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Biomedical subjects

N Rastogi

Publications and source records attributed to N Rastogi.

At least 145 records · Page 8Linked to original sources

Growth and cell division of Mycobacterium avium.

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.

Bacterial Proteins↗

Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for exclusion of antimicrobial agents?

Whole cells of Mycobacterium avium, characterized by their negative response in the nine biochemical tests used for mycobacterial identification in our laboratory, turned positive for nitrate reductase, Tween-80 hydrolysis, beta-glucosidase, acid phosphatase, alkaline phosphatase, penicillinase, and trehalase after their wall portion was removed to yield spheroplasts. This suggested that the negative results in most of the biochemical procedures were caused by the exclusion mechanism at the wall level. Preliminary transmission and scanning electron microscopic studies showed differences at wall level between laboratory-maintained opaque, dome-shaped (SmD) and host-recycled smooth, transparent (SmT) colony type variants of M. avium and suggested the presence of an outer regularly structured layer in SmT variants. Comparative ultrastructural studies utilizing different polysaccharide coloration methods confirmed the presence of an outer polysaccharide layer in SmT variants which was probably related to their enhanced pathogenicity for experimental animals and drug resistance as compared to that of SmD variants. These findings are discussed with respect to multiple drug resistance, virulence, and gene expression of M. avium.

Anti-Bacterial Agents↗

Action of antituberculous and beta-lactam drugs (including imipenem) against extra- and intra-cellularly growing Mycobacterium avium-intracellulare.

The extracellular and intracellular activities of 4 antituberculous drugs (rifampicin, streptomycin, ansamycin and kanamycin) and 7 beta-lactam antibiotics (penicillin G, amoxycillin, carbenicillin, ticarcillin, ampicillin, cloxacillin and imipenem) were determined against the bacteria of the Mycobacterium avium complex (MAC). The extracellular activities were determined against 14 strains by the broth-dilution method, whereas the intracellular activities were determined against type strain ATCC 15769 multiplying actively inside murine J-774 macrophages. Six of the beta-lactam drugs (except the beta-lactamase-stable drug imipenem) were also used in association with the beta-lactamase inhibitor clavulanic acid. Our results showed that the screening of MAC strains against beta-lactams may be useful, and that correlation of usual in vitro minimal inhibitory concentrations with the intracellular activities of the same drugs against pathogenic mycobacteria, whenever feasible, would be the method of choice to designate the therapeutic protocols against these intracellular pathogens.

Animals↗

Drug susceptibility testing in tuberculosis: a comparison of the proportion methods using Lowenstein-Jensen, Middlebrook 7H10 and 7H11 agar media and a radiometric method.

In the present investigation, the conventional drug susceptibility testing procedures using the 1% proportion method on Löwenstein-Jensen (LJ) and Middlebrook 7H10/7H11 agar media were compared with a recent radiometric method. The latter using "Bactec 460-TB" apparatus and a special broth medium (Becton and Dickinson Diagnostic Instrument Systems, Towson, MD, USA) gave drug susceptibility results within a week as compared to 4-5 weeks with the LJ method, and 3-4 weeks using 7H10/7H11 agar media. This report describes the critical concentrations retained for each of the methods and the methodology used. Also, the compatibility of the results obtained with the radiometric method with those obtained with conventional 7H11 agar method was perfect. In this report, we justify the choice of the 7H11 agar method instead of the LJ method as our conventional drug susceptibility procedure, and suggest also that radiometric drug susceptibility determination by Bactec 460-TB apparatus may be the method of choice for routine, rapid drug susceptibility determinations.

Colony Count, Microbial↗

Selective inhibition of the Mycobacterium tuberculosis complex by p-nitro-alpha-acetylamino-beta-hydroxypropio phenone (NAP) and p-nitrobenzoic acid (PNB) used in 7H11 agar medium.

The potential of p-nitrobenzoic acid (PNB) and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) for discriminating the Mycobacterium tuberculosis (M.tb) complex from other mycobacteria was evaluated in cases of clinical isolates. For this purpose, 500 micrograms/ml of PNB and 5 or 10 micrograms/ml of NAP were incorporated in 7H11 agar medium, and appropriate dilutions corresponding to about 10(6) viable units were then plated on PNB- or NAP-containing media. Results were reported as growth or no growth as compared to a parallel control. Our data reconfirmed the potential of PNB to discriminate M.tb complex bacteria. This study also showed for the first time that the NAP test (used until now in 7H12a broth only) can also be successfully applied using 7H11 agar. Also, preliminary data with 5 micrograms/ml of NAP on a limited number of strains suggested that it may discriminate the M.tb complex bacilli from M. xenopi and M. gastri better than PNB in 7H11 agar.

Antitubercular Agents↗

Rapid preliminary differentiation of species within the Mycobacterium tuberculosis complex: proposition of a radiometric method.

A radiometric method using the "Bactec 460-TB" apparatus now enables rapid obtaining of drug-susceptibility data and differentiation of clinical mycobacterial specimens into those belonging to the Mycobacterium tuberculosis complex (comprised of M. tuberculosis, M. bovis, M. bovis BCG and M. africanum) and non-tuberculous mycobacteria, using a specific inhibitor (p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone). In the present work, further differentiation of the M. tuberculosis complex into 4 individual species was achieved using the susceptibility of strains to 1.25 micrograms/ml of thiophene-2-carboxylic acid hydrazide, 40 micrograms/ml of D-cycloserine, and 100 micrograms/ml of pyrazinamide.

Cycloserine↗

Antimycobacterial spectrum of colistin (polymixin E).

Minimal inhibitory concentrations and minimal bactericidal concentrations of colistin (polymyxin E) were determined for the type strains of fifteen mycobacterial species. Colistin was found to be active against pathogenic species Mycobacterium xenopi, M. intracellulare, M. tuberculosis, M. fortuitum and also against the rapidly growing, non-pathogenic species M. phlei and M. smegmatis. The discriminatory potential of susceptibility to colistin as a test was investigated on 25 strains of the M. fortuitum/M. chelonei complex, and also on 11 strains of the M. avium/M. intracellulare complex. The experimental data indicated the potential of colistin susceptibility testing for discriminating M. fortuitum from M. chelonei.

Colistin↗

Biochemical and cultural analysis of mycobacterial recombinants obtained by spheroplast fusion.

Spheroplast fusion using polyethylene glycol 6000 resulted in mycobacterial recombinants with a frequency of about 10(-3). The parental strains were two carotenoid pigment mutants of Mycobacterium aurum which were obtained by chemical mutagenesis of the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine. Isolation of recombinant clones was based on their pigmentation on Löwenstein-Jensen medium, and genetically stable recombinants were analysed for their carotenoid pigments and their alpha- and beta-mycolic acids. They were then submitted to 14 biochemical tests as well as determination of their susceptibility to 13 drugs and their ability to grow at 30 and 42 degrees C. Recombinants were found to be positive for nitrate reductase (100%), gamma-glutamyl transferase (94%), urease (53%) and arylsulphatase (21%). Out of 34 recombinants tested, 5 grew both at 30 degrees and 42 degrees C, whereas 1 grew only at 37 degrees C. The results of drug susceptibility testing were inconclusive.

Anti-Bacterial Agents↗

The electron-transparent zone in phagocytized Mycobacterium avium and other mycobacteria: formation, persistence and role in bacterial survival.

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

Animals↗