BackgroundTuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.Methods and FindingsMicroscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.ConclusionAs nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

SummaryVery little is known about the biological functions of pili that have recently been found to be expressed by important Gram-positive pathogens such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1 S. pyogenes strain SF370 to both human tonsil epithelium and primary human keratinocytes, which represent the two main sites of infection by this human-specific pathogen. Mutants lacking minor pilus subunits retained the ability to express cell-surface pili, but these were functionally defective. In contrast to above, pili were not required for S. pyogenes adhesion to either immortalized HEp-2 or A549 cells, highlighting an important limitation of these extensively used adhesion/invasion models. Adhering bacteria were internalized very effectively by both HEp-2 and A549 cells, but not by tonsil epithelium or primary keratinocytes. While pili acted as the primary adhesin, the surface M1 protein clearly enhanced adhesion to tonsil, but surprisingly, had the opposite effect on adhesion to keratinocytes. These studies provide clear evidence that S. pyogenes pili display an adhesive specificity for clinically relevant human tissues and are likely to play a critical role in the initial stages of infection.

Alcohol binge-drinking, especially among adolescents and young adults, is a serious public health concern. The present study examined ethanol binge-like drinking by peri-adolescent [postnatal days (PNDs 30—72)] and adult (PNDs 90—132) alcohol-preferring (P) rats with a drinking-in-the-dark—multiple-scheduled-acces (DID-MSA) procedure used by our laboratory. Male and female P rats were provided concurrent access to 15% and 30% ethanol for three 1-hr sessions across the dark cycle 5 days/week. For the 1st week, adolescent and adult female P rats consumed 3.4 and 1.6 g/kg of ethanol, respectively, during the 1st hr of access, whereas for male rats the values were 3.5 and 1.1 g/kg of ethanol, respectively. Adult intakes increased to ~2.0 g/kg/hr and adolescent intakes decreased to ~2.5 g/kg/hr across the 6 weeks of ethanol access. The daily ethanol intake of adult DID-MSA rats approximated or modestly exceeded that seen in continuous access (CA) rats or the selection criterion for P rats (≥ 5g/kg/day). However, in general, the daily ethanol intake of DID-MSA peri-adolescent rats significantly exceeded that of their CA counterparts. BELs were assessed at 15-min intervals across the 3rd hr of access during the 4th week. Ethanol intake was 1.7 g/kg vs. 2.7 g/kg and BELs were 57 mg% vs. 100 mg% at 15- and 60-min, respectively. Intoxication induced by DID-MSA in female P rats was assessed during the 1st vs. 4th week of ethanol access. Level of impairment did not differ between the 2 weeks (106 vs. 97 sec latency to fall, 120 sec criterion) and was significant (vs. naïve controls) only during the 4th week. Overall, these findings support the use of the DID-MSA procedure in rats, and underscore the presence of age- and sex-dependent effects mediating ethanol binge-like drinking in P rats.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H 2 O 2 -tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519 , but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.