Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax

Recovery of a Burkholderia thailandensis-like isolate from an Australian water source

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia thailandensis</it>, a close relative of <it>Burkholderia pseudomallei</it>, has previously been reported only from Southeast Asia and North America. It is biochemically differentiated from <it>B. pseudomallei </it>by the ability to utilize arabinose. During the course of environmental sampling for <it>B. pseudomallei </it>in the Northern Territory of Australia, an isolate, MSMB 43, was recovered that is arabinose positive.</p> <p>Results</p> <p>Genetic analysis using 16S rDNA sequencing and DNA/DNA hybridization indicates that MSMB 43 is most similar to <it>B. thailandensis </it>although multi-locus sequence typing indicates that this isolate is divergent from both <it>B. pseudomallei </it>and other described <it>B. thailandensis</it>.</p> <p>Conclusion</p> <p>We report the isolation and initial characterization of strain MSMB 43, which is a <it>B. thailandensis</it>-like isolate recovered in Australia.</p

Characterization of Burkholderia rhizoxinica and B. endofungorum Isolated from Clinical Specimens

Eight isolates submitted to CDC from 1989 to 2006 from clinical specimens were initially identified as members of the genus Burkholderia based on preliminary cellular fatty acid analysis and/or 16S rRNA gene sequencing. With the recent descriptions of the new species B. rhizoxinica and B. endofungorum, which are considered endosymbiotic bacteria in Rhizopus microsporus fungi, we now identify seven of these clinical isolates as B. rhizoxinica and one as B. endofungorum based on biochemical testing, 16s rRNA, and DNA-DNA hybridization results. We also further characterize these isolates by assessing toxin production and/or by multiple locus sequence typing

Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon

Rhodococcus gordoniae sp. nov., an actinomycete isolated from clinical material and phenol contaminated soil.

The taxonomic relationships of two actinomycetes provisionally assigned to the genus Rhodococcus were determined using a polyphasic taxonomic approach. The generic assignment was confirmed by 16S rRNA gene similarity data, as the organisms, strains MTCC 1534 and W 4937T, were shown to belong to the Rhodococcus rhodochrous subclade. These organisms had phenotypic properties typical of rhodococci; they were aerobic, Gram-positive, weakly acid-fast actinomycetes that showed an elementary branching-rod–coccus growth cycle and contained meso-diaminopimelic acid, arabinose and galactose in whole-organism hydrolysates, N-glycolated muramic acid residues, dehydrogenated menaquinones with eight isoprene units as the predominant isoprenologue and mycolic acids that co-migrated with those extracted from the type strain of R. rhodochrous. The strains had identical phenotypic profiles and belong to the same genomic species, albeit one distinguished from Rhodococcus pyridinivorans, with which they formed a distinct phyletic line. They were also distinguished from representatives of all of the species classified in the R. rhodochrous 16S rRNA gene tree using a set of phenotypic features. The genotypic and phenotypic data show that the strains merit recognition as a novel species of Rhodococcus. The name proposed is Rhodococcus gordoniae sp. nov., with the type strain W 4937T (=DSM 44689T=NCTC 13296T)

Confirmation of Nontypeable Streptococcus pneumoniae-Like Organisms Isolated from Outbreaks of Epidemic Conjunctivitis as Streptococcus pneumoniae

Eleven isolates representing five distinct outbreaks of pneumococcal conjunctivitis were examined for phenotypic and genetic characteristics. None of the strains possessed capsules, and all strains were susceptible to optochin, bile soluble, and Gen-Probe AccuProbe test positive. All 11 isolates were confirmed as Streptococcus pneumoniae by DNA-DNA reassociation experiments

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y frederiksenii hybridization group 2 (unnamed genomospecies 2). This finding was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified

Nocardia wallacei sp. nov. and Nocardia blacklockiae sp. nov., Human Pathogens and Members of the “Nocardia transvalensis Complex”▿

Nocardia isolates that share the property of in vitro amikacin resistance are grouped together by some authors in the Nocardia transvalensis complex. Our examination of 13 isolates that are amikacin resistant has revealed the existence of three distinct species. Sequence analysis of the 16S rRNA, 65-kDa heat shock protein, and secA1 genes, coupled with DNA-DNA hybridization, indicated that “N. asteroides drug pattern IV,” “N. transvalensis new taxon 1,” and N. transvalensis sensu stricto should each be considered a distinct species. The phenotypic and molecular characteristics of the proposed new species Nocardia wallacei (N. asteroides drug pattern IV) and N. blacklockiae (N. transvalensis new taxon 1) are presented and compared with those of N. transvalensis sensu stricto. The relative genetic diversity of isolates best placed with the species N. blacklockiae is also discussed. Case studies demonstrating the pathogenicity of N. wallacei and N. blacklockiae are presented. The type strain of N. wallacei is ATCC 49873 (DSM 45136), and that of N. blacklockiae is ATCC 700035 (DSM 45135)