Effects of different methods of decontamination for successful cultivation of Mycobacterium tuberculosis.

Background & objectives: There has been an extensive invasion of tuberculosis at the global level by multidrug resistant as well as extensively drug resistant organisms. Attempts to recover the pathogen in pure culture have frequently failed since the specimens are often highly contaminated and also due to use of insufficient or over-active decontamination procedures. Hence in the present study different methods of decontamination were tested to evaluate their independent efficacies for culture of Mycobacterium tuberculosis. Methods: A total of 359 samples (241 sputum, 59 urine, 50 endometrium biopsy, 9 pus samples) from clinically suspected cases of tuberculosis were subjected to four different methods of decontamination followed by inoculation in Lowenstein-Jensen medium (LJM), and bilayered medium (BLM) and Kirchner’s liquid medium (KLM) to determine the influence of differential decontamination processes. Sputum scanty and positive specimens were graded and each sample was subjected to decontamination by four different techniques. Results: Treatment of specimens with 4 per cent NaOH yielded minimum recovery of pure cultures, while use of 2 per cent NaOH produced higher number of contaminants compared to other methods of decontamination. Addition of N-acetyl L-cystein (NALC) coupled with 2 per cent NaOH to the samples for decontamination provided fairly reasonable recovery, but the highest number of M. tuberculosis cultures could be obtained when the specimens were treated with tri-sodium phosphate and benzalkonium (TSPB). Among the sputum positive cases recovery of growth of M. tuberculosis was higher with greater number of bacilli present in the specimens. Regarding the influence of culture media, BLM produced not only rapid growth, but reasonably higher rate of isolation of M. tuberculosis. Interpretation & conclusions: Although use of TSPB was found to be an efficient method of decontamination for successful isolation of M. tuberculosis from contaminated samples, both NALC+ 2 per cent NaOH and TSPB also showed significant recovery of M. tuberculosis cultures in BLM that can facilitate early diagnosis and initiation of treatment.

Degradation of bacterial DNA by a natural antimicrobial agent with the help of biomimetic membrane system.

The antimicrobial efficacy of methylglyoxal (MG) against several gram-negative bacteria including Escherichia coli has been reported. To determine the mechanism of action of MG, molecular interactions between lipid and MG within the liposomal membrane were also investigated. Multilamellar and unilamellar vesicles were prepared from 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The effect of MG on DPPC liposomal membrane was studied by fluorescence spectroscopy and differential scanning calorimetry. The results indicate that MG interacts mainly with the DPPC head group that produces a significant increase in the fluidity of liposomal vesicles, which could be the cause of a fusion/aggregation effect in microbial cells. The agarose gel electrophoresis study with the genomic DNA extracted from E. coli ATCC 25922 revealed that addition of MG could completely degrade this DNA within 1 h, pointing out to their distinctly high degree of sensitivity towards MG. Further, the drug was able to cross the cell membranes, penetrating into the interior of the cell and interacting with DNA for demonstrating antibacterial activity of MG.

Antibacterial & antitoxic effects of the cardiovascular drug lacidipine in an animal model.

Background & objectives: Vibrio cholerae produces acute infection by liberating potent enterotoxin, called cholera toxin in human intestine. Cardiovascular drug lacidipine possessing powerful in vitro action against V. cholerae was tested to determine its possible activity against a toxigenic V. cholerae strain in an established animal model. Methods: In the rabbit intestine four loops were constructed, 3 of which were injected with over night grown V. cholerae 569B culture. Of these, two loops were simultaneously given graded doses (100, 200 μg) of lacidipine, one was left as such for a positive control. The first loop received sterile medium (negative control). After 18 h, contents of all the loops were examined for accumulation of fluid and number of viable cells. Results: Lacidipine when administrated with live V. cholerae 569B, caused a reduction in the number of viable bacteria along with amount of fluid in the loops. The amount of fluid and number of viable cells were much reduced in the loop that had 200 μg of lacidipine than the loop that received 100 μg of the drug. Interpretation & conclusions: Lacidipine has distinct inhibitory action against V. cholerae 569B with respect to both viability and production of cholera toxin in the rabbit ileum. Structural modifications of this compound may possibly lead to procurement of new potent antimicrobial drugs.

Distinct synergistic action of piperacillin and methylglyoxal against Pseudomonas aeruginosa.

The dicarbonyl compound methylglyoxal is a natural constituent of Manuka honey produced from Manuka flowers in New Zealand. It is known to possess both anticancer and antibacterial activity. Such observations prompted to investigate the ability of methylglyoxal as a potent drug against multidrug resistant Pseudomonas aeruginosa. A total of 12 test P. aeruginosa strains isolated from various hospitals were tested for their resistances against many antibiotics, most of which are applied in the treatment of P. aeruginosa infections. Results revealed that the strains were resistant to many drugs at high levels, only piperacillin, carbenicillin, amikacin and ciprofloxacin showed resistances at comparatively lower levels. Following multiple experimentations it was observed that methylglyoxal was also antimicrobic against all the strains at comparable levels. Distinct and statistically significant synergism was observed between methylglyoxal and piperacillin by disc diffusion tests when compared with their individual effects. The fractional inhibitory concentration index of this combination evaluated by checkerboard analysis, was 0.5, which confirmed synergism between the pair. Synergism was also noted when methylglyoxal was combined with carbenicillin and amikacin.

Comparison of a novel bilayered medium with the conventional media for cultivation of Mycobacterium tuberculosis.

Background & objectives: There is resurgence of tuberculosis in recent years in spite of availability of comprehensive multidrug therapy. Conventional culture media require a long time for the appearance of growth of Mycobacterium tuberculosis, while the other methods are expensive. Hence, a rapid low cost and safe bilayered medium was developed for early growth and sensitivity testing of M. tuberculosis and the results were compared with those on Lowenstein Jensen medium, Middlebrook 7H10 and Kirchner’s liquid media. Methods: A specially designed bilayered medium, consisting of a lower layer of Lowenstein Jensen medium without malachite green and a top layer of Middlebrook 7H 10 medium with added antibiotics and antifungal agents was prepared. Sputum from clinically suspected cases of tuberculosis, pleural fluid and pus samples were inoculated on the bilayered medium along with the inoculation on other conventional media after proper decontamination and concentration of the samples. Antibiotic sensitivity pattern was determined against a few rapidly growing control and test strains by disc diffusion technique and the results could be recorded by 3 to 7 days. Results: Statistically significant (P< 0.001) isolation rate was obtained on this bilayered medium when compared with the other three media, being 81.7 per cent growth by 7 days. Antibiotic sensitivity test could be recorded by 3 days in case of the rapidly growing strains on this medium, and by 7 days in case of M. tuberculosis strains. Interpretation & conclusions: Bilayered medium produced rapid growth earliest by 48 h, higher isolation rates were achieved as compared to the other conventional media and drug sensitivity testing could also be carried out successfully. Thus, the bilayered medium can be used for obtaining early culture report.

Effect of Mesua ferrea Linn. flower extract on Salmonella.

Based on its traditional uses in folk medicine, the whole flower extract of Mesua ferrea Linn. was tested for its in vitro antimicrobial efficacy against five different strains of Salmonella spp. All the strains were found to be highly sensitive to the extract, MIC of the extract against each organism being 50 microg/ml. The extract was tested in vitro for its mode of antibacterial activity against S. Typhimurium NCTC 74 and it was found to be bactericidal in action. In vivo studies of this extract offered significant protection to Swiss albino mice at doses approximately 2 and 4 mg/mouse when challenged with 50 median lethal dose of S. Typhimurium NCTC 74. Further, the extract caused statistically significant reduction in viable count of the strain in liver, spleen and heart blood of challenged mice.

Antimycobacterial activity of the antiinflammatory agent diclofenac sodium, and its synergism with streptomycin

Diclofenac sódico, um agente antiinflamatório, mostrou ação inibitória marcante contra isolados clínicos de Mycobacterium tuberculosis sensíveis e resistentes à drogas, bem como contra outras micobactérias. A droga foi testada in vitro contra 45 cepas diferentes de micobactérias, sendo que a maioria foi inibida pela droga na concentração de 10-25 µg/ml. Quando testado in vitro, diclofenac injetado em ratos albinos da linhagem Swiss, na proporção de 10 µg/g de peso corporal, provocou proteção significativa dos animais desafiados com metade da dose letal de M. tuberculosis H37 Rv 102. De acordo com o teste c2, os dados in vivo foram altamente significativos (p < 0,01). Diclofenac foi posteriormente testado quanto ao sinergismo com a droga antimicobacteriana convencional estreptomicina, frente a M. smegmatis 798. Quando comparado aos seus efeitos individuais, o sinergismo foi estatisticamente significativo (p < 0,05). Através da análise checkerboard, o índice fracional de concentração inibitória para essa combinação foi 0,37, confirmando o sinergismo.

In vitro and in vivo antimycobacterial activity of antiinflammatory drug, diclofenac sodium.

The non-steroidal antiinflammatory drug diclofenac sodium exhibited remarkable inhibitory action against both drug sensitive and drug resistant clinical isolates of Mycobacterium tuberculosis, as well as other mycobacteria. This agent was tested in vitro against 45 different strains of mycobacteria, most of which were inhibited by the drug at 10-25 microg/ml concentration. When tested in vivo, diclofenac, injected at 10 mg/kg body weight of a Swiss strain of white mice, could significantly protect them when challenged with a 50 median lethal dose of M. tuberculosis H37 Rv102. According to Chi-square test, the in vivo data were highly significant (P<0.01).

Antibacterial potential of an antispasmodic drug dicyclomine hydrochloride.

BACKGROUND & OBJECTIVES: Several compounds are known to possess antimicrobial activity in addition to their predesignated pharmacological actions. In the present study, dicyclomine hydrochloride, an antispasmodic drug, was tested for possible antimicrobial property in vitro and in vivo. METHODS: The minimum inhibitory concentration (MIC) of dicyclomine against the bacteria was determined by agar and broth dilution methods in vitro. The antibacterial activity of dicyclomine was confirmed by animal experiments. Toxicity and protective efficacy of the drug were tested in vivo. RESULTS: Dicyclomine inhibited most of the bacterial isolates tested at 25-100 microg/ml concentration, and a few were sensitive even at a lower concentration (10 microg/ml). Dicyclomine was found to be bacteriostatic in nature against Shigella dysenteriae 7, and bactericidal against S. aureus NCTC 6571, 8530, and 8531. When administered to Swiss white mice at doses of 30 and 60 microg/mouse, dicyclomine protected the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. INTERPRETATION & CONCLUSION: Dicyclomine showed inhibitory action against several pathogenic bacteria. It also offered significant protection to mice against the bacterial challange. As dicyclomine is in routine therapeutic use, it may be developed as a potent antimicrobial agent in many infections.

Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios.

Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT). It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice. Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios. Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media.