The effect of rifampicin onMycobacterium smegmatis

Rifampicin was found to inhibit the growth and incorporation of¹⁴C-adenine,¹⁴C-leucine and¹⁴C-glycine in exponentially growing cells ofM. smegmatis cultivated in Merrill’s synthetic medium. Increasing concentrations of the antibiotic inhibited respiration in resting cells, in the presence of glucose or 2-oxoglutarate as substrates in particular. In addition to the well-known interference of rifampicin with the biosynthesis of RNA, the effect on the energy metabolism should also be considered.     

Participation of various adenosine and guanosine derivatives in the abortive RNA synthesis initiation reaction: effect of Mg2+, Mn2+, and rifampicin

Adenosine, TMP, ADP, ATP and UpA along with guanosine and tis analogous derivatives have different reactivity towards [alpha-32P]UTP in abortive initiation reactions catalyzed by E. coli RNA polymerase on T2 DNA in the presence of Mg2+ or Mn2+. Rifampicin moderately inhibited almost all of the above mentioned reactions, except the ATP and the GTP which were even 2.5 times more reactive in the presence of this antibiotic.     

Interaction of rifampicin with hepatocyte organoids in rats and its intracellular distribution

Interaction of rifampicin with isolated subcellular fractions of the rat liver (binding and dissociation of the complexes) and intracellular distribution of the antibiotic were studied in the presence of the main organoids taken in the ratio close to the natural volume ratio of the hepatocyte cell components. The nuclei and mitochondria were most active during drug binding. The microsomes were less important. Rifampicin formed mobile complexes with organoids and was easily released from the subcellular fractions recovering its activity on washing. The intracellular distribution was the following: 37.7 per cent of rifampicin in the active form was accumulated in cytosol and the remaining amount was reversibly bound in the fractions of the nuclei, mitochondria and microsomes. The characteristic features of the cell pharmacokinetics of rifampicin, i.e. significant concentration in cytosol, possible deposition in the subcellular structures and at the same time the capacity for recovery of the activity might define the antimicrobial potential of this antibiotic in respect to the intracellular microorganisms.     

Action of tetracycline and rifampicin on Rickettsia prowazekii and Rickettsia sibirica

The effect of tetracycline and rifampicin on R. prowazekii, strain Breinl and R. sibirica, strain X1 was studied in the experiments with chick embryos exposed to the antibiotic mixture with the infection material. It was shown that tetracycline in doses of 0.1 and 1 mg/embryo had the rickettsiostatic and rickettsiocidic effects respectively on R. sibirica. Rifampicin had only the rickettsiostatic effect in a dose of 0.1 mg/embryo and no rickettsiocidic effect even in a dose of 2 mg/embryo. Higher doses were toxic for 100 per cent of the embryos. The rickettsiostatic and rickettsiocidic effects of tetracycline on R. prowazekii were evident in doses of 0.05 and 0.1 mg/embryo, respectively. Rifampicin in a dose of 0.05 mg/embryo had both the rickettsiostatic and the rickettsiocidic effects on R. prowazekii. Therefore, rifampicin was more active with respect to R. prowazekii and tetracycline was more active with respect to R. sibirica. In addition, R. sibirica was more resistant to both tetracycline and rifampicin as compared to R. prowazekii.     

An experimental model of chemotherapy on dormant tuberculous infection, with particular reference to rifampicin

Mice were infected intravenously with a streptomycin (SM)-dependent strain of tubercle bacilli which had been starved of the antibiotic. The inoculum persisted in the spleen for a fairly long period, especially keeping almost the initial level of viable counts in the first few weeks. Isoniazid (INH) administration exerted little effect on such fate of the infection at a bactericidal dose to the same strain multiplying under the supply of SM. Rifampicin (RFP) was, however, highly effective in either case. Similar results were obtained in the corresponding in vitro experiments. The data suggest that this animal model is a convenient system for screening drugs effective on the dormant tuberculous infection.     

The effect of rifampicin on the stability of the messenger ribonucleic acid of Bacillus amyloliquefaciens as determined by DNA:RNA hybridization.

Rifampicin at a concentration of 10 mug/ml completely inhibited protein synthesis in exponential-phase cultures of Bacillus amyloliquefaciens. At this same concentration the drug was shown to have no effect on the stability of mRNA, determined as the functional and hybridizable material, when compared with hybridizable mRNA in an uninhibited system. In each case, the half-life of the mRNA had a value in the range 5 +/- 1 min, at 30 degrees C.     

Lack of absorption and rapid elimination of Rifampicin as factors precluding tests against nuclear polyhedrosis in Spodoptera littoralis larvae

Rifampicin administered per os in artificial medium to first-instar larvae of Spodoptera littoralis together with nuclear polyhedrosis virus did not affect virus infection and resulted in total larval mortality. A pharmacokinetic study of this antibiotic was carried out by means of a microbiological assay. Using Bacillus thuringiensis var. thuringiensis as the test organism for assaying the antibiotic, as little as 0.5 μg of Rifampicin could be detected in the presence of hemolymph, and no such detectable amounts could be found in the hemolymph of sixth instar larvae which had been fed the antibiotic. Most if not all of the antibiotic was in the feces, excreted in active form. When given parenterally, Rifampicin was detected in the hemolymph 30 min after injection but not 24 hr thereafter, when active antibiotic was found concentrated in the first fecal pellets evacuated after injection. This disappearance is ascribed to excretion via the Malpighian tubules. The larval responses detailed above preclude the testing of Rifampicin as an antiviral drug against nuclear polyhedrosis.     

Transcription but not translation is required for EDTA-induced autolysis in Escherichia coli

Rifampicin, but not chloramphenicol or other inhibitors of translation, inhibited EDTA-induced autolysis in Escherichia coli. Inhibition of EDTA-induced autolysis in E. coli was also observed with nalidixic acid and novobiocin, inhibitors of topoisomerase II. Rifampicin or nalidixic acid-resistant mutants of E. coli were resistant to the inhibitory effect of the respective antibiotic on EDTA-induced autolysis. The implications of these studies in regard to our understanding of the regulation of autolysis in E. coli are discussed.     

Effects of rifampicin and doxycycline on the production of hydrogen peroxide by macrophages]

The influence of rifampicin and doxycycline on oxidative metabolism of macrophages was estimated in vitro by production of hydrogen peroxide. It was shown that low concentrations of rifampicin and doxycycline stimulated production of hydrogen peroxide by macrophages of guinea pigs. In concentrations of 1 to 10 micrograms/ml corresponding to the mean therapeutic ones doxycycline increased both the spontaneous and zymosan-induced production of hydrogen peroxide by the macrophages. The potentiating activity of doxycycline on the cells activated by opsonized zymosan was higher. The maximum increase in the induced production of hydrogen peroxide (by 40 per cent) was observed when the antibiotic concentration was 1 microgram/ml. Rifampicin in concentrations of 0.1 to 1 microgram/ml corresponding to the mean therapeutic ones stimulated the zymosan-induced production of hydrogen peroxide by the macrophages. The maximum increase in the production of hydrogen peroxide (by 22 per cent) was noted at the rifampicin concentration of 1 microgram/ml.     

Studies on alkaline phosphatase, catalase and z-galactosidase in Vibrio el tor under normal and rifampicin resistant conditions

Alkaline phosphate, catalase and beta-galactosidase activities of Vibrio et tor were decreased after acquisition of resistance towards rifampicin. Zn2+, Mn2+ and EDTA inhibited alkaline phosphatase which is most active with p-nitrophenylphosphate as substrate while Mg2+ was found to suppress alkaline phosphatase activity. Removal of EDTA however, restores the original activity. Rifampicin could not induce mutation of lactose nonfermenting Vibrio el for cells allowing them to grow on lactose as sole carbon source, z-galactosidase which is a constitutive enzyme in this case is repressed by glucose. This repression is overcome by cAMP.     

Interaction of rifampicin with nonprotein thiols in mycobacterium smegmatis

In this study, the interaction of Rifampicin (RIF) with cellular glutathione (GSH) in Mycobacterium smegmatis has been investigated. Minimum inhibitory concentration of RIF for M. smegmatis was demonstrated to be 17 micrograms ml-1 medium. Three subinhibitory concentrations viz. 5, 10 and 15 micrograms RIF ml-1 medium were used to study its interaction with cellular non protein thiols (NPSH). Maximum depletion (57.8%) in NPSH levels [5, 5'-dithiobis (2-nitrobenzoic acid) assay] was observed on second day when the cells were grown in the presence of 15 micrograms RIF ml-1 medium. When the same samples were assayed for GSH levels (glyoxylase assay) the depletion of GSH levels by RIF was still observed, confirming the earlier findings. GSH depletion paralleled with growth inhibition and reached to normal level on 5th day of growth. Cellular depletion of GSH was also observed when 3 day grown cells of M. smegmatis were exposed to various concentrations of RIF (20, 40 and 60 micrograms ml-1 medium) for different time intervals. Maximum depletion of NPSH levels was observed when 3 day grown cultures were treated with 60 micrograms RIF ml-1 medium for a period of 6 h. The results of this study clearly demonstrate that RIF depletes cellular GSH levels regardless of the fact that the drug is included in the medium before inoculating it or after the cells have been grown for a period of three days. The depletion of cellular GSH levels by RIF in M. smegmatis may contribute towards its antituberculous activity.     

A rapid and sensitive HPLC-MS method for the detection of plasma and cellular rifampicin

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC-MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze-thaw cycles, but were significantly affected by heat-inactivation (58 degrees C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8h period.     

Effect of rifampicin on tryptophanase induction in normal and rifampicin-resistantVibrio el tor

The induction of tryptophanase was not affected by rifampicin in the rifampicin-resistant mutant ofVibrio el tor while the antibiotic inhibited the induction of tryptophanase in the normal strain at level of ribonucleic acid and protein synthesis.     

Successful outcomes with oral fluoroquinolones combined with rifampicin in the treatment of Mycobacterium ulcerans: an observational cohort study

Background

The World Health Organization currently recommends combined streptomycin and rifampicin antibiotic treatment as first-line therapy for Mycobacterium ulcerans infections. Alternatives are needed when these are not tolerated or accepted by patients, contraindicated, or neither accessible nor affordable. Despite in vitro effectiveness, clinical evidence for fluoroquinolone antibiotic use against Mycobacterium ulcerans is lacking. We describe outcomes and tolerability of fluoroquinolone-containing antibiotic regimens for Mycobacterium ulcerans in south-eastern Australia.

Methodology/Principal Findings

Analysis was performed of prospectively collected data including all primary Mycobacterium ulcerans infections treated at Barwon Health between 1998 and 2010. Medical treatment involved antibiotic use for more than 7 days; surgical treatment involved surgical excision of a lesion. Treatment success was defined as complete lesion healing without recurrence at 12 months follow-up. A complication was defined as an adverse event attributed to an antibiotic that required its cessation. A total of 133 patients with 137 lesions were studied. Median age was 62 years (range 3–94 years). 47 (34%) had surgical treatment alone, and 90 (66%) had combined surgical and medical treatment. Rifampicin and ciprofloxacin comprised 61% and rifampicin and clarithromycin 23% of first-line antibiotic regimens. 13/47 (30%) treated with surgery alone failed treatment compared to 0/90 (0%) of those treated with combination medical and surgical treatment (p<0.0001). There was no difference in treatment success rate for antibiotic combinations containing a fluoroquinolone (61/61 cases; 100%) compared with those not containing a fluoroquinolone (29/29 cases; 100%). Complication rates were similar between ciprofloxacin and rifampicin (31%) and rifampicin and clarithromycin (33%) regimens (OR 0.89, 95% CI 0.27–2.99). Paradoxical reactions during treatment were observed in 8 (9%) of antibiotic treated cases.

Conclusions

Antibiotics combined with surgery may significantly increase treatment success for Mycobacterium ulcerans infections, and fluoroquinolone combined with rifampicin-containing antibiotic regimens can provide an effective and safe oral treatment option.     

Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.     

Cloning of genes that have environmental and clinical importance from rhodococci and related bacteria

Generalised and specialised transduction systems were developed for Rhodococcus by means of bacteriophage Q4. The latter was used in conjunction with DNA from an unstable genetic element of R. rhodochrous to construct resistance plasmids which replicate in strains of R. equi, R. erythropolis and R. rhodochrous. One of the plasmids, pDA21, was joined with Erythropolis coli suicide vector pEcoR251 to obtain shuttle plasmids maintained in both rhodococci and E. coli. Conjugation between these rhodococcal strains demonstrated all were interfertile with each other and that some of the determinants for this were located on the unstable genetic element. Plasmids derived from this element, such as pDA21, carried the conjugative and self- incompatibility capacities; deletion analysis revealed that DNA necessary for self-incompatibility overlapped with that for arsenic resistance.Rifampicin is one of the principal chemotherapeutic agents used to treat infections by rhodococci and related organisms. The genes responsible for two types of inactivation have been cloned. The sequence of the R. equi DNA responsible for decomposition of the antibiotic strongly resembled those of monooxygenases acting upon phenolic compounds, consistent with the presence of a naphthalenyl moiety in the rifampicin molecule. Antibiotic resistance conferred by the gene was surprisingly specific to the semisynthetic compounds rifampicin (150-fold increase) and rifapentine (70-fold). Similar specificity was observed with the other inactivation gene cloned, which ribosylates rifampicin at the 23-hydroxyl position. A 60-bp sequence upstream of the monooxygenase and ribosylation genes is strikingly similar suggesting a shared pattern of regulation.Rhodococcal arsenic resistance and azo dye degradation genes have been cloned and characterised.     

Effect of rifampicin on the infectivity of RNA bacteriophage f2.

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10-100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA.