The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.

A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.

Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drugscreening strategy. Mycobacterium tuberculosis remains a leading cause of morbidity, tuberculosis (TB), and mortality in the world. M. tuberculosis is intrinsically resistant to most classical antibiotics, partly because of its impermeable cell wall (1-6). Due to selective mutations in M. tuberculosis, almost one-third of new TB patients are now infected with first-line drug-resistant strains, monoresistant strains, or multidrug-resistant (MDR) strains. Consequently, second-line therapies are often implemented, leading to the appearance of extensively drug-resistant (XDR) strains (7,8). There is therefore a growing urgency in the need for new antimycobacterial therapies.The mycobacterial cell wall is composed of peptidoglycan covalently attached to arabinogalactan, which are in turn esterified by very-long-chain mycolic acids. Various noncovalently attached lipids are embedded at the outer surface and necessary for capsule formation. Among these lipids, two related waxes, phthiocerol dimycocerosates (PDIM) and phenolic glycolipids (PGL), are involved in virulence (9-11). PDIM and PGL are only or mostly, respectively, found in pathogenic mycobacteria, but their roles in antibiotic resistance remain unclear (12-16). In Mycobacterium marinum, a mild (2-to 10-fold) increase in antibiotic susceptibility was observed in PDIM-and PGL-deficient strains (14, 15). In contrast, in PDIM-and PGL-deficient M. tuberculosis, no change was detected (13).The present study aimed to understand how mycobacteria can become susceptible to glycopeptides. Using PDIM-negative and/or PGL-negative strains of Mycobacterium bovis BCG and M. tuberculosis, we investigated the correlation between the absence of PDIM and the glycopeptide susceptibility. Subsequently, we investigated whether vancomycin could synergistically inhibit MDR and XDR strains with a mycobacterial lipids synthesis inhibitor.We recently reported that the chaperonin Cpn60.1/GroEL-1/ Hsp60-1 of M. bovis BCG was necessary for the integrity of the cell wall as the ⌬cpn60.1 strain showed an abnormal mycobacterial cell wall with a lack of PDIM and mycolates with two more carbon atoms (17). We investigated the susceptibility of the wild-type (WT), ⌬cpn60.1, and complemented ⌬cpn60.1 M. bovis BCG (GL2 strain) strains to several antituberculosis drugs. We used the NCCLS agar proportion method (18) to determine the MIC scale range of each antibiotic. We inoculated equal quantities of s...

Microbiological cultures are moderately sensitive for diagnosing prosthetic joint infection (PJI). This study was conducted to determine whether amplificationbased DNA methods applied on intraoperative samples could enhance PJI diagnosis compared with culture alone in routine surgical practice. Revision arthroplasty was performed for suspected PJI (n ‫؍‬ 41) and osteoarthrosis control (n ‫؍‬ 28) patients , and a diagnosis of PJI was confirmed in 34 patients. Amplification by polymerase chain reaction was performed on both 16S ribosomal DNA universal target genes and femA Staphylococcus-specific target genes. Species identification was achieved through amplicon sequencing. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Microbiological and molecular assays identified a causative organism in 22 of 34 patients (64.7%) and in 31 of 34 patients (91.2%), respectively. In 18 of the 22 culture-positive patients, molecular and microbiological results were concordant for bacterial genus , species , and/or methicillin resistance. Bacterial agents were identified only by molecular methods in nine PJI patients , including seven who were receiving antibiotics at the time of surgery and one with recent but not concomitant antibiotherapy. DNA-based methods were found to effectively complement microbiological methods , without interfering with existing procedures for sample collection , for the identification of causative pathogens from intraoperative PJI samples , especially in patients with recent or concomitant antibiotherapy. (J Mol