Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates

BACKGROUND: Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR. However, we have identified 23 Bacillus spp. isolates that gave discrepant results when assayed by standard microbiological methods and PCR. We used multiple-locus variable-number tandem repeat analysis (MLVA), multiple-locus sequence typing (MLST), and phenotypic analysis to characterize these isolates, determine if they cluster phylogenetically and establish whether standard microbiological identification or PCR were associated with false positive/negative results. RESULTS: Six isolates were LRN real-time PCR-positive but resistant to gamma phage; MLVA data supported the identification of these isolates as gamma phage-resistant B. anthracis. Seventeen isolates were LRN real-time PCR-negative but susceptible to gamma phage lysis; these isolates appear to be a group of unusual gamma phage-susceptible B. cereus isolates that are closely related to each other and to B. anthracis. All six B. anthracis MLVA chromosomal loci were amplified from one unusual gamma phage-susceptible, motile, B. cereus isolate (although the amplicons were atypical sizes), and when analyzed phylogenetically, clustered with B. anthracis by MLST. CONCLUSION: MLVA and MLST aided in the identification of these isolates when standard microbiological methods and PCR could not definitely identify or rule out B. anthracis. This study emphasized the need to perform multiple tests when attempting to identify B. anthracis since relying on a single assay remains problematic due to the diverse nature of bacteria

Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing

<p>Abstract</p> <p>Background</p> <p><it>Bacillus cereus </it>is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype <it>B. cereus </it>and analysis has suggested a clonal or weakly clonal population structure for <it>B. cereus </it>and its close relatives <it>B. anthracis </it>and <it>B. thuringiensis</it>. In this study we used MLST to determine if <it>B. cereus </it>isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.</p> <p>Results</p> <p>A retrospective analysis of 55 clinical <it>B. cereus </it>isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27), gastrointestinal (GI) illness (n = 18), and associated isolates from food (n = 10) were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST) in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates.</p> <p>Conclusion</p> <p>STs of clinical <it>B. cereus </it>isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to <it>B. anthracis </it>and emetic <it>B. cereus </it>isolates.</p

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

Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia

<p>Abstract</p> <p>Background</p> <p>Brucellosis is primarily a zoonotic disease caused by <it>Brucella </it>species. There are currently ten <it>Brucella </it>spp. including the recently identified novel <it>B. inopinata </it>sp. isolated from a wound associated with a breast implant infection. In this study we report on the identification of an unusual <it>Brucella</it>-like strain (BO2) isolated from a lung biopsy in a 52-year-old patient in Australia with a clinical history of chronic destructive pneumonia.</p> <p>Results</p> <p>Standard biochemical profiles confirmed that the unusual strain was a member of the <it>Brucella </it>genus and the full-length 16S rRNA gene sequence was 100% identical to the recently identified <it>B. inopinata </it>sp. nov. (type strain BO1^T). Additional sequence analysis of the <it>recA, omp2a </it>and <it>2b </it>genes; and multiple locus sequence analysis (MLSA) demonstrated that strain BO2 exhibited significant similarity to the <it>B. inopinata </it>sp. compared to any of the other <it>Brucella </it>or <it>Ochrobactrum </it>species. Genotyping based on multiple-locus variable-number tandem repeat analysis (MLVA) established that the BO2 and BO1^Tstrains form a distinct phylogenetic cluster separate from the other <it>Brucella </it>spp.</p> <p>Conclusion</p> <p>Based on these molecular and microbiological characterizations, we propose that the BO2 strain is a novel lineage of the newly described <it>B. inopinata </it>species.</p

Genomic diversity of burkholderia pseudomalleiIsolates, Colombia

We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during 2016–2017. B. pseudomallei appears genetically diverse, suggesting it is well established and has spread across the region

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