Potential applications of lactic acid bacteria and bacteriocins in anti-mycobacterial therapy

Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (M. tuberculosis). WHO estimated that 10.4 million new (incident) TB cases worldwide in year 2016. The increased prevalence of drug resistant strains and side effects associated with the current anti-tubercular drugs make the treatment options more complicated. Hence, there are necessities to identify new drug candidates to fight against various sub-populations of M. tuberculosis with less or no toxicity/side effects and shorter treatment duration. Bacteriocins produced by lactic acid bacteria (LAB) attract attention of researchers because of its 'Generally recognized as safe' status. LAB and its bacteriocins possess an effective antimicrobial activity against various bacteria and fungi. Interestingly bacteriocins such as nisin and lacticin 3147 have shown antimycobacterial activity in vitro. As probiotics, LAB plays a vital role in promoting various health benefits including ability to modulate immune response against various infectious diseases. LAB and its metabolic products activate immune system and thereby limiting the M. tuberculosis pathogenesis. The protein and peptide engineering techniques paved the ways to obtain hybrid bacteriocin derivatives from the known peptide sequence of existing bacteriocin. In this review, we focus on the antimycobacterial property and immunomodulatory role of LAB and its metabolic products. Techniques for large scale synthesis of potential bacteriocin with multifunctional activity and enhanced stability are also discussed.

Adequacy of examining one sputum specimen in tuberculosis drug resistance surveys

Objective/background: Collection of one spot and one morning sputum specimen is recom-mended for tuberculosis [TB] drug resistance surveys. This was a retrospective analysis of Mycobacterium tuberculosis cultures isolated from two spot sputum specimens collected from smear positive TB patients in a TB drug resistance survey. It was conducted to under-stand the value of a second specimen

Methods: A TB drug resistance survey was conducted in the state of Tamil Nadu, India, to estimate the prevalence of drug resistance among new sputum smear-positive [NSP] and previously treated [PT] patients diagnosed in Revised National Tuberculosis Control Program microscopy centers. A total of 2425 patients [1524 NSP and 901 PT cases] were enrolled in the study. From these patients, two spot sputum specimens [C and D] were collected within a period of 2 h. No preservative was added to sputum. The samples were transported at ambient conditions without cold storage to the central laboratory for culture of M. tuberculosis. Culture yield from each sample was computed and analyzed. Results: The proportion of cultures retrieved from C and D specimens among NSP cases [89.3% and 89.7%] and PT cases [90.8% and 90.3%] were similar. The culture grades of C and D samples were comparable [chi-square test, 3560.135; p < .001] and the agreement was moderate [kappa test, 0.454]

Conclusion: The findings of the study reveal the adequacy of single spot sputum specimen from smear positive pulmonary TB patients for bacteriological examination in a quality-assured TB laboratory to determine precisely the level of drug resistance in a province of India

Exposure to cetyl pyridinium chloride and loss of integrity of cell wall of mycobacteria.

Background: Cetyl pyridinium chloride (CPC) liquefied sputum was shown to reduce AFB smear positivity presumably damaging cell wall of M. tuberculosis. Settings: National Institute for Research in Tuberculosis, Chennai, (Tamil Nadu). Objective: To assess the cell wall damage of mycobacteria in CPC liquefied sputum, by Transmission Electron Microscopy (TEM) and mycobacteriophage adsorption studies. Methods: Pooled sputum sample from smear positive pulmonary TB patients was homogenized and liquefied with CPC. It was examined in TEM daily for four days, to assess cell wall damage of M. tuberculosis, and photomicrographs were taken. M. smegmatis mc2155, treated with CPC, was infected with mycobacteriophage (phAE129) to study phage adsorption on cell wall and plaque formation. CPC untreated sputum and M. smegmatis formed controls. Results: Photomicrographs showed that cell wall of M. tuberculosis was intact in controls and damaged in CPC preserved sputum for 96 hours. Plaque formation was seen and absent respectively in CPC untreated and treated M. smegmatis cells. Conclusion: Exposure to CPC damaged the cell wall of M. tuberculosis within 96 hours. Mycobacteriophage failed to form plaques after M. smegmatis mc2155 was treated with CPC implying inhibition of phage adsorption on damaged cell wall and thus providing a clue for poor staining and smear positivity in microscopy.

Optimization of the conventional minimum inhibitory concentration method for drug susceptibility testing of ethionamide

Evaluation of newer methods and optimization of existing methods for the susceptibility testing of second-line drugs, especially ethionamide, are essential when treatment of multidrug-resistant tuberculosis [MDR-TB] is warranted. The ideal method must clearly demarcate sensitive from resistant strains. Hence, optimization of the conventional minimum inhibitory concentration [MIC] method was attempted using diluted inoculum. The optimized MIC method was evaluated using 206 Mycobacterium tuberculosis strains isolated from new and previously treated tuberculosis patients and were compared with the conventional MIC method and proportion sensitivity [PST] method. The sensitivity and specificity of the optimized MIC method in comparison with the PST method was 74% and 90%. Assessment of the optimized MIC method with the conventional MIC method gave a sensitivity of and specificity of 73% and 98%. Overall agreement between the 2 methods was found to be >/= 80%. Endowed with the ability to identify the resistant strains precisely, the optimized MIC method can be used for screening resistance to ethionamide

Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

OBJECTIVE@#To evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic.@*METHODS@#One hundred and ninety primary isolates on Lowenstein-Jensen medium were tested. Middlebrook 7H9 complete medium with and without rifampicin at 2 μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate. After incubation for 72 h, LRP was added. Following 4 h of further incubation, light output from both control and test was measured as relative light units. Strains exhibiting a reduction of less than 50% relative light units in the drug containing vial compared to control were classified as resistant. Results were compared with the conventional minimum inhibitory concentration method (MIC) of drug susceptibility testing.@*RESULTS@#The two methods showed high level of agreement of 97% (CI 0.94, 0.99) and P value was 0.000 1. The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91% (CI 0.75, 0.98) and 99% (CI 0.95, 1.00) respectively. Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method.@*CONCLUSIONS@#LRP assay with phAE85 is 99% specific, 91% sensitive and is highly reproducible. Thus the assay offers a simple procedure for drug sensitivity testing, within the scope of semi-automation.

Quality indicators in a mycobacteriology laboratory supporting clinical trials for pulmonary tuberculosis

Documentation of structured quality indicators for mycobacteriology laboratories supporting exclusively controlled clinical trials in pulmonary tuberculosis [PTB] is lacking. To document laboratory indicators for a solid [Lowenstein-Jensen medium] culture system in a mycobacteriology laboratory for a period of 4 years [2007-2010]. The sputum samples, collected from PTB suspects/patients enrolled in clinical trials, were subjected to fluorescence microscopy, culture and drug sensitivity testing [DST]. Data was retrospectively collected from TB laboratory registers and computed using pre-formulated Microsoft Office Excel. Laboratory indicators were calculated and analyzed. The number of samples processed in a calendar year varied from 6261 to 10,710. Of the samples processed in a calendar year, specimen contamination [4.8-6.9%], culture positives [78.4-85.1%] among smear positives, smear positives [71.8-79.0%] among culture positive samples, smear negatives among culture negative samples [95.2-96.7%], and average time to report DST results [76-97 days] varied as shown in parentheses. Values of quality indicators in mycobacteriology laboratories supporting exclusively clinical trials of PTB have to be defined and used for meaningful monitoring of laboratories.

Effect of bacteriophage lysin on lysogens

<p><b>OBJECTIVE</b>To study the effect of phage lysin on the growth of lysogens.</p><p><b>METHODS</b>Sputum specimens processed by modified Petroff's method were respectively treated with phagebiotics in combination with lysin and lysin alone. The specimens were incubated at 37 °C for 4 days. At the end of day 1, 2, 3 and day 4, the specimens were streaked on blood agar plates and incubated at 37 °C for 18-24 hours. The growth of normal flora observed after day 1 was considered as lysogens.</p><p><b>RESULTS</b>Sputum specimens treated with phagebiotics-lysin showed the growth of lysogens. When specimens treated with lysin alone, lysogen formation was avoided and normal flora was controlled.</p><p><b>CONCLUSIONS</b>Lysin may have no effect on the growth of lysogens.</p>

Effect of bacteriophage lysin on lysogens

Objective: To study the effect of phage lysin on the growth of lysogens. Methods: Sputum specimens processed by modified Petroff's method were respectively treated with phagebiotics in combination with lysin and lysin alone. The specimens were incubated at 37℃ for 4 days. At the end of day 1, 2, 3 and day 4, the specimens were streaked on blood agar plates and incubated at 37℃ for 18-24 hours. The growth of normal flora observed after day 1 was considered as lysogens.Results:When specimens treated with lysin alone, lysogen formation was avoided and normal flora was controlled. Conclusions: Lysin may have no effect on the growth of lysogens. Sputum specimens treated with phagebiotics-lysin showed the growth of lysogens.