In vitro synergistic activity between 8-methoxypsoralen and ethambutol, isoniazid, and rifampin when used in combination against Mycobacterium tuberculosis

8-Methoxypsoralen (8-MOP), a naturally occurring furocoumarin found in many plant species, has been reported to have antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In the present study, we further test the in vitro synergistic activity of 8-MOP and ethambutol (EMB), isoniazid (INH), or rifampin (RMP) against M. tuberculosis. This study showed that 8-MOP has antimycobacterial activity against two drug-sensitive and six drug-resistant clinical isolates of M. tuberculosis, with the minimum inhibitory concentrations of 100–200 and 200–400 μg/mL, respectively. A synergistic antimycobacterial effect between 8-MOP and EMB, INH, or RMP against six drug-resistant strains was observed, with the fractional inhibitory concentration indices (FICIs) of 0.093–0.156, 0.138–0.285 and 0.093–0.262, respectively. The combination of 8-MOP/EMB, 8-MOP/INH, and 8-MOP/RMP displayed either synergistic activity or had no interaction when tested against the two clinical drug-sensitive strains and the standard strain. No antagonism was observed for any drug combination against any of the strains tested. To our knowledge, this is first report that 8-MOP has synergistic activity with first-line antimycobacterial agents.     

Determination of Drug Resistance of Mycobacterium tuberculosis Cultures

A total of 3,303 strains of Mycobacterium tuberculosis were tested for sensitivity to streptomycin (SM), isoniazid (INH), and p-aminosalicylic acid (PAS) by the Steenken modified minimal inhibitory concentration (MIC) test. A simultaneous double blind comparison was carried out on 277 selected strains by the Steenken MIC test and the Canetti proportion method. Agreement between the results for the two tests was 82% for SM, 95% for INH, and 89% for PAS. A small number of strains appeared to be sensitive when tested by one method but resistant by the other. MIC determinations were carried out on 83 strains by using Steenken-Smith, Lowenstein-Jensen, and Middlebrook 7H10 media containing a more extended range of concentrations of the test drugs. The MIC values for both SM and dihydrostreptomycin increased on Steenken-Smith medium compared with the other two. INH did not show any medium effect, whereas PAS showed increased MIC values in 7H10 agar. The significance of the comparisons of the MIC values on the various media is discussed in terms of possible changes in the drug sensitivity testing methods used at present in this laboratory.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

New Approach for the Evaluation of Antimycobacterial Drug Combinations In Vitro (the Laboratory Model Man)

An attempt was made to study quantitatively the antimicrobial effect of combinations of commercially available antituberculosis drugs and antibiotics on the growth of multiple drug resistant strains of Mycobacteriunt intracellulare under simulated in vivo conditions. Combinations of erythromycin, isomiazid, methenamine, or exacillin eliminated populations of M. intracellulare when drug combinations in concentrations achievable in man were kept in contact with the organism for 10 hr daily. Although combinations of INH and rifampin failed to eliminate populations of M. intracellulare this pair seemed to be the most effective two-drug combination available. The requirement for successful treatment of drug-resistant mycobacterioses is the selection of an effective drug regimen and the maintenance of combined action of all drugs in the serum for approximately 10 hr daily. An in vitro model is described which enables the bacteriologist to design an effective combination of drugs and to measure its efficiency under simulated in vivo conditions.     

Drug-Resistant Tuberculosis among HIV-Infected Patients Starting Antiretroviral Therapy in Durban, South Africa

Objective

To estimate the prevalence of drug-resistant tuberculosis (TB) and describe the resistance patterns in patients commencing antiretroviral therapy (ART) in an HIV clinic in Durban, South Africa.

Design

Cross-sectional cohort study.

Methods

Consecutive HIV-infected adults (≥18y/o) initiating HIV care were enrolled from May 2007–May 2008, regardless of signs or symptoms of active TB. Prior TB history and current TB treatment status were self-reported. Subjects expectorated sputum for culture (MGIT liquid and 7H11 solid medium). Positive cultures were tested for susceptibility to first- and second-line anti-tuberculous drugs. The prevalence of drug-resistant TB, stratified by prior TB history and current TB treatment status, was assessed.

Results

1,035 subjects had complete culture results. Median CD4 count was 92/µl (IQR 42–150/µl). 267 subjects (26%) reported a prior history of TB and 210 (20%) were receiving TB treatment at enrollment; 191 (18%) subjects had positive sputum cultures, among whom the estimated prevalence of resistance to any antituberculous drug was 7.4% (95% CI 4.0–12.4). Among those with prior TB, the prevalence of resistance was 15.4% (95% CI 5.9–30.5) compared to 5.2% (95% CI 2.1–8.9) among those with no prior TB. 5.1% (95% CI 2.4–9.5) had rifampin or rifampin plus INH resistance.

Conclusions

The prevalence of TB resistance to at least one drug was 7.4% among adults with positive TB cultures initiating ART in Durban, South Africa, with 5.1% having rifampin or rifampin plus INH resistance. Improved tools for diagnosing TB and drug resistance are urgently needed in areas of high HIV/TB prevalence.     

Paradoxical effect of Tween 80 between the susceptibility to rifampicin and streptomycin and the susceptibility to ethambutol and sulfadimethoxine in the Mycobacterium avium-Mycobacterium intracellulare complex.

The susceptibility to rifampicin and streptomycin of the Mycobacterium avium-Mycobacterium intracellulare complex was augmented by the addition of Tween 80 into 7H10 agar medium with OADC, whereas the susceptibility to ethambutol and sulfadimethoxine was either not changed or reduced by the addition of Tween 80. In 7H10 agar medium without OADC, however, susceptibilities to both rifampicin and sulfadimethoxine were reduced by the addition of Tween 80 to the medium. A number of hypotheses are made to explain these phenomena.     

In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase

Agrobacterium tumefaciens-transformed tobacco suspension-cultured cells (TSCC) exhibit no significant quantitative changes of cell wall invertase protein (CWI) during a culture period of 40 days, whereas CWI activity decreases strongly between 10 and 30 days after cell transfer to fresh medium. Western blot analysis revealed that the apoplasmic invertase inhibitor (INH) is equally expressed throughout the entire culture period. When apoplasmic protein fractions from 4 and 28 days old cell cultures are chromatographed on Concanavalin A(ConA)-Sepharose, the non-glycosylated INH always coelutes with the ConA-bound fraction, suggesting that (i) INH and the glycosylated CWI form a complex in the apoplasmic space, and (ii) INH binding is not sufficient for CWI inhibition. The high specificity of INH binding to CWI was confirmed by native cathodic polyacrylamide gel electrophoresis. Expression analysis of CWI and INH indicates that, at least during certain stages of plant development (seedlings, roots of adult plants), CWI activity may be modulated by INH, the latter operating as a regulatory switch.     

Application of Genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volum laboratory in Southern China

The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104). Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics. In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530–533 and 57.9% (11/19) were the S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches.     

The effect of tube closures and media on the enrichment of Campylobacter jejuni

Enrichment of C. jejuni in metal, Morton-style capped tubes gave no growth. Cotton wool or sponge rubber plugged tubes yielded enrichment of C. jejuni to 1.6 × 10³ cfu per ml from an inoculation of 0.18 cfu per ml in 10 ml medium.Enrichment of C. jejuni from egg melange in cotton plugged tubes and bottles showed that the ratio of egg melange to broth should not exceed 4:1 in bottles and 2:3 in tubes. When enriching from incubating fertile eggs infected by C. jejuni a decreasing quantitative and qualitative recovery was experienced with increasing time of egg incubation. Five enrichment broths and two selective plating media were compared in this experiment. The medium of Doyle and Roman (Appl. Environ. Microbiol. 43, 1343–1353 (1982)) and a routine enrichment broth plus rifampin (brucella broth containing per litre: 50 ml lysed horse blood, 10 mg rifampin, 5000 IU polymyxin B, 10 mg vancomycin, 5 mg trimethoprim lactate and the reductants of George et al. (J. Clin. Microbiol. 8, 36–41 (1978)) were superior to BNP broth. All media showed decreased enrichment of C. jejuni with increasing time of egg incubation, when growth was only of the order of 2 × 10² cfu per ml at day 15 of egg incubation. Rifampin was required to suppress contamination by Proteus species and Gram-positive cocci.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

Seawater requirement for the production of lipoxazolidinones by marine actinomycete strain NPS8920

A novel marine actinomycete strain NPS8920 produces a new class of 4-oxazolidinone antibiotics lipoxazolidinone A, B and C. Lipoxazolidinone A possesses good potency (1-2 microg/mL) against drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Strain NPS8920 exhibits different morphologies in both agar and submerged cultures. The ability of strain NPS8920 to sporulate on saline-based agar media but not on deionized water-based agar medium supported that strain NPS8920 is a marine actinomycete. While strain NPS8920 does not require seawater for growth, the production of lipoxazolidinones by strain NPS8920 can only be detected in the seawater-based media. The optimal production of lipoxazolidinones was observed in the natural seawater-based medium. Strain NPS8920 produced 10-20% of lipoxazolidinones in the synthetic sea salt Instant Ocean-based medium and no production in the sodium chloride-based and deionized water-based media.     

Rifampin

Rifampin is a potent antituberculous drug. In the treatment of drug-resistant tuberculosis it is highly effective provided it is given in combination with other drugs to which the patient''s organisms are sensitive. Rifampin and ethambutol is a particularly powerful combination and will achieve almost 100% sputum conversion. It seems likely that rifampin will replace streptomycin, and ethambutol will replace PAS in first-treatment cases. Optimum first-line treatment will thus consist of rifampin, INH and ethambutol, with the probability of almost 100% success and the possibility also that the total duration of treatment may be considerably reduced. Rifampin is well tolerated but it may give rise to liver dysfunction and thrombocytopenia in a small proportion of patients. Patients treated with rifampin must be kept under close supervision because of the risk of side effects and, more important, because irregular treatment may lead to the development of rifampin-resistant organisms.     

The Effects of Nutrient Concentration,Addition of Thickeners,and Agitation Speed on Liquid Fermentation of Steinernema feltiae

Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.     

Mechanism of antibiotic resistance in Mycobacterium intracellulare

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.     

Sensitivity of Rhizobium species to certain fungicides

Summary Thirty-three strains of different species of rhizobia were seeded into medium 79 agar. Discs impregnated with 3, 0.3, 0.03 and 0.003%, corresponding to 10, 1, 1/10 and 1/100 the recommended rate of application, of fungicides TMTD, Rhizoctole, Phygon, Ceresan and Orthocide 75 were placed on the seeded agar. After 120 hrs, zones of inhibition were measured. The 3, 0.3 and 0.03% concentrations of the five fungicides were inhibitory to most rhizobia tested in different degrees. The 0.003% level of these fungicides while toxic to certain strains, was not so for others.Deceased August 6, 1968.     

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare , like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H₂O₂ and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H₂O₂ and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.     

Flavanoids in Pollen and Stigma of Brassica oleracea and Their Effects on Pollen Germination In Vitro

Brassica oleracea pollen was applied to a basic medium of 1.5per cent agar and 15 per cent sucrose to which flavanoids wereadded at three concentrations. Two types of agar were used;with agar 1, quercetin at a concentration of 0.5 x 10^–3per cent gave an increase in percentage germinating grains.With agar 2, an increase in germination occurred with kaempferoland naringin at concentrations of 0.5 x 10^–3 and 0.5 x10^–1 per cent respectively. Increase in pollen tube lengthoccurred with agar 2 and quercetin at a concentration of 0.5x 10^–3 per cent. The stigma tissue of B. oleracea contains at least three andthe pollen at least one glycoside of quercetin. The sugars inthe glycosides were not identified. Pollen germination and pollentube extension were not stimulated exclusively by the flavanoidspresent in the stigma. The flavanoid composition of the stigmadid not vary amongst five different S-allele genotypes, indicatingthat flavanoids are probably not directly involved in the incompatibilityreaction of B. oleracea.     

Bacillus subtilis tolerance of moderate concentrations of rifampin involves the sigma(B)-dependent general and multiple stress response.

Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. L-[(35)S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The sigma(B)-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the beta subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the sigma(D) regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the sigma(B) signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.     

在培养基中加入抗生素防止万年青茎段培养污染的研究

以玛丽安万年青茎段为试验材料,将浓度为50mg·L1的青霉素、氯霉素、利福平、庆大霉素、先锋霉素6号、链霉素和卡拉霉素7种抗生素分别经过过滤灭菌后加入到MS基本培养基,从中筛选出防止污染效果较好的两种抗生素,分别按50、100、150mg·L1的浓度加入到MS培养基中培养,以筛选出最适合浓度;然后再筛选出利福平和氯霉素的最佳浓度组合。结果表明:7种抗生素中以利福平和氯霉素防止污染效果较好,未污染率分别为68.42%和58.33%,成活率分别为65.79%和52.78%,利福平和氯霉素的浓度均为150mg·L1时防止污染的效果最佳;25mg·L1利福平+50mg·L1氯霉素和50mg·L1利福平+50mg·L1氯霉素组合,未污染率分别达到78.46%、85.18%,极显著高于50mg·L1利福平单独处理的效果。     

Rifampin-resistant RNA polymerase in spirochetes

Abstract Various free-living and host-associated spirochetes isolated by methods not involving rifampin were resistant to relatively high concentrations of this antibiotic. The lowest concentrations of rifampin that were inhibitory for the spirochetes ranged from 50 to more than 200 μ g/ml, depending on the species. The spirochete strains examined were at least 10-fold more resistant to rifampin than Escherichia coli and 10 000-fold more resistant than Staphylococcus aureus . The results support the conclusion that rifampin resistance is a general characteristic of spirochetes. Resistance of Spirochaeta aurantia to rifampin was not the result of detoxification of the antibiotic in the culture medium. The activity of spirochete DNA-dependent RNA polymerase in vitro was completely resistant to 10 μg of rifampin per ml, a concentration that totally inhibited E. coli RNA polymerase. Higher concentrations decreased the spirochetal activity. Thus, rifampin resistance may be due to a low affinity of spirochete RNA polymerase for the antibiotic.