Inactivation of rifampin by Nocardia brasiliensis

Abstract

Rifampin was glycosylated by a pathogenic species of Nocardia, i.e., Nocardia brasiliensis. The structures of two glycosylated compounds (RIP-1 and RIP-2) isolated from the culture broth of the bacterium were Rifamycins are members of the naphthalenic ansamycin group of antibiotics characterized by a 17-membered ansa chain (1,5,11,17 (Becton Dickinson, Cockeysville, Md.). One loopful of spores or mycelial fragments from the slant cultures of Nocardia spp. was inoculated into a 10-ml Erlenmeyer shake flask containing 5 ml of a seed medium (2% glucose-enriched brain heart infusion medium; Difco Laboratories, Detroit, Mich.) with 4-mm glass beads intended to reduce aggregation of the nocardial cells (21). The inoculated flasks were shaken at 250 rpm (5.8-cm stroke) for 4 days at 27°C. The mature seed cultures were used as inocula for the inactivation or MIC experiments. Other bacterial inocula were prepared by the method previously described (21).MIC determination. The MIC, defined as the lowest drug concentration causing complete inhibition of visible growth, was determined by an agar dilution method using MuellerHinton II agar medium to give final concentrations from 0.39 to 400 ,ug/ml. Drug-free plates were included as a bacterial growth control. The inoculum size of a test organism was adjusted to 107 CFU/ml for Nocardia spp. and 106 CFU/ml for other bacteria. The plates were spotted with a multipoint inoculator (A 400; Denly Instruments, Ltd., Sussex, England) that delivered 0.005 ml, resulting in a spot inoculum of approximately 5 x 104 CFU.Time course of rifampin inactivation. The mature seed cultures were inoculated at a 10% rate into 100-ml Erlenmeyer flasks containing 50 ml of brain heart infusion medium. Methanol-sterilized rifampin was added aseptically at a final concentration of 20 or 200 ,ug/ml. For determination of the rifampin concentration, 2 ml of the flask cultures was harvested at various time intervals and filtered. The pH of the filtrate was adjusted to 2.0 with 2 N HCl, and the filtrate was then extracted with 2 ml of ethyl acetate. After concentration of the extract in vacuo to dryness, 100 ,ul of methanol was added and 50 ,ul of the mixture was tested for antibacterial activity. The rifampin concentration was measured by the size of the inhibition zone on a lawn of Bacillus subtilis PCI 219 grown on nutrient agar (Difco) (15). Inactivation products were monitored by thin-layer chromatography (TLC) using CHCl3-MeOH (4:1) as a developing solvent. Although rifampin or its inactivation products were easily detected by their characteristic reddish brown spots on untreated TLC plates, the inactivation products were usually monitored additionally by a dual-wavelength TLC scanner (CS-91OM; Shimadzu Seisakujyo, Tokyo, Japan) at 238 nm. Growth was determined by measuring the dry weight of mycelia: specifically, 5 ml of the culture was harvested at 1313

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

“…At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl.…”

“…Covalent modification of RIF hydroxyls with ADP-ribose or phosphate results in high level resistance in Escherichia coli, such as a 64-fold increase in the RIF minimal inhibition concentration (6,10). Modification of RIF by Rgt results in a 4-fold increase of Streptomyces speibonae cultures (7) and markedly reduces the activity against Gram-positive bacteria (8).…”

“…1) is a potent frontline antibiotic against tuberculosis and other mycobacterial infections, but extensive usage of RIF and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity (1, 2). In addition to the point mutations in RNA polymerase that are responsible for resistance in mycobacteria (3, 4), some bacterial species, such as soil actinomycetes and parasitic bacteria, employ secondary enzyme-mediated inactivation mechanisms that chemically modify RIF to less active forms or degradation products (5).At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl.…”

Monooxygenation of rifampicin catalyzed by the rox gene product of Nocardia farcinica: structure elucidation, gene identification and role in drug resistance

“…1 As to rifampicin (RIF), the rpoB2 gene, a duplicated copy of the RNA polymerase b-subunit gene containing RIF-resistant amino acid substitutions, has been predicted to be a resistance determinant and its contribution to the RIF resistance of N. farcinica has been demonstrated experimentally. 2 On the other hand, decolorization of RIF by N. farcinica has been reported 3,4 and the sequenced strain IFM 10152 decolorizes RIF in prolonged culture (unpublished observation). This observation implies the participation of decolorization in RIF resistance.…”

Biotransformation of Bile Acids by Pathogenic Actinomycetes Nocardia otitidiscaviarum and Amycolatopsis sp. Strains

“…Our data showed that this inactivation is a species-specific phenomenon in this bacterium [6]. We also reported unique inactivation mechanisms of pathogenic Nocardia against clinically useful antibiotics such as rifampicin and erythromycin [7,8]. The present biotransformation of sterol-type compounds is commonly observed in pathogenic Nocardia species (data not shown), but our preliminary study indicates that other Nocardia species have a different transformation mechanism against bile acids (1, 2 and 3).…”