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Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser och 1 patent.

<p>Mycobacterium abscessus (Mab) is a non-tuberculous mycobacterium that is highly resistant to many antibiotics. Mab causes pulmonary infections in immunocompromised individuals. The presence of efflux pumps to pump out antibiotics and its ability to form biofilms makes Mab a virulent pathogen. Studies have been done on Mab antibiotic tolerance but there are still a lot of gaps in knowledge about the effects of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis in this bacterium during biofilm formation. In this study, we investigated the effects of the EPIs chlorpromazine (CPZ), 1-(1-naphthylmethyl)-piperazine (NMP), thioridazine (TRZ), Phenylalanine-arginine β-naphthylamide (PBN) and plumbagin (PLU) on antibiotic resistance, efflux, biofilm formation and lipid biosynthesis associated with log-phase growth and biofilm formation in Mab. We used the resazurin assay to determine the minimum inhibitory concentration (MIC) of the EPIs. We investigated the effects of the EPIs during biofilm-forming growth conditions on the MICs of antibiotics such as clarithromycin, amikacin, cefoxitin, ciprofloxacin which are the frontline antibiotics used to treat non-tuberculous mycobacterial infections. We also assessed the effects of the EPIs on the accumulation and efflux activities of the Mab cells through ethidium bromide (EtBr) assay. Furthermore, we evaluated the effects of the EPIs at sub-MIC concentrations on Mab biofilm formation under normoxic and hypoxic conditions. We utilized metabolic radiolabeling methods using 14C-palmitic acid and 14C- acetic acid which are precursors of lipid biosynthesis and analyzed lipids by silica-thin layer chromatography and autoradiography. We observed that Mab cells developed higher tolerance to the EPIs in a biofilm-stimulating medium. Furthermore, a decrease in the MICs of antibiotics was observed in the presence of the EPIs. Also, in the presence of the EPIs, there was less efflux activity within the Mab cells. In addition, EPIs inhibited biofilm formation significantly. We also noticed that NMP and PBN inhibited 14C-palmitic acid and 14C- acetic acid incorporation into polar lipids such as glycopeptidolipids, trehalose monomycolate, phosphatidylethanolamine, phosphatidylglycerol/cardiolipin, phosphatidylinositol mannosides at specific tested conditions. Our findings suggest that the EPIs inhibited the activities of the efflux pumps associated with the efflux of the antibiotics and lipid biosynthesis involved in biofilm formation. In conclusion, the results from this study gives insights on possible therapeutic opportunities.</p>

Tuberculosis remains one of the oldest diseases known to mankind but still persists as a very major risk. Discovery of several antimycobacterials was marked by a steady decline in the active cases of pulmonary tuberculosis. However, in the recent past there has been a surge in its clinical incidence. The reasons for this failure are the emergence of multi drug resistance and the ability of the organism to survive under extreme condition or for a very long period of time known as ‘persistence’. The latter one established itself as a major hindrance in the effective control of tuberculosis. The latent bacilli are confined for a very long period after the infection in caseous cavities, called granulomma, inside the host and gets reactivated any time when the host becomes immuno-compromised. Latency has been successfully simulated in vitro by various models which mimic the in vivo condition by depleting O2 (Wayne, 1977), nutrients (Nyka, 1974) or the carbon source (Ojha et al., 2000). Stationary phase is exemplary of a stage in bacterial growth where the organism is exposed to various stresses like nutrient starvation, accumulation of toxic metabolites, low temperature, high osmolarity and acidity to name a few. Some evidences suggest that cells survive in nutrient deprived stationary phase. The present investigation was pursued with an objective to further our understanding on the mechanism of adaptation that the persistent mycobacterium may undertake to survive during the stationary phase of growth. The fast growing M.smegmatis, a nonpathogenic member in the non-tuberculous genera, however, with a genetic and metabolic similarity to M.tuberculosis has been used as a model for this study. Chapter 1 introduces the challenges in tuberculosis therapy and discusses the reason for drug tolerance and persistence of M.tuberculosis and M.avium complexes that were uncovered recently. It focuses on the intricate lipid rich cell wall which forms the first barrier for drug delivery with an emphasis on the cell surface antigenic glycolipids, the glycopeptidolipids. A detail account of their structure, biosynthetic pathway, intracellular function and their implications on biofilm formation has been provided. The evolution of the genetic approaches currently used in mycobacterial research is highlighted. The transcription apparatus and the regulation of gene expression in mycobacteria at different environmental condition and stages of growth are also discussed. The need for a detail investigation of the stationary phase RNAP in mycobacteria is stressed. Chapter 2 observes the changes in the cell surface of M.smegmatis at different ambience of growth. It focuses on the composition of glycopeptidolipid, one of the non-covalently attached free lipids and the mycolic-acids which are covalently attached to the inner layer of the cell wall. Composition of the mycobacterial cell wall with respect to the glycopeptidolipids and mycolic acids during biofilm mode of growth is also addressed. This chapter examines the conditional synthesis of a novel class of polar glycopeptidolipid in carbon starved cultures of M.smegmatis and determines their molecular structure. Chapter 3 revisits the biosynthetic pathway of the glycopeptidolipids and justifies a need for a fresh perspective. It identifies a glycosyltransferase responsible for the transfer of an extra rhamnose to the existing rhamnose linked to the terminal alaninol in the new polar glycopeptidolipid. This chapter also identifies a conserved Polyketide synthase in the glycopeptidolipid biosynthetic cluster. Characterization of the domains present in its two distinct modules with a correlation to the structure of the fatty acylchain together with the formation of a hydroxy fatty acyl chain is delineated. Chapter 4 deals with the construction of a suicide vector which when recombines to the mc2155 genome, incorporates a hexa-histidine tag at the C’ of the β΄ subunit of the RNAP. It details the strategy for the construction of the strain and established the genetic exchange event both genotypically and phenotypically. A single step procedure for purification of the holo-RNAP is also described in this chapter. In chapter 5 the role of the mycobacterial principal likes sigma factor, SigB, at the stationary phase of growth is highlighted. An approach of expression proteomics involving differential display of the total protein was undertaken to investigate the genes that are under the SigB regular during the stationary phase of growth. This chapter also examines the stationary phase induced changes in the RNAP. Various proteins that interact with the assembly are identified and their role in conferring rifampicin resistance to the RNAP is described. Appendix 1 details the preparation of the partially methylated deoxy monosaccharide using the esoteric reactions of organic synthesis. They were used extensively for glycosyl linkage analysis of the glycopeptidolipids by mass spectrometry, where they acted as standards.