Shared Mycobacterium avium Genotypes Observed among Unlinked Clinical and Environmental Isolates

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event

Biosynthetic enhancement of the detection of bacteria by the polymerase chain reaction.

Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices

Molecular Detection of Viable Bacterial Pathogens in Water by Ratiometric Pre-rRNA Analysis ▿ †

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples

Molecular viability testing in milk.

<p>Spiked milk suspensions were incubated at 4°C and samples were taken on days 0, 1, and 2 after addition of bacteria (D0, D1, and D2). After the day 2 sample, the spiked suspensions were pasteurized at 63°C for 45 minutes then cooled on ice. Samples were taken from the pasteurized milk suspensions immediately after cooling (D2+P) and one day later (D3+P). Dark bars, MVT results. Light bars, static DNA results. Triangles, viable plate counts. NTC, milk with no spiked bacteria.</p

Lower limits of detection of MVT and static qPCR.

<p>¹ NAE, flat-glass Nucleic Acid Extraction cards (BCSI); Qiagen, Qiagen AllPrep Total Nucleic Acid kit.</p><p>² TSB, trypticase soy broth; 7H9, Middlebrook 7H9 broth with ADC enrichment. All stimulations at 37°C except <i>A. hydrophila,</i> which was stimulated at 30°C.</p><p>³ LOD, limit of detection. The LOD of MVT was the lowest density of spiked bacteria to exhibit a positive result, defined as ΔCt <u>></u> 1 in three out of three replicates. MVT was always negative in the absence of spiked bacteria.</p><p>⁴ LOD, limit of detection. The LOD of qPCR was the lowest density of spiked bacteria to exhibit a positive result, defined as a stronger signal (lower Ct value) than all three no-bacteria controls, in three out of three replicates.</p><p>⁵ Experiment depicted graphically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086433#pone-0086433-g001" target="_blank">Figure 1</a>.</p

Toward Low-Cost Affinity Reagents: Lyophilized YeastscFv Probes Specific for Pathogen Antigens

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeastscFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigenspecific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2–3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewabl

Sample adequacy controls for infectious disease diagnosis by oral swabbing.

Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota (Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden's index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with "air swabs" collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlockTM swabs collected slightly more human mtDNA than did OmniSwabsTM (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally, which makes it a useful SAC for oral swab-based diagnosis

Chronic exposure to ambient traffic-related air pollution (TRAP) alters gut microbial abundance and bile acid metabolism in a transgenic rat model of Alzheimer's disease.

BackgroundTraffic-related air pollution (TRAP) is linked to increased risk for age-related dementia, including Alzheimer's disease (AD). The gut microbiome is posited to influence AD risk, and an increase in microbial-derived secondary bile acids (BAs) is observed in AD patients. We recently reported that chronic exposure to ambient TRAP modified AD risk in a sex-dependent manner in the TgF344 AD (TG) rat.ObjectivesIn this study, we used samples from the same cohort to test our hypothesis that TRAP sex-dependently produces gut dysbiosis and increases secondary BAs to a larger extent in the TG rat relative to wildtype (WT) controls.MethodsMale and female TG and age-matched WT rats were exposed to either filtered air (FA) or TRAP from 28 days up to 15 months of age (n&nbsp;=&nbsp;5-6). Tissue samples were collected after 9 or 14months of exposure.ResultsAt 10 months of age, TRAP tended to decrease the alpha diversity as well as the beneficial taxa Lactobacillus and Ruminococcus flavefaciens uniquely in male TG rats as determined by 16&nbsp;S rDNA sequencing. A basal decrease in Firmicutes/Bacteroidetes (F/B) ratio was also noted in TG rats at 10 months. At 15 months of age, TRAP altered inflammation-related bacteria in the gut of female rats from both genotypes. BAs were more affected by chronic TRAP exposure in females, with a general trend of increase in host-produced unconjugated primary and microbiota-produced secondary BAs. Most of the mRNAs of the hepatic BA-processing genes were not altered by TRAP, except for a down-regulation of the BA-uptake transporter Ntcp in males.ConclusionIn conclusion, chronic TRAP exposure produced distinct gut dysbiosis and altered BA homeostasis in a sex and host genotype-specific manner

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL−1, comparable to a more labor-intensive fluorescence detection method reported previously

Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix

<div><p>Assays for bacterial ribosomal RNA precursors (pre-rRNA) have been shown to distinguish viable from inactivated bacterial cells in drinking water samples. Because the synthesis of pre-rRNA is rapidly induced by nutritional stimulation, viable bacteria can be distinguished from inactivated cells and free nucleic acids by measuring the production of species-specific pre-rRNA in samples that have been briefly stimulated with nutrients. Here, pre-rRNA analysis was applied to bacteria from serum, a human sample matrix. In contrast to drinking water, serum is rich in nutrients that might be expected to mask the effects of nutritional stimulation. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were used to detect pre-rRNA of four bacterial species: <em>Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus,</em> and the <em>Mycobacterium tuberculosis</em> complex. These species were chosen for their clinical significance and phylogenetic diversity (Proteobacteria, Firmicutes, and Actinobacteria). To maximize resolving power, pre-rRNA was normalized to genomic DNA of each pathogen. When viable cells were shifted from serum to bacteriological culture medium, rapid replenishment of pre-rRNA was always observed. Cells of <em>P. aeruginosa</em> that were inactivated in the presence of serum exhibited no pre-rRNA response to nutritional stimulation, despite strong genomic DNA signals in these samples. When semi-automated methods were used, pre-rRNA analysis detected viable <em>A. baumannii</em> cells in serum at densities of ≤100 CFU/mL in <5.5 hours. Originally developed for rapid microbiological analysis of drinking water, ratiometric pre-rRNA analysis can also assess the viability of bacterial cells derived from human specimens, without requiring bacteriological culture.</p> </div