Dithiothreitol

Mycobacterium tuberculosis (Mtb) forms biofilms harbouring antibiotic-tolerant bacilli in vitro, but the factors that induce biofilm formation and the nature of the extracellular material that holds the cells together are poorly understood. Here we show that intracellular thiol reductive stress (TRS) induces formation of Mtb biofilms in vitro, which harbour drug-tolerant but metabolically active bacteria with unchanged levels of ATP/ADP, NAD(+)/NADH and NADP(+)/NADPH. The development of these biofilms requires DNA, RNA and protein synthesis. Transcriptional analysis suggests that Mtb modulates only ∼7% of its genes for survival in biofilms. In addition to proteins, lipids and DNA, the extracellular material in these biofilms is primarily composed of polysaccharides, with cellulose being a key component. Our results contribute to a better understanding of the mechanisms underlying Mtb biofilm formation, although the clinical relevance of Mtb biofilms in human tuberculosis remains unclear.

BackgroundOxidative potential (OP) has been suggested to be a more health-relevant metric than particulate matter (PM) mass. Land use regression (LUR) models can estimate long-term exposure to air pollution in epidemiological studies, but few have been developed for OP.ObjectivesWe aimed to characterize the spatial contrasts of two OP methods and to develop and evaluate LUR models to assess long-term exposure to the OP of PM2.5.MethodsThree 2-week PM2.5 samples were collected at 10 regional background, 12 urban background, and 18 street sites spread over the Netherlands/Belgium in 1 year and analyzed for OP using electron spin resonance (OP(ESR)) and dithiothreitol (OP(DTT)). LUR models were developed using temporally adjusted annual averages and a range of land-use and traffic-related GIS variables.ResultsStreet/urban background site ratio was 1.2 for OP(DTT) and 1.4 for OP(ESR), whereas regional/urban background ratio was 0.8 for both. OP(ESR) correlated moderately with OP(DTT) (R2 = 0.35). The LUR models included estimated regional background OP, local traffic, and large-scale urbanity with explained variance (R2) of 0.60 for OP(DTT) and 0.67 for OP(ESR). OP(DTT) and OP(ESR) model predictions were moderately correlated (R2 = 0.44). OP model predictions were moderately to highly correlated with predictions from a previously published PM2.5 model (R2 = 0.37-0.52), and highly correlated with predictions from previously published models of traffic components (R2 > 0.50).ConclusionLUR models explained a large fraction of the spatial variation of the two OP metrics. The moderate correlations among the predictions of OP(DTT), OP(ESR), and PM2.5 models offer the potential to investigate which metric is the strongest predictor of health effects.CitationYang A, Wang M, Eeftens M, Beelen R, Dons E, Leseman DL, Brunekreef B, Cassee FR, Janssen NA, Hoek G. 2015. Spatial variation and land use regression modeling of the oxidative potential of fine particles. Environ Health Perspect 123:1187-1192; http://dx.doi.org/10.1289/ehp.1408916.

BackgroundA goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.ResultsElectrotransformation efficiencies of up to 105 transformants per mug DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 +/- 52.5 x 107 transformants per mug DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 +/- 0.5 x 105 transformants per mug DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.ConclusionThis investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.

A substantial proportion of symptomatic patients with urinary tract infections (UTIs) have negative or low-count urine cultures despite clinically compatible presentations. Biofilm-associated bacterial aggregation and the presence of viable bacteria not recovered by standard culture may contribute to this diagnostic gap. Flow cytometry (FACS) can detect these viable cells, but culturability remains essential for species identification and antimicrobial susceptibility testing. We evaluated whether low-concentration dithiothreitol (DTT) can safely disperse urinary biofilm-associated aggregates and improve bacterial recovery from urine samples. Seventy-two clinical urine samples were processed in parallel (untreated vs 0.1% DTT-treated). Viability was quantified using SYTO9/PI-based flow cytometry with bead-normalized bacterial counts. Culturability was assessed on chromogenic media. Comparative analyses included paired statistics, Bland-Altman agreement, and correlation between viable and culturable fractions. DTT (0.1%) did not alter bacterial viability (P = 0.47), confirming its non-toxicity at this concentration. In contrast, DTT significantly increased CFU recovery (median: 10 to 5.5 × 10³ CFU/mL; P < 0.0001), converting several previously negative or low-count samples into positive cultures. Agreement between viable and culturable fractions improved after DTT treatment, primarily through reduced systematic bias in Bland-Altman analysis, while correlation coefficients remained similar (r: 0.44 vs 0.45). A persistent discrepancy between FACS-derived viable counts and culture-based counts suggests the presence of viable bacterial populations not fully recovered by standard culture conditions, findings that are consistent with but do not directly demonstrate the presence of VBNC subpopulations. Low-dose DTT is a safe and effective pre-analytical treatment that markedly improves culture sensitivity without compromising bacterial viability. These findings support further investigation of DTT-assisted processing as a potential strategy to reduce culture underestimation in urinary diagnostics and to better characterize viable bacterial populations not detected by standard culture methods.IMPORTANCEStandard urine culture may fail to detect the full spectrum of bacteria present in urinary tract infections, particularly when microorganisms are embedded within mucus, biofilm-like aggregates, or enter a viable but non-culturable state. These hidden bacterial populations may contribute to persistent symptoms or recurrent infections while remaining undetected by routine culture methods. Our findings suggest that pre-treating urine samples with a low concentration of dithiothreitol may help disperse biofilm-like aggregates while preserving bacterial viability. When combined with flow cytometry, this approach may allow a more comprehensive estimation of the viable bacterial fraction and, in some cases, may increase the likelihood of bacterial recovery by culture in samples that would otherwise appear negative. Because the procedure relies on inexpensive reagents and techniques compatible with routine laboratory workflows, it may represent a practical adjunct to conventional diagnostics. Further studies are needed to confirm its clinical impact.