B-Lactamase of mycobacterium smegmatics strains ATCC 27204 and 19979: physiological, biochemical and genetic studies.

Abstract

The mechanisms of resistance to lactam antibiotics have been studied in both Gram positive and Gram negative bacteria. Lactamases play an important role in causing lactam resistance. They render the drugs antibiotically inactive by hydrolysing the lactam ring. The physiological, biochemical and genetic aspects of lactamase mediated resistance have been well documented only in a few genera. Mycobacteria are Gram positive organisms which include important pathogens such as M. tuberculosis and M. leprae, as well as several saprophytic species. Although lactam resistance is known in these organisms, the role of lactamase has not been established. The physiological, biochemical and genetic aspects of lactamase mediated resistance have been undertaken in the present study. The investigations are mostly confined to two non pathogenic and fast growing organisms, viz., M. smegmatis SN2 and M. butyricum ATCC 19979. M. smegmatis SN2 and M. butyricum ATCC 19979 showed resistance to a variety of lactams tested. The size of the inoculum or permeability barrier did not appear to contribute to the lactam resistance. The drug resistance was evidently mediated through the constitutive production of lactamase in both organisms. The levels of enzyme produced were comparable to those of Gram negative bacteria. Lactamase was not released on spheroplasting of M. smegmatis SN2 and was not accessible to the antibodies raised against it, suggesting that the enzyme was not located on the outer surface of the cell membrane. Culture supernatants of M. smegmatis SN2 and M. butyricum contained high levels of extracellular lactamase activity. The extracellular enzyme level varied with growth conditions like additional carbon source and pH of the medium. Cell wall damage and/or autolysis was responsible for the extracellular lactamase activity. Lactamases from M. smegmatis SN2 and M. butyricum ATCC 19979 have been purified by column chromatographic and gel filtration techniques to homogeneity. The molecular weights (30,000 and 29,000 respectively) and isoelectric points of the enzymes have been determined. Both the lactamases showed similar substrate profiles and resembled the lactamases mediated by Escherichia coli TEM type factors commonly encountered in Gram negative bacteria. On the basis of substrate profile, these enzymes appeared to fit best with the class III lactamases. With respect to isoelectric point, the above enzymes were different from mycobacterial lactamases reported previously or other plasmid mediated Gram negative enzymes. These enzymes were also insensitive to inhibition by p chloromercuribenzoate and sodium chloride. The antibodies raised against purified lactamase from M. smegmatis SN2 stimulated the enzyme activity by nearly 100%. The enzymes from M. butyricum ATCC 19979 and Escherichia coli HB101 (pBR322) were also stimulated by the antibody by 37% and 12% respectively. Immunoprecipitation of these enzymes could be accomplished by the antibody, indicating that they shared common antigenic determinants. Treatment of M. smegmatis SN2 with acridine orange resulted in decreased resistance against benzylpenicillin. These cells produced only one fourth the amount of lactamase compared to the parental cells. On the contrary, acridine orange treatment of M. butyricum ATCC 19979 did not result in the loss of lactam resistance. The observations were suggestive of the presence of plasmid coded lactamase in M. smegmatis SN2. In order to screen for the presence of any plasmid in these organisms, an efficient and gentle method applicable in general for the lysis of different mycobacterial species was established. The method essentially involved glycine sensitization of mycobacterial cells followed by treatment with a combination of lysozyme and lipase at 40°C to lyse them. Addition of EDTA and detergent released the supercoiled plasmid DNAs. Using this method, the presence of plasmids has been demonstrated in M. smegmatis SN2, M. butyricum ATCC 19979 and M. tuberculosis H37Rv. Under these conditions, however, no plasmid could be detected in M. tuberculosis H37Ra. The molecular weight of the plasmids detected in the above organisms was estimated to be in the range of 2-4 × 10 . A 2.9 Kb plasmid present in M. smegmatis SN2 showed sequence homology to the 5' end of the bla gene of Tn3 transposon. This plasmid was absent in the partially resistant variants isolated after acridine orange treatment. Even though the ~4 Kb plasmid present in the parental M. butyricum ATCC 19979 could not be detected in cells treated with acridine orange, they retained their lactam resistance to the same level. These results suggested that lactamase in M. smegmatis SN2 was partially mediated by plasmid, whereas in M. butyricum ATCC 19979 it was chromosomally mediated.