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 2μg/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%h(CI 0.75,0.98)and 99ct(CI0.95,1.00)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 die scope of semi-automation.

Potential applications of lactic acid bacteria and bacteriocins in antimycobacterial 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.

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 at37 ℃ for 18-24 hours.The growth of normal flora observed after day 1 was considered as lysogens.Results: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.Conclusions:Lysin may have no effect on the growth of lysogens.

Bioactivity of selected seaweeds from gulf of Mannar,South-east cost of India

Objective:To evaluate antimicrobial and antimycobacterial activity of three seaweeds[Gelidiella acerosa(G.acerosa),Turbinaria conoides(T.conoides)and Sargassum wightii(S.wightii)]from Gulf of Manner.Methods:Three seaweeds G.acerosa,T.conoides and S.wightii were collected from Gulf of Mannar,South-East Coast of India.Solvent extraction of the selected seaweeds was done using hexane and ethanol.These extracts were tested for antibacterial activity against four bacterial strains(Escherichia coli,Pseudomonas aeruginosa,Bacillus subtilis and Staphylococcus aureus)by disc diffusion method.The active extracts of T.conoides were tested for antimycobacterial activity by luciferase reporter phage assay at two different concentrations(100μg/mL and 500μg/mL).Results:Among the tested seaweeds,ethanol extract of T.conoides showed maximum antibacterial activity against Bacillus subtilis and Staphylococcus aureus followed by hexane extract at concentration of 1 mg/disc.The remaining two seaweeds(G.acerosa and S.wightii)did not show any activity.The ethanol extract of T.conoides exhibited maximum antimycobacterial activity(87.33%)followed by hexane extract(74.68%)against Mycobacterium tuberculosis H37Rv at 500μg/mL.Conclusions:Ethanol extract of T.conoids showed both antibacterial and antimycobacterial activity.Further photochemical studies are needed to identify the active antimycobacterial agents.