Acidovorax anthurii sp. nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium.

The bacterial leaf-spot of anthurium emerged during the 1980s, in the French West Indies and Trinidad. This new bacterial disease is presently wide spread and constitutes a serious limiting factor for commercial anthurium production. Twenty-nine strains isolated from leaf-spots of naturally infected anthurium were characterized and compared with reference strains belonging to the Comamonadaceae family, the genera Ralstonia and Burkholderia, and representative fluorescent pseudomonads. From artificial inoculations 25 out of 29 strains were pathogenic on anthurium. Biochemical and physiological tests, fatty acid analysis, DNA-DNA hybridization, 16S rRNA gene sequence analysis, DNA-16S RNA hybridization were performed. The 25 pathogenic strains on anthurium were clustered in one phenon closely related to phytopathogenic strains of the genus Acidovorax. Anthurium strains were 79-99% (deltaTm range 0.2-1.6) related to the strain CFBP 3232 and constituted a discrete DNA homology group indicating that they belong to the same species. DNA-rRNA hybridization, 16S rRNA sequence and fatty acid analysis confirmed that this new species belongs to the beta-subclass of Proteobacteria and to rRNA superfamily III, to the family of Comamonadaceae and to the genus Acidovorax. The name Acidovorax anthurii is proposed for this new phytopathogenic bacterium. The type strain has been deposited in the Collection Française des Bactéries Phytopathogènes as CFBP 3232T.

Acidovorax anthurii sp. nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium

The bacterial leaf-spot of anthurium emerged during the 1980s, in the French West Indies and Trinidad. This new bacterial disease is presently wide spread and constitutes a serious limiting factor for commercial anthurium production. Twenty-nine strains isolated from leaf-spots of naturally infected anthurium were characterized and compared with reference strains belonging to the Comamonadaceae family, the genera Ralstonia and Burkholderia, and representative fluorescent pseudomonads. From artificial inoculations 25 out of 29 strains were pathogenic on anthurium. Biochemical and physiological tests, fatty acid analysis, DNA-DNA hybridization, 16S rRNA gene sequence analysis, DNA-16S RNA hybridization were performed. The 25 pathogenic strains on anthurium were clustered in one phenon closely related to phytopathogenic strains of the genus Acidovorax. Anthurium strains were 79-99% (deltaTm range 0.2-1.6) related to the strain CFBP 3232 and constituted a discrete DNA homology group indicating that they belong to the same species. DNA-rRNA hybridization, 16S rRNA sequence and fatty acid analysis confirmed that this new species belongs to the beta-subclass of Proteobacteria and to rRNA superfamily III, to the family of Comamonadaceae and to the genus Acidovorax. The name Acidovorax anthurii is proposed for this new phytopathogenic bacterium. The type strain has been deposited in the Collection Fran‡aise des Bact‚ries PhytopathogŠnes as CFBP 3232T.

Whipple's syndrome (uveitis, B27-negative spondylarthropathy, meningitis, and lymphadenopathy) associated with Arthrobacter sp. infection.

OBJECTIVE: To report an unusual case of Whipple's disease, including uveitis, seronegative spondylarthropathy, meningitis, and lymphadenopathy, associated with an Arthrobacter sp. infection. DESIGN: Interventional case report. PATIENT AND INTERVENTION: A 60-year-old white man presenting with severe chronic uveitis and systemic inflammatory manifestations was treated efficiently for Whipple's disease after histopathologic analysis of vitreous and inguinal adenopathy biopsy specimens. The authors performed a retrospective, laboratory-based evaluation of stored tissue specimens. MEASUREMENTS: Molecular analysis based on 16S ribosomal RNA gene amplification was applied to pretreatment biopsy specimens of inguinal lymph node to identify a causative bacterial agent. RESULTS: Tropheryma whippelii genome was not detected in these specimens. However, an amplification product was obtained after the first polymerase chain reaction run and subsequently was sequenced. It corresponded to an Arthrobacter sp., a gram-positive agent presenting diagnostic patterns and therapeutic management similar to those of Whipple's disease caused by T. whippelii. CONCLUSION: The absence of T. whippelii identification by molecular amplification during a clinically and histologically oriented Whipple's syndrome should not rule out the diagnosis. Arthrobacter infection may represent a new bacterial etiology of systemic inflammatory disorders involving the eye and associated with periodic acid-Schiff-positive inclusions.

Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.

Salmonellae often have the ability to express two different flagellar antigen specificities (phase 1 and phase 2). At the cell level, only one flagellar phase is expressed at a time. Two genes, fliC, encoding phase-1 flagellin, and fljB, encoding phase-2 flagellin, are alternatively expressed. Flagellin genes from 264 serovars of Salmonella enterica were amplified by two phase-specific PCR systems. Amplification products were subjected to restriction fragment length polymorphism (RFLP) analysis by using endonucleases HhaI and HphI. RFLP with HhaI and HphI yielded 64 and 42 different restriction profiles, respectively, among 329 flagellin genes coding for 26 antigens. The phase-1 gene showed 46 patterns with HhaI and 30 patterns with HphI. The phase-2 gene showed 23 patterns with HhaI and 17 patterns with HphI. When the data from both enzymes were combined, 116 patterns were obtained: 74 for fliC, 47 for fljB, and 5 shared by both genes. Of these combined patterns, 80% were specifically associated with one flagellar antigen and 20% were associated with more than one antigen. Each flagellar antigen was divided into 2 to 18 different combined patterns. In the sample of strains used, determination of the phase-1 and phase-2 flagellin gene RFLP, added to the knowledge of the O antigen, allowed identification of all diphasic serovars. Overall, the diversity uncovered by flagellin gene RFLP did not precisely match that evidenced by flagellar agglutination.

Isolation from the rumen of a new acetogenic bacterium phylogenetically closely related to Clostridium difficile.

Five strains of filamentous acetogenic bacterium were isolated from high dilutions of ruminal content of newborn lambs. These Gram-positive spore-forming bacteria grew either chemolithotrophically with H2+ CO2 or chemo-organotrophically with glucose, cellobiose, fructose, maltose, mannose and syringic acid. The DNA base composition of the five strains were between 29.1 and 31.3 mol% G + C. Their temperature and pH optimum for growth were 35-40 degrees C and 6.5-7.0, respectively. The full 16S rRNA gene sequence analysis of the reference strain indicated that it was most closely related to Clostridium difficile. The sequence similarity value between the 16S rRNA gene of the reference strain and this pathogenic strain was 99.7%.

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy.

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.

Strategy for detection and identification of bacteria based on 16S rRNA genes in suspected cases of Whipple's disease.

The 16S ribosomal RNA (rRNA) gene of the phylogenetic subdivision containing gram-positive bacteria with a high G + C content was detected specifically in clinical specimens from patients suspected of having Whipple's disease. The primary structure of 16S rDNA amplified from clinical samples was determined by cloning and sequencing. Two sorts of sequences were identified: one corresponded exactly to the rRNA sequence of Tropheryma whippelii (GenBank accession no. M87484) while the other was related to that of members of the genus Corynebacterium. No sequence related to Mycobacterium spp. or Rhodococcus equi was observed. Exhaustive examination of negative specimens with broad-range eubacterial primers detected one sequence related to Enterobacteriaceae and another related to Enterococcus spp. To speed identification of T. whippelii, a nested amplification method was devised. A first amplification specific for the gram-positive bacteria subdivision was performed, followed by a second amplification with T. whippelii-specific primers. The amplified T. whippelii product was checked by digestion with AvaII, StuI, and PstI endonucleases. These techniques were applied to DNA extracted from seven intestinal biopsy samples, two cerebrospinal fluid samples and one articular fluid from patients suspected of having Whipple's disease. T. whippelii 16S rDNA was found in two of the biopsy samples, one of the cerebrospinal fluid samples and in the articular fluid.

Acetogenic coccoid spore-forming bacteria isolated from the rumen.

Thirteen strains of a new acetogenic bacterium were isolated from the rumen contents of lambs, llamas and bisons. This paper is the first report of Gram-positive coccoid spore-forming bacteria occurring in chains and able to use H2 + CO2 as energy source and produce acetate from this gas mixture. One of them, chosen as the reference strain for its efficiency in utilizing H2/CO2 likely via the acetyl-CoA pathway, was characterized in detail. The G + C ratio of the DNA of the organism was 46.5 mol%. The temperature and pH optimum were 37 degrees-40 degrees C and 6.3-6.8, respectively. Numerous organic substrates including some o-methylate aromatic compounds were used heterotrophically. The full 16S rRNA gene sequence was determined. The phylogeny, physiology, morphology and numerous features described here are sufficiently different from those of any bacteria described today to justify the definition of a new species. The name "New acetogenic bacterium" is temporarily proposed, awaiting a future taxonomic revision of the genus Clostridium.

Identification of Bartonella henselae and B. quintana 16s rDNA sequences by branch-, genus- and species-specific amplification.

Given the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B. quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16S rRNA from the Proteobacteria alpha subgroup was developed. The primary structure of amplified 16S rDNA was determined by cloning and sequencing. Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B. henselae); the second was related to, but distinct from, GenBank accession number Z11684 (referred to as 'B. henselae variant'); and a third sequence was identical with GenBank accession number M73228 (B. quintana). No sequence corresponding to Afipia spp. was found. To speed identification and reduce the cost of analysis, a nested amplification method for B. henselae and B. quintana was devised. These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis. B. henselae or B. henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease. B. quintana was not associated with cat scratch disease, but a B. quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients. B. henselae 16S rDNA sequences were not found in bacillary angiomatosis specimens.

Bacillary angiomatosis in HIV-infected patients: report of three cases with different clinical courses and identification of Rochalimaea quintana as the aetiological agent.

Three cases of cutaneous bacillary angiomatosis in HIV-infected patients are reported. They differed profoundly with respect to the extent of the lesions and the clinical course. In two cases, Rochalimaea quintana was identified by direct sequencing of the DNA amplified with the polymerase chain reaction (PCR), whereas an easy, rapid method based on the restriction length of polymorphism analysis of PCR products (PCR-RFLP) was used in the third case. This report illustrates the variations in clinical presentations and evolutive profiles in patients with bacillary angiomatosis, and confirms the causal role of R. quintana in this disease.

Balneatrix alpica gen. nov., sp. nov., a bacterium associated with pneumonia and meningitis in a spa therapy center.

In 1987, an outbreak of pneumonia and meningitis caused by an unknown bacterium occurred in a spa therapy centre. Nine isolates of this pathogen constituted a tight DNA hybridization group. rRNA-DNA hybridization and 16S rRNA sequencing showed that the studied bacteria represented a new branch in superfamily II (= gamma subclass) of the Proteobacteria, close to the genus Oceanospirillum. The new bacterium was highly polymorphic and, in young cultures, had curved Gram-negative cells, motile by polar single flagella. The new bacterium differed from the genus Oceanospirillum by its lacking the NaCl requirement and by reducing nitrate into nitrite, producing indole from tryptophan and producing acid from carbohydrates. The name Balneatrix alpica gen. nov., sp. nov. is proposed for the studied organism. The type strain is strain 4-87 (= CIP 103589).

Desulfovibrio longus sp. nov., a sulfate-reducing bacterium isolated from an oil-producing well.

A novel type of sulfate-reducing bacteria with unusual morphology was isolated from an oil-producing well in the Paris Basin. The cells of this bacterium, strain SEBR 2582T (T = type strain), are long, thin, flexible rods, contain desulfoviridin, and are physiologically similar to members of the genus Desulfovibrio. On the basis of 16S rRNA sequence data, this strain should be included in the genus Desulfovibrio. However, strain SEBR 2582T differs from other members of this genus morphologically, physiologically, and phylogenetically. Thus, a new species, Desulfovibrio longus sp. nov., is proposed for this organism.

Nucleotide sequences of 16S rRNA from ten Serratia species.

Comparison of 16S ribosomal ribonucleic acid (rRNA) sequences has emerged as a powerful tool for bacterial phylogeny. However, earlier studies often only included one or a few species per genus, and it is not sure whether the rRNA sequences could discriminate closely related species. The genus Serratia is composed of ten species, some being up to 60% related by DNA hybridization. The reverse transcriptase/primer extension method was used to determine 1,492 to 1,509 nucleotides in each of ten Serratia 16S rRNA sequences. All rRNA sequences determined were unique. The phylogenetic tree obtained with the neighbour-joining method showed a cluster of Serratia species distinct from both Escherichia coli and Proteus vulgaris. S. fonticola--whose position in the genus Serratia is questioned--was clearly included in the Serratia branch and grouped within the psychrophilic Serratia species. Variable regions in the Serratia rRNA molecules were identified and could serve as the basis for a specific probe design.