Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed

African Lineage Brucella melitensis Isolates from Omani Livestock

Brucellosis is a common livestock disease in the Middle East and North Africa, but remains poorly described in the region both genetically and epidemiologically. Traditionally found in goats and sheep, Brucella melitensis is increasingly recognized as infecting camels. Most studies of brucellosis in camels to date have focused on serological surveys, providing only limited understanding of the molecular epidemiology of circulating strains. We genotyped B. melitensis isolates from Omani camels using whole genome SNP assays and VNTRs to provide context for regional brucellosis cases. We identified a lineage of B. melitensis circulating in camels as well as in goats, sheep, and cattle in Oman. This lineage is genetically distinct from most genotypes from the Arabian Peninsula and from isolates from much of the rest of the Middle East. We then developed diagnostic assays that rapidly identify strains from this lineage. In analyses of genotypes from throughout the region, Omani isolates were genetically most closely related to strains from brucellosis cases in humans and livestock in North Africa. Our findings suggest an African origin for B. melitensis in Oman that has likely occurred through the trade of infected livestock. Moreover, African lineages of B. melitensis appear to be undersampled and consequently are underrepresented in genetic databases for Brucella. As we begin to more fully understand global genomic diversity of B. melitensis, finding and characterizing these unique but widespread lineages is essential. We predict that increased sampling of humans and livestock in Africa will reveal little known diversity in this important zoonotic pathogen

Performance evaluation of Active Melioidosis Detect-Lateral Flow Assay (AMD-LFA) for diagnosis of melioidosis in endemic settings with limited resources

Melioidosis is a fatal infection caused by the soil saprophyte Burkholderia pseudomallei. Early diagnosis and befitting medical management can significantly influence the clinical outcomes among patients with melioidosis. Witnessing an annual increment in the number of melioidosis cases, over the past few years, mainly from the developing tropical nations, the present study was undertaken to evaluate the diagnostic utility of Active Melioidosis Detect (TM) LateralFlow Assay (AMD-LFA), in comparison with enrichment culture and PCR. A total of 206clinical specimens obtained from 175 patients with clinical suspicion of melioidosis were considered for the evaluation. Positivity for B.pseudomallei using enrichment culture, PCR and AMD-LFA were observed among 63 (30.5%), 55 (26.6%) and 63 (30.5%) specimens respectively. The AMD-LFA failed to detect melioidosis from 9 culture-confirmed cases (6 whole blood specimens, 2 pus samples, and one synovial fluid). Further the test gave faint bands from 9 urine samples which were negative by culture and PCR. AMD-LFA demonstrated a sensitivity, specificity, of 85.71%(CI:74.61% to 93.25%) and 93.62% (Cl:88.23% to 97.04%), with positive predictive value of 85.71% (CI: 75.98% to 91.92%) and negative predictive value of 93.62% (Cl:88.89% to 96.42%). The test needs further evaluation in view of the faint bands from negative urine samples, for incorporating the test as a point of care assay. In view of its rapidity and ease of testing AMD-LFA might be useful in early diagnosis of melioidosis at resource constraint settings