Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5 ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.Tuberculosis (TB) caused by members of Mycobacterium tuberculosis complex is one of the common human diseases, causing 3,000,000 deaths per year worldwide (23). While the disease is associated with economic impoverishment, TB is on the rise in many industrialized nations. The spread of TB is due to immigration, the emergence of drug-resistant strains (24), and the AIDS epidemics. The increasing number of drug-resistant M. tuberculosis strains has made the rapid identification of susceptibility clinically important because those patients suffering from these strains require specialized antibiotic treatment.Recently, a number of genetic changes leading to rifampin, isoniazide, pyrazinamide (PZA), streptomycin, and kanamycin resistances have been characterized. Nearly 95% of rifampinresistant M. tuberculosis strains carry mutations in a certain region of the beta subunit of the RNA polymerase encoded by the rpoB gene (20, 21). The mutations correlated to isoniazide resistance were found on the katG (25), inhA (1), and ahpC/oxyR (26) genes. Ethambutol-resistant M. tuberculosis strains carried mutations on a certain part of the gene coding for alabinosyl transferase (2, 17). Mutations on the rrs and rpsL genes coding for the 16S ribosomal RNA and ribosome protein S12, respectively, confer streptomycin resistance (8, 11). A part of the kanamycin-resistant phenotype can be detected by identifying the mutations on the rrs gene (19). Missense mutations in the DNA gyrase gene (gyrA) have been reported to be associated with increased levels of resistance to fluoroquinolones (5).PZA is one of the most important drugs for anti-TB shortcourse chemotherapy. PZA is converted to pyrazinoic acid by mediation of pyr...

Metagenomic screening and conventional cultivation have been used to exploit microbial lipolytic enzymes in nature. We used an indigenous forest soil (NS) and oil-fed enriched soil (OS) as microbial and genetic resources. Thirty-four strains (17 each) of lipolytic bacteria were isolated from the NS and OS microcosms. These isolates were classified into the (sub)phyla Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria, all of which are known to be the main microbial resources of commercially available lipolytic enzymes. Seven and 39 lipolytic enzymes were successfully retrieved from the metagenomic libraries of the NS and OS microcosms, respectively. The screening efficiency (a ratio of positive lipolytic clones to the total number of environmental clones) was markedly higher in the OS microcosm than in the NS microcosm. Moreover, metagenomic clones encoding the lipolytic enzymes associated with Alphaproteobacteria, Deltaproteobacteria, Acidobacteria, Armatimonadetes, and Planctomycetes and hitherto-uncultivated microbes were recovered from these libraries. The results of the present study indicate that functional metagenomics can be effectively used to capture as yet undiscovered lipolytic enzymes that have eluded the cultivation-based method, and these combined approaches may be able to provide an overview of lipolytic organisms potentially present in nature.

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