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Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic

ABSTRACT: Majority of the cells in the bacterial populations exposed to lethal concentrations of antibiotics for prolonged duration succumbs to the antibiotics’ sterilizing activity. The remaining cells survive by diverse mechanisms that include reduced permeability of the antibiotics. However, in the cells surviving in the continued presence of lethal concentrations of antibiotics, it is not known whether any cell surface alterations occur that in turn may reduce permeability of the antibiotics. Here we report the presence of a highly negatively charged, hydrophilic, thickened capsular outer layer (TCOL) on a small proportion of the rifampicin surviving population (RSP) of Mycobacterium tuberculosis (Mtb) cells upon prolonged continuous exposure to bactericidal concentrations of rifampicin in vitro. The TCOL reduced the intracellular entry of 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), a fluorochrome-conjugated rifampicin permeability probe of negligible bacteriocidal activity but comparable properties. Gentle mechanical removal of the TCOL enabled significant increase in the 5-FAM-rifampicin permeability. Zeta potential measurements of the cells’ surface charge and hexadecane assay for cell surface hydrophobicity showed that the TCOL imparted high negative charge and polar nature to the cells’ surface. Flow cytometry using the MLP and RSP cells, stained with calcofluor white, which specifically binds glucose/mannose units in ? (1 ? 4) or ? (1 ? 3) linkages, revealed the presence of lower content of polysaccharides containing such residues in the TCOL. GC-MS analyses of the TCOL and the normal capsular outer layer (NCOL) of MLP cells showed elevated levels of ?-D-glucopyranoside, mannose, arabinose, galactose, and their derivatives in the TCOL, indicating the presence of high content of polysaccharides with these residues. We hypothesize that the significantly high thickness and the elevated negative charge of the TCOL might have functioned as a physical barrier restricting the permeability of the relatively non-polar rifampicin. This might have reduced intracellular rifampicin concentration enabling the cells’ survival in the continued presence of high doses of rifampicin. In the context of our earlier report on the de novo emergence of rifampicin-resistant genetic mutants of Mtb from the population surviving under lethal doses of the antibiotic, the present findings attain clinical significance if a subpopulation of the tubercle bacilli in tuberculosis patients possesses TCOL.

SUBMITTER: Sebastian J

PROVIDER: S-EPMC7773709 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Bacteria undergo adaptive morphological changes to survive under stress conditions. The present work documents the morphological changes in Mycobacterium tuberculosis (Mtb) cells cultured under hypoxic condition using Wayne's in vitro hypoxia model involving non-replicating persistence stages 1 and 2 (NRP stage 1 and NRP stage 2) and reveals their physiological significance. Transmission electron microscopy of the NRP stage 2 cells showed uneven but thick outer layer (TOL), unlike the evenly thin outer layer of the actively growing mid-log phase (MLP) cells. On the contrary, the saprophytic Mycobacterium smegmatis NRP stage 2 cells lacked TOL. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) of the Mtb NRP stage 2 cells confirmed the rough uneven surface unlike the smooth surface of the MLP cells. Zeta potential measurements showed high negative charge on the surface of NRP stage 2 cells and polysaccharide specific calcofluor white (CFW) staining of the cells revealed high content of polysaccharide in the TOL. This observation was supported by the real-time PCR data showing high levels of expression of the genes involved in the synthesis of sugars, such as trehalose, mannose and others, which are implicated in polysaccharide synthesis. Experiments to understand the physiological significance of the TOL revealed restricted entry of the biologically low-active 5-carboxyfluorescein-rifampicin (5-FAM-RIF), at concentrations equivalent to microbicidal concentrations of the unconjugated biologically active rifampicin, into the NRP stage 2 cells, unlike in the MLP cells. Further, as expected, mechanical removal of the TOL by mild bead beating or release of the NRP stage 2 cells from hypoxia into normoxia in fresh growth medium also significantly increased 5-FAM-RIF permeability into the NRP stage 2 cells to an extent comparable to that into the MLP cells. Taken together, these observations revealed that Mtb cells under hypoxia develop TOL that helps in restricting rifampicin entry, thereby conferring rifampicin tolerance.

Project description:BackgroundBuruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Previous minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug over time. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a time-consuming method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against M. ulcerans clinical isolates by TKA, while comparing four methodologies: CFU enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification.Methodology/principal findingsTKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified with different methodologies after 1, 3, 7, 10, 14, 21 and 28 days of treatment. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combinations were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies except IS2404 quantitative PCR provided similar results with a good correlation with CFU enumeration. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA.Conclusions/significanceOur study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment.

Project description:BackgroundAccumulating data indicate that higher rifampicin doses are more effective and shorten tuberculosis (TB) treatment duration. This study evaluated the safety, tolerability, pharmacokinetics, and 7- and 14-day early bactericidal activity (EBA) of increasing doses of rifampicin. Here we report the results of the final cohorts of PanACEA HIGHRIF1, a dose escalation study in treatment-naive adult smear-positive patients with TB.MethodsPatients received, in consecutive cohorts, 40 or 50 mg·kg-1 rifampicin once daily in monotherapy (day 1-7), supplemented with standard dose isoniazid, pyrazinamide and ethambutol between days 8 and 14.ResultsIn the 40 mg·kg-1 cohort (n=15), 13 patients experienced a total of 36 adverse events during monotherapy, resulting in one treatment discontinuation. In the 50 mg·kg-1 cohort (n=17), all patients experienced adverse events during monotherapy, 93 in total; 11 patients withdrew or stopped study medication. Adverse events were mostly mild/moderate and tolerability rather than safety related, i.e. gastrointestinal disorders, pruritis, hyperbilirubinaemia and jaundice. There was a more than proportional increase in the rifampicin geometric mean area under the plasma concentration-time curve from time 0 to 12 h (AUC0-24 h) for 50 mg·kg-1 compared with 40 mg·kg-1; 571 (range 320-995) versus 387 (range 201-847) mg·L-1·h, while peak exposures saw proportional increases. Protein-unbound exposure after 50 mg·kg-1 (11% (range 8-17%)) was comparable with lower rifampicin doses. Rifampicin exposures and bilirubin concentrations were correlated (Spearman's ρ=0.670 on day 3, p<0.001). EBA increased considerably with dose, with the highest seen after 50 mg·kg-1: 14-day EBA -0.427 (95% CI -0.500- -0.355) log10CFU·mL-1·day-1.ConclusionAlthough associated with an increased bactericidal effect, the 50 mg·kg-1 dose was not well tolerated. Rifampicin at 40 mg·kg-1 was well tolerated and therefore selected for evaluation in a phase IIc treatment-shortening trial.

Project description:BACKGROUND: Cattle and other ruminants have evolved the ability to derive most of their metabolic energy requirement from otherwise indigestible plant matter through a symbiotic relationship with plant fibre degrading microbes within a specialised fermentation chamber, the rumen. The genetic changes underlying the evolution of the ruminant lifestyle are poorly understood. The BPI-like locus encodes several putative innate immune proteins, expressed predominantly in the oral cavity and airways, which are structurally related to Bactericidal/Permeability Increasing protein (BPI). We have previously reported the expression of variant BPI-like proteins in cattle (Biochim Biophys Acta 2002, 1579, 92-100). Characterisation of the BPI-like locus in cattle would lead to a better understanding of the role of the BPI-like proteins in cattle physiology RESULTS: We have sequenced and characterised a 722 kbp segment of BTA13 containing the bovine BPI-like protein locus. Nine of the 13 contiguous BPI-like genes in the locus in cattle are orthologous to genes in the human and mouse locus, and are thought to play a role in host defence. Phylogenetic analysis indicates the remaining four genes, which we have named BSP30A, BSP30B, BSP30C and BSP30D, appear to have arisen in cattle through a series of duplications. The transcripts of the four BSP30 genes are most abundant in tissues associated with the oral cavity and airways. BSP30C transcripts are also found in the abomasum. This, as well as the ratios of non-synonymous to synonymous differences between pairs of the BSP30 genes, is consistent with at least BSP30C having acquired a distinct function from the other BSP30 proteins and from its paralog in human and mouse, parotid secretory protein (PSP). CONCLUSION: The BPI-like locus in mammals appears to have evolved rapidly through multiple gene duplication events, and is thus a hot spot for genome evolution. It is possible that BSP30 gene duplication is a characteristic feature of ruminants and that the BSP30 proteins contribute to an aspect of ruminant-specific physiology.

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Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic

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