons of 16S rDNA sequences of pseudomonads revealed three hypervariable (hv) regions : hv1 (positions 71–95), hv2 (positions 455–475) and hv3 (positions 998–1043) (3).

Bacteria isolates phenotypically related to Pseudomonas corrugata have frequently been isolated from the rhizosphere of Arabidopsis thaliana and Brassica napus grown on different soils. 16S rDNA (rrs) gene sequencing, DNA-DNA hybridization, biochemical characterization and siderophore typing showed that these isolates belong to two different species that are distinct from other species of the genus Pseudomonas, including P. corrugata. A description of properties of these two new species is given based on the study of 16 isolates. Proposed names are Pseudomonas brassicacearum (10 strains studied) and Pseudomonas thivervalensis (6 strains studied). The type strain of Pseudomonas brassicacearum is CFBP 11706T and that of Pseudomonas thivervalensis is CFBP 11261T.

Thirty-three fluorescent Pseudomonas strains isolated from tomato pith necrosis (FPTPN strains) and 89 Pseudomonas corrugata strains were studied by numerical taxonomy. In the dendrogram of distances, the P. corrugata strains constituted a single phenon (phenon 1), whereas 17 of the 33 FPTPN strains clustered in a separate phenon (phenon 2). The other 16 FPTPN strains were included in phena consisting of well-characterized fluorescent Pseudomonas species or were isolated phenotypes. Phena 1 and 2 were distinguished by fluorescence on King B medium, accumulation of poly-beta-hydroxybutyrate, production of levan, and assimilation of sorbitol. DNA-DNA hybridization showed that P. corrugata is a true genomic species (66 to 100% DNA relatedness) and that the FPTPN strains of phenon 2 were divided into three genomic groups. Genomic groups 1 and 2 were not distinct from each other phenotypically, and genomic group 3 could be distinguished from genomic groups 1 and 2 only on the basis of assimilation of citraconate and laevulinate. Genomic groups 1 and 2 are related to P. corrugata (40 to 55% DNA relatedness), whereas genomic group 3 is less closely related to P. corrugata (20 to 23% DNA relatedness). The lipopolysaccharide patterns on electrophoresis gels and fatty acid profiles of strains belonging to genomic group 1 through 3 are different from each other and from the lipopolysaccharide patterns and fatty acid profiles of P. corrugata. However, cross-reactions were observed between P. corrugata and the FPTPN strain genomic groups, indicating that there are common epitopes of the lipopolysaccharides. The three FPTPN strain genomic groups were not named as species but were designated Pseudomonas genomospecies FP1, FP2, and FP3.

Types 5 and 8 Straphylococcus aureus capsular polysaccharides are now considered as potential candidates for vaccination against human and animal infections. In order to get sufficient amounts of capsular polysacccharide for this use, we describe a new simple efficient purification method of type 5 capsular polysacccharide. After extraction of capsular polysaccharide from S. aureus Reynolds strain by autoclaving, the successive steps were ultrafiltration with a 30 kDa cutoff hollowfiber cartridge, treatment by sodium metaperiodate, ultrafiltration with a 100 kDa cutoff hollowfiber cartridge and high-performance size exclusion liquid chromatography. Approximatively 85% of the polysaccharide initially extracted was recovered and contaminating components represented 4.2% of the purified fraction.

Assimilation of 99 carbon sources by eighty-eight Pseudomonas savastanoi strains isolated from oleander (41), olive (32), and various other host plants was performed. The results of numerical analysis showed that the majority of the olive and oleander strains were clustered in one phenon. Strains from other hosts such as Myrtus sp., Ligustrum sp., Phillyrea sp. and Retama sphaerocarpa, were grouped into a different phenon together with five oleander strains.

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox+). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox−). These Tox− isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox+ and Tox− isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox+ isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox− isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox− isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox+, but not among Tox− isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox+ and Tox− P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.