Investigations on the clonality of isolates of Pasteurella gallinarum obtained from turkeys in Germany.

Pasteurella gallinarum has been considered an opportunistic pathogen rather than a primary pathogen for chickens. As P. gallinarum has been found to have a high genotypic diversity, one would expect a polyclonal distribution among isolates from different farms if this organism is a secondary invader. The aims of this investigation were to genetically characterize isolates obtained from outbreaks affecting several turkey farms to confirm the existence of the infection in turkeys and to investigate the genetic relationship between isolates from affected farms. A total of 17 isolates from 14 outbreaks of respiratory disease in Germany were subjected to extended phenotypic and genotypic characterization. All isolates were of the same phenotype, typical of P. gallinarum. Ribotyping of three isolates using either HpaII or HindIII showed that they had identical profiles and indicated that the isolates all originated from the same clone. Comparison with HpaII ribotypes from a previous study showed that the pattern was identical to that obtained with isolates from a Zimbabwean outbreak in chickens during 1999 to 2000. Restriction endonuclease analysis typing of 14 isolates from all 14 farms showed that they had identical profiles but these differed from those obtained with isolates from the Zimbabwean outbreak. Sequencing of the 16S rRNA gene and sequence comparisons with other Pasteurellaceae confirmed their classification as P. gallinarum. Identification of the same clone of P. gallinarum from 14 outbreaks of acute respiratory disease in turkeys within a time period of 2 months suggests a common source of infection, and that P. gallinarum probably played a primary role rather than a secondary role in the outbreaks.     

Analysis of secA1 gene sequences for identification of Nocardia species

Molecular methodologies, especially 16S rRNA gene sequence analysis, have allowed the recognition of many new species of Nocardia and to date have been the most precise methods for identifying isolates reliably to the species level. We describe here a novel method for identifying Nocardia isolates by using sequence analysis of a portion of the secA1 gene. A region of the secA1 gene of 30 type or reference strains of Nocardia species was amplified using secA1-specific primers. Sequence analysis of 468 bp allowed clear differentiation of all species, with a range of interspecies similarity of 85.0% to 98.7%. Corresponding 16S rRNA gene sequences of a 1,285-bp region for the same isolates showed a range of interspecies similarity of 94.4 to 99.8%. In addition to the type and reference strains, a 468-bp fragment of the secA1 gene was sequenced from 40 clinical isolates of 12 Nocardia species previously identified by 16S rRNA gene sequence analysis. The secA1 gene sequences of most isolates showed >99.0% similarity to the secA1 sequences of the type or reference strain to which their identification corresponded, with a range of 95.3 to 100%. Comparison of the deduced 156 amino acid sequences of the SecA1 proteins of the clinical isolates showed between zero and two amino acid residue differences compared to that of the corresponding type or reference strain. Sequencing of the secA1 gene, and using deduced amino acid sequences of the SecA1 protein, may provide a more discriminative and precise method for the identification of Nocardia isolates than 16S rRNA gene sequencing.     

Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene

The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodA(int)) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodA(int) sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodA(int) sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodA(int) sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (> 98% sodA(int) sequence identity) but shared only 92% sodA(int) identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.     

Phenotypic and genetic characterization of clinical isolates of CDC coryneform group A-3: proposal of a new species of Cellulomonas, Cellulomonas denverensis sp. nov

CDC coryneform group A-3 bacteria are rare human pathogens. In this study, six group A-3 isolates (two from blood, one from cerebrospinal fluid, and one each from homograft valve, lip wound, and pilonidal cyst) were compared to the type strains of phenotypically related organisms, Cellulomonas fimi, Cellulomonas hominis, Oerskovia turbata, and Sanguibacter suarezii, and characterized by phenotypic, chemotaxonomic, and genotypic studies. DNA-DNA reassociation analysis identified two genomic groups, and phylogenetic analysis of the 16S rRNA gene sequence identified the taxonomic positions of these groups to genus level. Two groups were defined, and both were more closely related to Cellulomonas species: one group of three strains, for which we propose the new species Cellulomonas denverensis sp. nov., with the type strain W6929 (ATCC BAA-788(T) or DSM 15764(T)), was related to C. hominis ATCC 51964(T) (98.5% 16S rRNA gene sequence similarity), and the second group of three strains was related to C. hominis ATCC 51964(T) (99.8 to 99.9% 16S rRNA gene sequence similarity). The definition of this new Cellulomonas species and the confirmation of three strains as C. hominis serve to further clarify the complex taxonomy of CDC coryneform group A-3 bacteria and will assist in our understanding of the epidemiology and clinical significance of these microorganisms.     

Characterization of sucrose-negative Pasteurella multocida variants, including isolates from large-cat bite wounds

To validate the identification of Pasteurella multocida-like bacteria negative for acid formation from sucrose, including isolates from bite wounds caused by large cats, 17 strains were phenotypically and genotypically characterized. Phylogenetic analysis of partially sequenced rpoB and infB genes showed the monophyly of the strains characterized and the reference strains of P. multocida. The sucrose-negative strains formed two groups, one related to reference strains of P. multocida and the other related to a separate species-like group (taxon 45 of Bisgaard). DNA-DNA hybridization further documented the species-like nature of this group. Ribotyping showed the heterogeneity of all strains except four strains that shared the same ribotype and that were isolated from bovine lungs. Phylogenetic analysis by 16S rRNA sequence comparison showed the monophyly of the strains characterized and the reference strains of P. multocida. Two strains isolated from leopard bite wounds were related to the type strain of P. dagmatis; however, they represented a new taxon (taxon 46 of Bisgaard), in accordance with their distinct phenotypic and genotypic identifications. The present study documents that sucrose-negative strains of P. multocida-like bacteria belong to two genotypically distinct groups. The study further confirms the phenotypic heterogeneity of P. multocida strains and documents two new species-like taxa of Pasteurella related to P. multocida. Until diagnostic tools have been further elaborated, special care should be taken in the identification of Pasteurella-like bacteria isolated from bite wounds caused by large cats. The evidence of phenotypic and genotypic divergence calls for the further development of PCR tests and DNA sequencing to document doubtful isolates.     

Phenotypic,Genotypic, and Antimicrobial Characteristics of Streptococcus halichoeri Isolates from Humans,Proposal To Rename Streptococcus halichoeri as Streptococcus halichoeri subsp. halichoeri,and Description of Streptococcus halichoeri subsp. hominis subsp. nov., a Bacterium Associated with Human Clinical Infections

Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324^T, 97.9% similarity to S. canis ATCC 43496^T, and 97.8% similarity to S. ictaluri ATCC BAA-1300^T. A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844^T = CCUG 67100^T = LMG 28801^T.     

Plague pandemics investigated by ribotyping of Yersinia pestis strains.

Yersinia pestis is the causative agent of plague, a disease which has caused the deaths of millions of people and which persists now in endemic foci. The rRNA gene restriction patterns (i.e., ribotypes) of 70 strains of Y. pestis, isolated on the five continents over a period of 72 years, were determined by hybridization with a 16S-23S rRNA probe from Escherichia coli. The combination of the EcoRI and EcoRV patterns resulted in the elucidation of 16 ribotypes. Two of them (B and O) characterized 65.7% of the strains studied, while the 14 other ribotypes were found in no more than three strains each. A relationship was established between biovars and ribotypes: strains of biovar Orientalis were of ribotypes A to G, those of biovar Antiqua were of ribotypes F to O, and those of biovar Medievalis were of ribotypes O and P. Great heterogeneity in rRNA restriction patterns was found among strains isolated in Africa; this heterogeneity was less pronounced among Asian isolates and was completely absent from the American strains. Pulsed-field gel electrophoresis was performed on the DNAs of some strains, but it appeared that different colonies from the same strain displayed different pulsed-field gel electrophoresis patterns and therefore that this technique was not suitable for comparison of Y. pestis isolates. In contrast, the ribotypes of individual colonies within a given strain were stable and were not modified after five passages in vivo. A clear correlation between the history of the three plague pandemics and the ribotypes of the strains could be established.     

Ribotypes of Salmonella enterica serovar Gallinarum biovars gallinarum and pullorum

Ninety-four isolates of Salmonella enterica serovar Gallinarum biovar pullorum and forty-one isolates of biovar gallinarum were ribotyped using the enzymes, HindIII, EcoRI and SmaI, and a digoxigenin-labelled E. coli-derived rRNA probe. Using HindIII, 13 profile types were observed within biovar gallinarum and 12 within biovar pullorum. The most common types accounted for 39% of biovar pullorum isolates and 47% for biovar gallinarum. EcoRI digests revealed two profile types within biovar pullorum, one accounting for 96% of the isolates, and three EcoRI profiles within biovar gallinarum, with 81% of the isolates belonging to the dominant type. Using SmaI, biovar pullorum showed two profile types with 94% of the isolates belonging to the dominant one, while SmaI igestion revealed only one ribotype within biovar gallinarum. The SmaI ribotype of biovar gallinarum was identical to the less common of the two SmaI types in biovar pullorum. Two of the HindIII profile types were seen in both biovars. The two biovars did not share any of the EcoRI profiles. Numerical analysis based on the ribotypes revealed a 94% similarity between the two biovars, but they clustered separately in a similarity dendrogram, underlining the existence of two different biovars. The results indicate that ribotyping, especially using EcoRI, may be useful in separating biovars gallinarum and pullorum. The ribotypes obtained with isolates from different countries indicated that different clones of biovar gallinarum might exist in different regions.     

Genomic DNA restriction site heterogeneity in bovine Pasteurella multocida serogroup A isolates detected with an rRNA probe.

A total of 81 Pasteurella multocida isolates from healthy and diseased dairy and beef cattle originating from various geographical locations was examined by rRNA gene restriction site polymorphism analysis (ribotyping), restriction endonuclease analysis (REA), SDS-PAGE analysis of whole-cell (WCP) and outer-membrane (OMP) proteins, and capsule and somatic serotyping. Bacterial strains were isolated from nose, lung and in one case testicle, of Holstein and cross-bred beef cattle. The isolates represented for the most part serogroup A3 (88%). Ribotyping was performed on DNA digested with HaeII, electrophoresed and then hybridised with 32P-labelled 16S-23S rRNA from Escherichia coli. Six ribotypes (R1-R6) and 10 REA types were found among the 81 isolates with similar discrimination index (DI) of c. 0.60. Protein profiles revealed reproducibility and high levels of polymorphisms among lung isolates. Isolates were compared according to their geographical habitat, their isolation from dairy or from beef cattle and from nasal cavities or lungs. No correlation was apparent between geographical locations and ribotypes. Overall, isolates obtained from dairy cattle were predominantly R1, whereas those obtained from beef cattle were equally distributed between R1 and R2. R1 was more representative of lung isolates. For some strains, particularly the single isolate ribotypes, good correlation was achieved between WCP analysis, REA types and ribotypes. For others, REA to some extent and WCP profiles were able to discriminate among isolates within ribotypes. The data suggest that a combination of ribotyping, REA and WCP analysis is useful for investigating the epidemiology of bovine P. multocida serogroup A.     

Distinct genotypes of human and canine isolates of Campylobacter upsaliensis determined by 16S rRNA gene typing and plasmid profiling.

The utility of combined 16S rRNA (rrs) gene restriction fragment length polymorphism and plasmid profiles to differentiate between and within Campylobacter upsaliensis of human and canine origin was examined. Fourteen distinct rrs gene restriction fragment length polymorphs consisting of bands sized between 1.9 and 4.8 kb were observed. The copy number of the 16S rRNA gene was three in most strains of C. upsaliensis. Plasmids were found in almost 60% of the strains; ranging in size from 1.5 to 100 kb, they gave 15 distinct plasmid profiles. All isolates from humans contained one or more plasmids, as did strains isolated from dogs with sporadic diarrhea. The two commonest 16S ribotypes were divided into eight and nine subgroups by plasmid profiling. The genotyping of canine isolates from three veterinary surveys detected both multiple infections and reinfection of dogs. Except for one, each of the isolates from humans constituted a single and unique 16S ribotype, and these more frequently carried plasmids than did canine strains. Ribotypes of human strains were not found among canine isolates. These results suggest that host-specific genotypic differences may exist among strains of C. upsaliensis, for example, intraspecific clones or clone complexes pathogenic for humans.     

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction.

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.     

Genotyping of Mycoplasma pneumoniae clinical isolates reveals eight P1 subtypes within two genomic groups

Three methods for genotyping of Mycoplasma pneumoniae clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniae isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.     

Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds.

One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.     

Nocardia veterana as a pathogen in North American patients

The molecular methodologies used in our laboratories have allowed us to define a group of Nocardia isolates from clinical samples which resemble the type strain of Nocardia veterana. Three patient isolates and the type strain of N. veterana gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veterana type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia nova type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veterana type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veterana type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana. Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veterana type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova, indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veterana is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veterana from the related species Nocardia africana and N. nova because of the very high degree of 16S rRNA gene similarity among them.     

16S ribosomal DNA sequence analysis distinguishes biotypes of Streptococcus bovis: Streptococcus bovis Biotype II/2 is a separate genospecies and the predominant clinical isolate in adult males

We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to S. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as S. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.     

Analysis of multiple differing copies of the 16S rRNA gene in five clinical isolates and three type strains of Nocardia species and implications for species assignment

Five clinical isolates of Nocardia that showed ambiguous bases within the variable region of the 16S rRNA gene sequence were evaluated for the presence of multiple copies of this gene. The type strains of three Nocardia species, Nocardia concava, Nocardia ignorata, and Nocardia yamanashiensis, which also showed ambiguous bases in the variable region, were also examined. Cloning experiments using an amplified region of the 16S rRNA that contains the variable region showed that each isolate possessed 16S rRNA genes with at least two different sequences. In addition, hybridization studies using a 16S rRNA gene-specific probe and extracted genomic DNA of the patient isolates and of the type strain of N. ignorata showed that each isolate possessed at least three copies of the gene. These multiple differing copies of the 16S rRNA gene and the results of DNA-DNA hybridization studies indicate problems of species definition and identification for such isolates. A broader species concept than that currently in vogue may be required to accommodate such organisms.     

Molecular Characterization of the Genera Proteus, Morganella, and Providencia by Ribotyping

The so-called Proteus-Providencia group is constituted at present by three genera and 10 species. Several of the recognized species are common opportunistic pathogens for humans and animals. Different methods based on the study of phenotypic characters have been used in the past with variable levels of efficiency for typing some species for epidemiological purposes. We have determined the rRNA gene restriction patterns (ribotypes) for the type strains of the 10 different species of the genera Proteus, Morganella, and Providencia. Visual inspection of EcoRV- and HincII-digested DNA from the type strains showed remarkably different patterns for both enzymes, but EcoRV provided better differentiation. Both endonucleases were retained to study a large number of wild and collection strains belonging to the different species. Clinical isolates of Proteus mirabilis, Proteus penneri, Morganella morganii, and Providencia heimbachae showed patterns identical or very similar to those of the respective type strains, so that groups of related patterns (ribogroups) were found to correspond to the diverse species. On the contrary, distinct ribogroups were detected within Providencia alcalifaciens (two ribogroups with both enzymes), Providencia rettgeri (four ribogroups with EcoRV and five with HincII), Providencia stuartii (two ribogroups with EcoRV), Providencia rustigianii (two ribogroups with HincII), and Proteus vulgaris (two ribogroups with both enzymes). The pattern shown by the ancient P. vulgaris type strain NCTC 4175 differed considerably from both P. vulgaris ribogroups as well as from the newly proposed type strain ATCC 29905 and from any other strain in this study, thus confirming its atypical nature. Minor differences were frequently observed among patterns of strains belonging to the same ribogroup. These differences were assumed to define ribotypes within each ribogroup. No correlation was observed between ribogroups or ribotypes and biogroups of P. vulgaris, P. alcalifaciens, P. stuartii, and P. rettgeri. Since, not only different species showed different rRNA gene restriction patterns, but also different ribogroups and ribotypes have been found in the majority of the species, ribotyping would be a sensitive method for molecular characterization of clinical isolates belonging to the genera Proteus, Morganella, and Providencia.     

Comprehensive ribotyping scheme for heat-stable serotypes of Campylobacter jejuni.

Strains from diverse sources belonging to all 47 heat-stable Penner serotypes of Campylobacter jejuni were examined for polymorphism around the 16S rRNA genes. Penner serotype reference strains and a group of nonserotypeable isolates were included in the study. Complete typeability was obtained; 30 distinct PstI and 42 HaeIII polymorphisms were found. Three bands were detected in almost all strains with these enzymes, confirming that three copies of the 16S rRNA gene are typical for C.jejuni. By combination of the two enzyme polymorphisms, 77 16S ribotypes were defined among the 261 strains analyzed. With two exceptions, no specific association was observed between these ribotypes and heat-stable serotypes. Nine serotypes were homogeneous with respect to the 16S ribotype. Most nonserotypeable strains belonged to ribotypes defined elsewhere in the study. The 16S ribotypes of C.jejuni described here were not found in strains of Campylobacter coli, and vice versa.     

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.     

Amoebal coculture of "Mycobacterium massiliense" sp. nov. from the sputum of a patient with hemoptoic pneumonia

A nonphotochromogenic, rapidly growing Mycobacterium strain was isolated in pure culture from the sputum and the bronchoalveolar fluid of a patient with hemoptoic pneumonia by using axenic media and an amoebal coculture system. Both isolates grew in less than 7 days at 24 to 37 degrees C with an optimal growth temperature of 30 degrees C. The isolates exhibited biochemical and antimicrobial susceptibility profiles overlapping those of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium immunogenum, indicating that they belonged to M. chelonae-M. abscessus group. They differed from M. abscessus in beta-galactosidase, beta-N-acetyl-beta-glucosaminidase, and beta-glucuronidase activities and by the lack of nitrate reductase and indole production activities, as well as in their in vitro susceptibilities to minocycline and doxycycline. These isolates and M. abscessus differed from M. chelonae and M. immunogenum by exhibiting gelatinase and tryptophane desaminase activities. Their 16S rRNA genes had complete sequence identity with that of M. abscessus and >99.6% similarity with those of M. chelonae and M. immunogenum. Further molecular investigations showed that partial hsp65 and sodA gene sequences differed from that of M. abscessus by five and three positions over 441 bp, respectively. Partial rpoB and recA gene sequence analyses showed 96 and 98% similarities with M. abscessus, respectively. Similarly, 16S-23S rRNA internal transcribed spacer sequence of the isolates differed from that of M. abscessus by a A-->G substitution at position 60 and a C insertion at position 102. Phenotypic and genotypic features of these two isolates indicated that they were representative of a new mycobacterial species within the M. chelonae-M. abscessus group. Phylogenetic analysis suggested that these isolates were perhaps recently derived from M. abscessus. We propose the name of "Mycobacterium massiliense" for this new species. The type strain has been deposited in the Collection Institut Pasteur as CIP 108297(T) and in Culture Collection of the University of Goteborg, Goteborg, Sweden, as CCUG 48898(T).