Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.     

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.     

Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition

Clostridium difficile is a major enteric pathogen responsible for antibiotic-associated diarrhea. Host susceptibility to C. difficile infections results partly from inability of the intestinal microbiota to resist C. difficile colonization. During early infancy, asymptomatic colonization by C. difficile is common and the intestinal microbiota shows low complexity. Thus, we investigated the potential relationship between the microbiota composition and the implantation of C. difficile in infant gut. Fecal samples from 53 infants, ages 0 to 13 months, 27 negative and 26 positive for C. difficile, were studied. Dominant microbiota profiles were assessed by PCR-temporal temperature gradient gel electrophoresis (TTGE). Bacterial signatures of the intestinal microbiota associated with colonization by C. difficile were deciphered using principal component analysis (PCA). Resulting bands of interest in TTGE profiles were excised, sequenced, and analyzed by nucleotide BLAST (NCBI). While global biodiversity was not affected, interclass PCA on instrumental variables highlighted significant differences in dominant bacterial species between C. difficile-colonized and noncolonized infants (P = 0.017). Four bands were specifically associated with the presence or absence of C. difficile: 16S rRNA gene sequences related to Ruminococcus gnavus and Klebsiella pneumoniae for colonized infants and to Bifidobacterium longum for noncolonized infants. We demonstrated that the presence of C. difficile in the intestinal microbiota of infants was associated with changes in this ecosystem's composition. These results suggest that the composition of the gut microbiota might be crucial in the colonization process, although the chronology of events remains to be determined.     

Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of Bartonella henselae Isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene

Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.     

Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of Pseudomonas aeruginosa and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis

We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002-3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish P. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.     

PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.     

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.     

Nocardia kruczakiae sp. nov., a pathogen in immunocompromised patients and a member of the "N. nova complex"

Molecular methodologies have become useful techniques for the identification of pathogenic Nocardia species and for the recognition of novel species that are capable of causing human disease. Two isolates recovered from immunocompromised patients were characterized as Nocardia nova by biochemical and susceptibility testing results. The restriction fragment length polymorphism (RFLP) patterns obtained by restriction endonuclease analysis (REA) of an amplified portion of the heat shock protein gene were identical to those obtained with the type strain of N. nova. REA of an amplified portion of the 16S rRNA gene showed RFLP patterns that were unlike those obtained for the type strain of N. nova but that were similar to those obtained for the type strains of N. africana and N. veterana. Subsequent sequencing of a portion of the 16S rRNA gene produced identical results for the two patient isolates. Sequence analysis of 1,352-bp portions of the 16S rRNA gene indicated that these isolates were 99.8% similar to the recently described species N. veterana but were only 99.3, 98.1, and 98.1% similar to the type strains of N. africana, N. nova, and N. vaccinii, respectively. DNA-DNA hybridization studies confirmed that the two patient isolates belonged to the same species but were not closely related to N. africana, N. nova, N. vaccinii, or N. veterana. The patient isolates have been designated N. kruczakiae sp. nov. Because N. africana, N. veterana, and the new species are not readily differentiated from N. nova by phenotypic methods alone, the designation "N. nova complex" can be used to designate isolates such as these that phenotypically resemble N. nova but that have not been definitively characterized by 16S rRNA gene sequencing or DNA-DNA hybridization.     

Intragenomic and intraspecific heterogeneity of the 16S rRNA gene in seven bacterial species from the respiratory tract of cystic fibrosis patients assessed by PCR-Temporal Temperature Gel Electrophoresis

16S rRNA gene-based cultivation-independent methods are increasingly used to study the diversity of microbiota during health and disease. One bias of these methods is the variability of 16S rRNA gene that may exist among strains of a same species (intraspecific heterogeneity) or between rrs copies in a genome (intragenomic heterogeneity). We evaluated the level of intraspecific and intragenomic 16S rDNA variability in seven species frequently encountered in respiratory tract samples in cystic fibrosis (CF). A total of 179 strains were subjected to V3 region 16S rDNA PCR-TTGE. Using this easy-to-perform and rapid method, different levels of V3 region rrs heterogeneity were demonstrated. No intraspecific and intragenomic rrs heterogeneity was demonstrated for Moraxella catarrhalis (n=16), Pseudomonas aeruginosa (n=31) and Streptococcus pneumoniae (n=14) showing a single PCR-TTGE band characteristic of the species. Low level of intraspecific heterogeneity was observed for Staphylococcus aureus (n=30), Stenotrophomonas maltophilia (n=29) and Achromobacter xylosoxidans (n=28), and 17%, 38% and 96% of these strains showed intragenomic heterogeneity (two to four different rrs copies), respectively. Haemophilus influenzae (n=31) displayed the higher level of intraspecific variability with 23 different PCR-TTGE patterns and 61% of the strains showed intragenomic rrs heterogeneity (two to four different rrs copies). Although only one hypervariable region of the 16S rRNA gene was explored, intraspecific and intragenomic rrs heterogeneity was frequently observed in this study and should be taken into consideration for a better interpretation of 16S rRNA gene-based diversity profiles in denaturing gels and to avoid any overestimation of the respiratory microbiota diversity in CF.     

Ribotyping of Mycoplasma gallisepticum strains with a 16S ribosomal RNA gene probe

Ribotyping with a 16S ribosomal RNA gene (rDNA) probe was applied to 25 Mycoplasma gallisepticum strains composed of nine originally isolated in the US and UK, and 16 field isolates from Japan. Four distinct ribotypes were identified among the M. gallisepticum strains on the basis of sizes of the bands produced by digesting genomic DNA with restriction enzymes HindIII, EcoRI, BglII and EcoRV. Three ribotypes were recognized among the 16 Japanese isolates. The original and vaccine F strains were classified as the same ribotype, although they produced different banding patterns with BglII, suggesting that the vaccine strain may be a mutant of the original. Other avian mycoplasma species produced banding patterns different from each other and from M. gallisepticum strains. Characterization of M. gallisepticum strains by ribotyping with a 16S rDNA probe may benefit surveillance and epidemiological investigations on M. gallisepticum infection.     

Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers.

Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.     

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.     

Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerase chain reaction and restriction fragment length polymorphism analysis of 16S ribosomal DNA.

Streptococcus uberis type II has been proposed recently as a separate species designated Streptococcus parauberis (A. M. Williams and M. D. Collins, J. Appl. Bacteriol. 68:485-490, 1990). Differentiation of S. parauberis from S. uberis has been possible only by DNA-DNA hybridization or 16S rRNA sequencing, since the biochemical and serological characteristics of the two species are indistinguishable. A simple and reliable technique was developed for differentiating S. parauberis (S. uberis type II [ATCC 13386]) from S. uberis (S. uberis type I [ATCC 9927, ATCC 13387, and ATCC 27958]) by restriction fragment length polymorphism (RFLP) analysis of 1.4-kb 16S ribosomal DNA (rDNA). Oligonucleotide primers complementary to 16S rRNA genes were used to amplify 16S ribosomal gene fragments from genomic DNA by polymerase chain reaction. The 1.4-kb 16S rDNA fragment was digested with ScaI, NspI, DdeI, and AvaII restriction endonucleases. Restriction fragments produced by all four restriction endonucleases were characteristic for each species. RFLP analysis of 16S rDNA from 24 "S. uberis" isolates obtained from mammary secretions of dairy cows indicated that all 24 isolates were indeed S. uberis.     

Diagnostics of neisseriaceae and moraxellaceae by ribosomal DNA sequencing: ribosomal differentiation of medical microorganisms

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.     

Amplified fragment length polymorphism fingerprinting is an effective technique to distinguish streptococcus pneumoniae from other Streptococci and an efficient alternative to pulsed-field gel electrophoresis for molecular typing of pneumococci

Amplified fragment length polymorphism versus pulsed-field gel electrophoresis was used for fingerprinting of 85 macrolide-resistant pneumococcal isolates identified by using primarily phenotypic methods. Confirmation of identification by 16S rRNA sequencing revealed that 27 isolates were actually nonpneumococci. Amplified fragment length polymorphism but not pulsed-field gel electrophoresis offered simultaneous and accurate discrimination between pneumococci and nonpneumococcal species.     

Detection of genomic variation in Providencia stuartii clinical isolates by analysis of DNA restriction fragment length polymorphisms containing rRNA cistrons.

Chromosomal DNA from 26 strains of Providencia stuartii isolated mainly in hospitals in the United Kingdom and reference strains of P. stuartii, P. rustigianii, and Proteus vulgaris were digested with the restriction endonucleases EcoRI and HindIII. After electrophoresis in agarose gels, the fragments were subjected to Southern blot hybridization analysis with a biotin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNA from P. stuartii NCTC 11800T. The pattern of bands (the rDNA fingerprint), which depended on restriction fragment length polymorphisms containing rRNA genes, was used as a measure of minor genomic variation within and between species. The P. stuartii clinical isolates had similar total digest patterns, but the rDNA fingerprints revealed some heterogeneity between strains, with EcoRI digests providing better strain discrimination than HindIII. Such rDNA fingerprints comprised between five and seven bands with sizes in the range of 5 to 28 kilobases. The 11 different EcoRI patterns were compared by numerical analysis, and several groups or subgroups of strains were identified. Over half (15 of 26) of the urease-negative isolates (subgroups Aa and Ab) had patterns that differed only by the presence or absence of a 25-kilobase band. Urease-negative strains from other clinical material were more heterogeneous in their patterns. No correlation was apparent between strain pattern group and urease production or geographic location of isolate. The P. stuartii rDNA fingerprints were quite distinct from those of allied Providencia and Proteus species and provided a more sensitive measure of minor genomic differences than total DNA digests did.     

Identification of clinically relevant viridans group streptococci by sequence analysis of the 16S-23S ribosomal DNA spacer region

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29 reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for the score for S. gordonii strains. The interspecies similarity scores for the ITS sequences varied from 0.31 to 0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMérieux Vitek, Marcy l'Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing for identification of VS was verified by 16S rDNA sequencing for all strains except strains of S. oralis and S. mitis, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.     

Limited diversity among human isolates of Bartonella henselae

A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28). Either of two distinct alleles of both gltA and 16S ribosomal DNA (rDNA) was found in all isolates, with a high level of congruity between 16S and gltA inheritance among proven human pathogens. Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I, with the same gltA allele as the type strain (Houston-1). Comparable feline isolates were more commonly 16S type II, with less congruity of inheritance between 16S and gltA alleles. Previously described arbitrarily primed PCR and EagI-HhaI infrequent restriction site PCR fingerprinting techniques separated Bartonella species effectively but lacked discriminating power within B. henselae. Examination of the 16-23S intergenic spacer region revealed for several strains several point mutations as well as a repeat sequence of unknown significance which is readily detected by HaeIII restriction fragment length polymorphism analysis. The bacteriophage-associated papA gene was present in all isolates. Enterobacterial repetitive intergenic consensus PCR proved to be a useful and robust typing tool and clearly separated human isolates (including imported strains) from the majority of feline isolates. Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains. They strengthen arguments for careful exploration of genotype-phenotype relationships and for the development of a multilocus enzyme electrophoresis and multilocus sequence typing-based approach to the phylogeny of B. henselae.     

Genotypic identification of erythromycin-resistant campylobacter isolates as helicobacter species and analysis of resistance mechanism

The correct identification of Campylobacter species remains cumbersome, especially when conventional biochemical tests and antimicrobial susceptibility patterns are used for a phenotypical identification. Correct identification is important for epidemiological purposes and for studying changes in antimicrobial resistance patterns. Six erythromycin-resistant campylobacter strains were investigated by 16S ribosomal DNA (rDNA) sequencing, 23S rDNA sequencing, and restriction fragment length polymorphism analysis of a putative heme-copper oxidase domain described as being specific for thermophilic Campylobacter species. Three erythromycin-resistant isolates from feces of human immunodeficiency virus (HIV)-seropositive patients with diarrhea and one blood isolate of from HIV-seropositive patient with cellulitis were identified by 16S rDNA analysis as Helicobacter cinaedi, whereas 23S rDNA sequencing suggested Wolinella succinogenes. The 16S rDNA sequence data of fecal isolates of two patients with travelers diarrhea revealed Helicobacter pullorum and were also in contrast with 23S rDNA sequencing. Of 4 H. cinaedi isolates, 1 contained the putative heme-copper oxidase gene thought to be specific for thermophilic species. The six erythromycin-resistant Helicobacter species had a similar point mutation A2143G in 23S rDNA resembling the macrolides resistance in Helicobacter pylori. We conclude that 16S rDNA sequencing should be preferred to 23S rDNA analysis and that macrolide-resistant campylobacter strains should be investigated by this approach for a correct identification.