Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation

Abstract

Rifampin is an important chemotherapeutic agent for use against tuberculosis, leprosy, and infections by organisms related to those causing these diseases (3, 7). The antimicrobial activity of rifampin is due to its inhibition of DNA-dependent RNA polymerase, and most rifampin-resistant bacteria have been reported to have an alteration in the ␤-subunit of this enzyme (2). Such a resistance mechanism has been reported for Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium africanum, and Mycobacterium leprae (14, 16). During our studies of fast-growing mycobacterial strains, we found that several strains inactivate rifampin (1, 5). We analyzed the inactivated antibiotics and found them to differ in mass spectrum and chromatographic mobility from those previously reported, i.e., the glycosylated (glucosylated) or phosphorylated compounds produced by pathogenic Nocardia spp. (11,17). These results prompted us to determine the detailed structures of the inactivated compounds. In this paper the isolation, structures, and antimicrobial activities of the inactivated compounds are reported.Rifampin was generously provided by CIBA-GEIGY Pharmaceuticals, Basel, Switzerland. MICs were determined by an agar dilution method with brain heart infusion agar (Difco Laboratories, Detroit, Mich.) medium. The inoculum size of each test organism was adjusted to 10 6 CFU/ml. The plates were spotted with a multipoint inoculator (A 400; Denly Instruments, Ltd., Sussex, England) that delivered 0.005 ml of inoculum, resulting in a spot inoculum of approximately 5 ϫ 10 4 CFU. Inactivation of rifampin was monitored by a bioassay method with Bacillus subtilis PCI 219 as a test organism (17). Inactivated compounds were monitored with a thin-layer chromatography scanner (CS-910; Shimadzu Seisakusho, Kyoto, Japan) (17, 18) at 238 nm.One loopful of the culture from slant cultures of Mycobacterium smegmatis DSM 43756 was inoculated into a 100-ml Erlenmeyer shake flask containing 20 ml of a seed culture medium (2% glycerol-enriched brain heart infusion medium; Difco Laboratories) with 4-mm-diameter glass beads to reduce aggregation of the mycobacterial cells (18). The inoculated flasks were shaken at 250 rpm (5.8-cm stroke) for 4 days at 33ЊC. For large-scale preparation of the inactivation products, the seed culture was used as an inoculum. Ten milliliters of seed culture was inoculated into 500-ml shake flasks containing 100 ml of the same medium. After 24 h of incubation at 33ЊC, rifampin (stock solution in methanol at 100 mg/ml) was added to a final concentration of 50 g/ml, and the mixture was incubated for 3 days. Following separation from mycelia by centrifugation at 6,000 rpm (5,800 ϫ g), the supernatant was adjusted to pH 2.0 with 1 N HCl and extracted three times with an equal volume of ethylacetate. The extracts were combined, dehydrated with Na 2 SO 4 , and concentrated in vacuo to dryness. The dried material was purified by silica gel column chromatography (column size, 3.5 by 15 cm) by using 75 g of Wakogel C-200 (Wako Pu...

A Rifampin-Hypersensitive Mutant Reveals Differences between Strains of Mycobacterium smegmatis and Presence of a Novel Transposon, IS 1623

“…Drug resistance can also be affected by rifampin modification. Bacteria, including some actinomycetes, are able to inactivate rifampin via glucosylation, phosphorylation, decolorization, and ribosylation (5,24). Atypical mycobacteria, especially rapidly growing species such as M. smegmatis, are naturally more resistant to rifampin (20).…”

“…Rifampin ribosylation has been described as a mechanism of mycobacterial drug resistance in several fast-growing species, including M. smegmatis, Mycobacterium chelonae, and Mycobacterium parafortuitum (5,12,19). Homologs of arr are not present in M. tuberculosis, M. leprae, M. avium, or other slowly growing species for which genome data are available.…”

Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

“…These enzymes use a number of strategies to overcome the toxic properties of antibiotics, including destruction of the antibiotic for example by hydrolysis, as well as drug modification by kinases, acyltransferases, adenylyltransferases, and glycosyltransferases. To this impressive arsenal of antibiotic countermeasures, research by Dabbs and colleagues has added ADP-ribosylation (3)(4)(5)(6)(7)(8)(9)(10).…”

“…Although well established as a protein modification mechanism of significant importance, small molecule ADPribosylation is unknown with one exception: a reported ADPribosylation of the antibiotic rifampin that results in resistance in bacterial pathogens (5,6,9,10). Rifampin is a semisynthetic derivative of the natural product rifamycin B derived over 40 years ago from Amycolatopsis mediterranei ( Fig.…”

Transposon Mutagenesis of the Obligate Intracellular Pathogen Rickettsia prowazekii

“…However, to our knowledge, such mutants have not been isolated. In this report, we describe an additional antibiotic selection, rifampin resistance generated by ribosylation of the antibiotic (8,18,23), that, when coupled with an efficient transposon mutagenesis system (10,11), provides a functional mechanism for the insertional mutagenesis of R. prowazekii and the identification of nonessential rickettsial genes.…”