Development of a protocol for the isolation of Listeria monocytogenes from sludge.

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.

Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin.

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.

Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster.

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.

Phenotypic and genomic evidence for the revision of Pseudomonas corrugata and proposal of Pseudomonas mediterranea sp. nov.

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%.

Ramlibacter tataouinensis gen. nov., sp. nov., and Ramlibacter henchirensis sp. nov., cyst-producing bacteria isolated from subdesert soil in Tunisia.

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.

Erwinia toletana sp. nov., associated with Pseudomonas savastanoi-induced tree knots.

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 6631(T) (=A37(T)=ATCC 700880(T)=CECT 5263(T)).