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The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis. Conventional susceptibility testing for the front-line tuberculosis drug pyrazinamide (PZA) is difficult, because of the requirement for acid pH for the drug to show activity. Resistance to PZA in M. tuberculosis is caused by mutations in the pncA gene, and detection of pncA mutations can be an indicator of PZA resistance. In this study, we examined the feasibility of a microarray-based approach exploiting short overlapping oligonucleotides (sliding-frame array) to rapidly detect pncA mutations (substitutions, deletions, and insertions) in multiple strains of PZA-resistant M. tuberculosis. The genetic mapping of these mutations is necessary to link the gene sequence to the protein function defined by mutant phenotype. Microarray analysis was performed in a blind manner using 57 isolates of M. tuberculosis for which the sequence of the pncA gene was previously determined. Our results showed that all mutations could be unambiguously detected, suggesting that microarray can be a routine and valuable tool for rapid identification of drug-resistant M. tuberculosis isolates. We expect that mutation mapping with a sliding-frame microarray will accelerate the molecular analysis of drug-resistant M. tuberculosis bacteria and the microorganism populations.

Background: Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high in vivo steril- izing activity but poor in vitro activity. This separation in pyrazinamide activity reflects differences between the in vivo tissue environment and in vitro culture conditions. The well-known acid pH requirement for pyrazin- amide activity was discovered previously based on such reasoning but does not completely explain the discrepancy between in vivo and in vitro activity of pyrazinamide. This study examined the effect of iron, which could potentially be elevated in local inflammatory lesions, on pyrazinamide activity in vitro. Materials and methods: The effect of iron on the activity of pyrazinamide or its active derivative pyrazinoic acid against Mycobacterium tuberculosis was assessed in liquid medium in a drug exposure assay or in solid medium with pyrazinamide plus iron or pyrazinamide alone. The effect of iron on pyrazinamide or pyrazinoic acid was expressed as percentage of growth inhibition. Results: We have shown that iron enhances the activity of pyrazinamide and pyrazinoic acid against M. tuberculosis in both liquid and solid media at acid pH 5.6. Iron enhanced the activity of pyrazinoic acid but not pyrazinamide against the naturally pyrazinamide-resistant Mycobacterium bovis BCG. Other metal ions such as magnesium, calcium and zinc did not enhance the activity of pyrazinamide or pyrazinoic acid. Conclusions: Iron increased the activity of pyrazinamide or pyrazinoic acid against M. tuberculosis in vitro. These findings may have implications for the study of mechanism of action of pyrazinamide and possible iron supplement for improving the activity of pyrazinamide.

The capsule of Pasteurella multocida serotype A strain ATCC 11039 is composed of hyaluronic acid and is an important virulence factor. Repeated subculturing of certain capsular serotype A strains results in dissociation from a capsulated to a noncapsulated phenotype with a concomitant loss of virulence. Although noncapsulated variants have been thought to arise as a result of mutation, the molecular mechanisms underlying this event are unknown. In this study, we demonstrate that restoration of the capsulated phenotype occurs in vivo subsequent to intraperitoneal inoculation of BALB/c mice with a noncapsulated variant. Moreover, reverse transcription polymerase chain reaction analysis revealed the capsule locus to be under transcriptional control. Cloning and sequencing of a 290-bp fragment within the promoter containing intergenic region of the capsule locus of 11039/iso revealed no significant alterations occurred subsequent to subculturing. These results demonstrate that serotype A P. multocida strain ATCC 11039 regulates capsule expression in response to an unidentified environmental factor(s), thereby providing insights into the molecular mechanisms underlying colonial dissociation.

Serotype A extracellular polysaccharide produced by Pasteurella multocida is composed largely of hyaluronic acid and is an important virulence factor in avian pasteurellosis. Hyaluronidase, mechanical shearing, and serial subculturing were employed to decapsulate strains exhibiting disparate degrees of capsulation, thereby allowing an investigation of the role serotype A capsulation plays in outer cell envelope physiology and virulence of avian isolates. Experimental loss of capsular material occurred concomitantly with an overall increase in cell surface hydrophobicity values, which approached those of naturally-occurring noncapsulated strains. The electronegativity of capsulated cell surfaces was found to be significantly greater than that of both noncapsulated and experimentally decapsulated cells. Naturally-occurring noncapsulated variants were readily phagocytized by mouse peritoneal macrophages, whereas capsulated strains were not. A moderately capsulated variant was phagocytized less effectively than noncapsulated strains. Experimental decapsulation of capsulated variants did not significantly hinder their ability to resist phagocytosis. Moreover, small amounts of residual capsular material appeared to remain after experimental decapsulation as detected with the aid of light and transmission electron microscopy. These data suggest that while cell surface hydrophobicity, charge, and susceptibility to phagocytosis are influenced by the degree to which cells are capsulated with serotype A extracellular polysaccharide, other factors which are unaffected by experimental decapsulation may also be involved in protection from phagocytosis in capsulated avian isolates of P . multocida.

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn 10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn 10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017—1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn 10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn 10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Background Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high sterilizing in vivo activity but poor in vitro activity. Pyrazinamide is thought to act by the entrapment of pyrazinoic acid in the bacterial cell, leading to acidification and membrane damage. Consequently, the effects of weak acids and molecules affecting membranes added to pyrazinamide were studied. Objectives To examine the effects of weak acids, UV, oxidative stress and additional energy inhibitors on pyrazinamide activity in vitro against Mycobacterium tuberculosis as well as the effect of pyrazinamide on Escherichia coli ampicillin persisters. Methods Drug exposure experiments followed by cfu counts were performed to determine the effects of the above various factors on pyrazinamide activity in vitro against M. tuberculosis. Results Some weak acids such as benzoic acid, sorbic acid and propyl hydroxybenzoic acid could enhance the activity of pyrazinamide in vitro against old tubercle bacilli but not young bacilli whereas other weak acids such as salicylic acid and lactic acid did not appear to enhance pyrazinamide activity. While energy inhibitors carbonylcyanide m-chlorophenylhydrazone and dinitrophenol enhanced pyrazinamide activity for old tubercle bacilli but not young bacilli, valinomycin and KCN increased pyrazinamide activity for both young and old bacilli. Oxidative stresses due to H2O2 and menadione did not have a significant effect on pyrazinamide activity. UV, which presumably damages the membrane, enhanced the activity of pyrazinamide. Pyrazinamide, which otherwise has no activity against actively growing E. coli bacteria, could kill non-growing starved E. coli and also ampicillin-tolerant persisters. Conclusions Some weak acids, UV and various energy inhibitors were found to enhance the activity of pyrazinamide in vitro against M. tuberculosis. Pyrazinamide shows preferential activity against both M. tuberculosis and E. coli persisters over the growing forms.

Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen.

Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.

Tuberculosis is a worldwide health problem posing increasing threat with the spread of HIV infection and drug resistant Mycobacterium tuberculosis strains. Consequently, control of this disease has become a significant challenge despite the availability of chemotherapy and BCG vaccine. Drug resistance for all first-line anti-tuberculosis agents and some second-line agents has been observed. Moreover, the occurrence of strains of M. tuberculosis resistant to multiple anti-tuberculosis drugs is increasing. Mechanisms of action and resistance of major anti-tuberculosis drugs are reviewed. In addition, the phenotypic drug resistance such as dormant or persistent tubercle bacilli and its importance are also emphasized. In order to combat the threat of drug resistant tuberculosis and to more effectively control the disease, an understanding of the mechanisms underlying drug resistance is necessary. This knowledge could be used for the development of molecular tests for rapid detection of drug resistant bacilli and future anti-tuberculosis drugs.