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Oral swab analysis (OSA) has been shown to detect Mycobacterium tuberculosis (MTB) DNA in patients with pulmonary tuberculosis (TB). In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93% sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs. A qPCR targeting conserved bacterial ribosomal rRNA gene (rDNA) sequences was used to quantify bacterial biomass in samples. There was no detectable reduction in total bacterial rDNA signal over the course of 10 rapidly repeated tongue samplings, indicating that swabs collect only a small portion of the biomass available for testing. Copan FLOQSwabs collected ~2-fold more biomass than Puritan PurFlock swabs, the best brand used previously (p = 0.006). FLOQSwabs were therefore evaluated in patients with possible TB in Uganda. A FLOQSwab was collected from each patient upon enrollment (Day 1) and, in a subset of sputum GeneXpert Ultra-positive patients, a second swab was collected on the following day (Day 2). Swabs were tested for MTB DNA by manual IS6110-targeted qPCR. Relative to sputum GeneXpert Ultra, single-swab sensitivity was 88% (44/50) on Day 1 and 94.4% (17/18) on Day 2. Specificity was 79.2% (42/53). Among an expanded sample of Ugandan patients, 62% (87/141) had colony-forming bacilli in their tongue dorsum swab samples. These findings will help guide further development of this promising TB screening method.

Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations. [ABSTRACT FROM AUTHOR]

The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, 'transposome' insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M. avium genome.

Results demonstrate that transparent derivatives of red opaque and White opaque clones of Mycobacterium avium preserve their Congo red binding properties. Data suggest that manifestation of opaque-transparent morphotype is independent of the red-white switch. Transparent derivatives of White opaque coloniesare are more resistant to antibiotics.

A study was conducted to determine whether a biological stain utilized to evaluate cell-surface differences in bacteria could advance novel information on cell-surface variation in the Mycobacterium avium-intracellulare complex. Results indicated that the white segregants of M avium were more flocculent and more resistant to ciprofloxacin and rifamycin drugs than the red segregants.

Research was conducted to investigate the characterization of pre-ribosomal RNA (rRNA) in toxigenic Pseudo-nitzschia species. Probe hybridization methods were used to determine the nucleotide sequences of external and internal transcribed spacers, and characterized pre-rRNA pools in cultured P. multiseries cells. Results indicated that pre-rRNA sequences included conserved regions in secondary structures required for rRNA maturation and that the extent of pre-rRNA depletion in starved Escherichia coli cells varied with conditions.

Growth state phases in Escherichia coli are analyzed by pre-rRNA-targeted probes to find out how bacterial growth activity proceeds. Results show that that rRNA processing persists even after growth as is the case with many eukaryotic cells and that the extent of depletion of pre-16S rRNA relies on specific circumstances on what substance happened to be inadequate. Thus, carbon-energy-starved cells tended to be more prone to depletion than those cells that lacked nitrogen.

We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms (LSPs) comprising over 700 kb that distinguish Mr. avium subsp. avium from M. avium subsp, paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains the in vitro mycobactin dependence of M. avium subsp, paratuberculosis.

Rationale: Nontuberculous mycobacteria (NTM), including Mycobacterium avium complex (MAC), are emerging pathogens that can opportunistically cause debilitating pulmonary disease in susceptible human hosts. Potential sources of exposure in homes include point-of-use water sources, such as taps and showerheads, as well as gardening soils. The relative human health impacts of NTM in these home environments remain poorly understood.Objectives: This study tested associations between MAC pulmonary disease and NTM colonization of five potential point-of-use sources of pathogen exposure in homes.Methods: A case-control study was conducted of Washington and Oregon residents who had been diagnosed with MAC pulmonary disease, and population controls were matched by age, sex, and geography. Samples were collected from bathroom faucets, kitchen faucets, shower aerosols, indoor soil, and outdoor soil. Mycobacteria in environmental samples were identified in a blinded fashion by using bacteriological culture combined with polymerase chain reaction. The isolation of NTM from case homes (n = 56) versus control homes (n = 51) was quantitatively compared using conditional logistic regression models with adjustment for potential confounding variables.Results: NTM were isolated from shower aerosols collected in case homes more often than in control homes. An adjusted conditional logistic regression analysis showed that NTM isolation from shower aerosols had a high odds ratio associated with disease (odds ratio, 4.0; 95% confidence interval, 1.2-13). Other home environmental samples (tap water, soils) did not exhibit this association.Conclusions: The results implicate shower aerosols as uniquely significant sources of NTM exposure in homes. [ABSTRACT FROM AUTHOR]

Ratiometric pre-rRNA analysis (RPA) has detected the replenishment of rRNA precursors occurring rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA has distinguished viable from inactivated bacteria and it can be used as a molecular viability test for pathogens in water and other environmental samples.

A study is conducted to examine the effectiveness of two major ultraviolet (UV) technologies against a highly prevalent species of Mycobacterium avium complex. The observation showed M. avium to be more resistant to UV irradiation than most waterborne pathogens and is one of the rarest microorganisms that are highly resistant to both chemical and UV disinfection in water.