Dodecanoic acid & palmitic acid disarms rifampicin resistance by putatively targeting mycobacterial efflux pump Rv1218c.

Background & objectives: Drug-resistant tuberculosis (TB) jeopardizes the treatment process with poor outcomes. Efflux pumps (EPs) belonging to the ABC transporter family in Mycobacterium tuberculosis confer resistance to rifampicin (RMP) besides genetic mutations thus serving as a target for a potential adjunct therapeutic inhibitory molecule. Rv1218c is one such pump that was previously reported to be active in multidrug-resistant TB clinical isolates. Methods: In this study, the inhibition potential of Rv1218c-EP was tested on 8 molecules that were shortlisted by in silico methods. These molecules were subjected to the minimum inhibitory concentration (MIC) determination, checkerboard drug combination assay, ethidium bromide-DNA binding assay, and in vitro and ex vivo cytotoxicity assay. Results: Based on the outcome of the study, two molecules dodecanoic acid (DA) and palmitic acid (PA) were found to be potential enough to decrease the MIC of RMP by 8 to 1000 folds against multidrug-resistant clinical isolates and Rv1218c expressing recombinant Mycobacterium smegmatis. Interpretation & conclusions: These molecules were also found to reduce the time taken by RMP to kill these drug-resistant Mycobacteria to 48 h, unlike control isolates that survived more than 240 h of RMP exposure. The functional concentration of both molecules was non-toxic to the epithelial and blood mononuclear cells. With further comprehensive scientific validation, PA and DA could be recommended as adjunct therapeutic molecules with first-line anti-TB drugs to treat drug-resistant TB.

The EcpD Tip Adhesin of the Escherichia coli Common Pilus Mediates Binding of Enteropathogenic E. coli to Extracellular Matrix Proteins.

The attachment of enteropathogenic Escherichia coli (EPEC) to intestinal epithelial cells is facilitated by several adhesins; however, the individual host-cell receptors for pili-mediated adherence have not been fully characterized. In this study, we evaluated the hypothesis that the E. coli common pilus (ECP) tip adhesin protein EcpD mediates attachment of EPEC to several extracellular matrix (ECM) glycoproteins (fibronectin, laminin, collagens I and IV, and mucin). We found that the ΔecpA mutant, which lacks production of the EcpA filament but retains EcpD on the surface, adhered to these glycoproteins below the wild-type levels, while the ΔecpD mutant, which does not display EcpA or EcpD, bound significantly less to these host glycoproteins. In agreement, a purified recombinant EcpD subunit bound significantly more than EcpA to laminin, fibronectin, collagens I and IV, and mucin in a dose-dependent manner. These are compelling data that strongly suggest that ECP-producing EPEC may bind to host ECM glycoproteins and mucins through the tip adhesin protein EcpD. This study highlights the versatility of EPEC to bind to different host proteins and suggests that the interaction of ECP with the host's ECM glycoproteins may facilitate colonization of the intestinal mucosal epithelium.

Mycobacterium tuberculosis strain lineage in mixed tribal population across India and Andaman Nicobar Island.

In India, the tribal population constitutes almost 8.6% of the nation's total population. Despite their large presence, there are only a few reports available on Mycobacterium tuberculosis (M. tb) strain prevalence in Indian tribal communities considering the mobile nature of this population and also the influence of the mainstream populations they coexist within many areas for their livelihood. This study attempts to provide critical information pertaining to the TB strain diversity, its public health implications, and distribution among the tribal population in eleven Indian states and Andaman & Nicobar (A&N) Island. The study employed a population-based molecular approach. Clinical isolates were received from 66 villages (10 states and Island) and these villages were selected by implying situation analysis. A total of 78 M. tb clinical isolates were received from 10 different states and A&N Island. Among these, 16 different strains were observed by spoligotyping technique. The major M. tb strains spoligotype belong to the Beijing, CAS1_DELHI, and EAI5 family of M. tb strains followed by EAI1_SOM, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U strains. Drug-susceptibility testing (DST) results showed almost 15.4% of clinical isolates found to be resistant to isoniazid (INH) or rifampicin (RMP) + INH. Predominant multidrug-resistant (MDR-TB) isolates seem to be Beijing strain. Beijing, CAS1_DELHI, EAI3_IND, and EAI5 were the principal strains infecting mixed tribal populations across India. Despite the small sample size, this study has demonstrated higher diversity among the TB strains with significant MDR-TB findings. Prevalence of Beijing MDR-TB strains in Central, Southern, Eastern India and A&N Island indicates the transmission of the TB strains.

Of tuberculosis and non-tuberculous mycobacterial infections - a comparative analysis of epidemiology, diagnosis and treatment.

Pulmonary diseases due to mycobacteria cause significant morbidity and mortality to human health. In addition to tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), recent epidemiological studies have shown the emergence of non-tuberculous mycobacteria (NTM) species in causing lung diseases in humans. Although more than 170 NTM species are present in various environmental niches, only a handful, primarily Mycobacterium avium complex and M. abscessus, have been implicated in pulmonary disease. While TB is transmitted through inhalation of aerosol droplets containing Mtb, generated by patients with symptomatic disease, NTM disease is mostly disseminated through aerosols originated from the environment. However, following inhalation, both Mtb and NTM are phagocytosed by alveolar macrophages in the lungs. Subsequently, various immune cells are recruited from the circulation to the site of infection, which leads to granuloma formation. Although the pathophysiology of TB and NTM diseases share several fundamental cellular and molecular events, the host-susceptibility to Mtb and NTM infections are different. Striking differences also exist in the disease presentation between TB and NTM cases. While NTM disease is primarily associated with bronchiectasis, this condition is rarely a predisposing factor for TB. Similarly, in Human Immunodeficiency Virus (HIV)-infected individuals, NTM disease presents as disseminated, extrapulmonary form rather than as a miliary, pulmonary disease, which is seen in Mtb infection. The diagnostic modalities for TB, including molecular diagnosis and drug-susceptibility testing (DST), are more advanced and possess a higher rate of sensitivity and specificity, compared to the tools available for NTM infections. In general, drug-sensitive TB is effectively treated with a standard multi-drug regimen containing well-defined first- and second-line antibiotics. However, the treatment of drug-resistant TB requires the additional, newer class of antibiotics in combination with or without the first and second-line drugs. In contrast, the NTM species display significant heterogeneity in their susceptibility to standard anti-TB drugs. Thus, the treatment for NTM diseases usually involves the use of macrolides and injectable aminoglycosides. Although well-established international guidelines are available, treatment of NTM disease is mostly empirical and not entirely successful. In general, the treatment duration is much longer for NTM diseases, compared to TB, and resection surgery of affected organ(s) is part of treatment for patients with NTM diseases that do not respond to the antibiotics treatment. Here, we discuss the epidemiology, diagnosis, and treatment modalities available for TB and NTM diseases of humans.

Induction of T7 Promoter at Higher Temperatures May Be Counterproductive.

Bacterial expression of recombinant proteins is the most popular and convenient method for obtaining large quantities of pure protein. The induction of T7 promoter with isopropyl-ß-d-thiogalactopyranoside (IPTG) is widely used for expression of large quantities of proteins in Escherichia coli. It has been reported that basic T7 promoter is leaky and expresses cloned genes without induction. The effect of T7 promoter induction on expression of proteins at different temperature using flow cytometry has not yet been investigated. Green fluorescent protein (GFP) as a non-peptide tag can be used for protein solubility screening and for high-throughput optimization of expression conditions using flow cytometry. Therefore, flow cytometry was used to study the effect of induction on the expression of T7 promoter driven emerald GFP (emGFP) at various temperatures. We noticed that percentage of emGFP positive cells decreased instead of increasing upon induction at higher temperatures. Western blot analysis confirmed that the amount of total and soluble emGFP decreased in induced cells compared uninduced cells at higher temperatures. Our results indicate that induction of basic T7 promoter at higher temperature may not necessarily increase protein expression. While using a basic T7 promoter it is highly recommended to analyze the effect of induction on protein expression at various temperatures.

Anti-biofilm and Antibacterial Activity of Allium sativum Against Drug Resistant Shiga-Toxin Producing Escherichia coli (STEC) Isolates from Patient Samples and Food Sources.

Escherichia coli (E. coli) colonizes human intestinal tract and is usually harmless to the host. However, several strains of E. coli have acquired virulent genes and could cause enteric diseases, urinary tract and even brain infections. Shiga toxin producing Escherichia coli (STEC) is an enterohaemorrhagic E. coli (EHEC) which can result in bloody diarrhoea and could potentially lead to deadly heamolytic uremic syndrome (HUS). STEC is one of the important food borne pathogens that causes food poisoning leading to diarrhoea and number of STEC outbreaks have occurred across the world. The use of standard antibiotics to treat STEC infection is not recommended as it increases the production of shiga toxin which could lead to HUS. Therefore, use of alternative approaches which include use of plant products to treat STEC infections have been gaining attention. The objective of this study was to evaluate the antibacterial and anti-biofilm activity of garlic (Allium sativum) against STEC strains isolated from various patient and food samples using in vitro assays. The microbiological isolation of STEC from various patient and food samples resulted in eight STEC isolates of which seven strains were multidrug resistant. Antibacterial assay results indicated that all the strains exhibited dose dependent sensitivity towards garlic with zone of inhibition diameters ranging from 7 to 24 mm with 15 µl of fresh garlic extract (FGE). Minimum inhibitory  concentration (MIC) of FGE for isolates ranged from 30 to 140 µl/ml. Interestingly, the biofilm formation of all isolates in presence of 4% of FGE decreased by 35 to 59%. FTIR analysis indicated that treatment with 1% FGE results in compositional and content changes in the biofilm. In addition, the total carbohydrate content of biofilm was reduced by 40% upon 1% FGE treatment. The results of the present study report for the first time the antibacterial and anti-biofilm activity of garlic against STEC. The findings will enable development of novel garlic organosulfide based drugs for the prevention and treatment of STEC infections.

PknG supports mycobacterial adaptation in acidic environment.

Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), has the remarkable ability to adapt to the hostile environment inside host cells. Eleven eukaryotic like serine-threonine protein kinases (STPKs) are present in Mtb. Protein kinase G (PknG) has been shown to promote mycobacterial survival inside host cells. A homolog of PknG is also present in Mycobacterium smegmatis (MS), a fast grower, non-pathogenic mycobacterium. In the present study, we have analyzed the role of PknG in mycobacteria during exposure to acidic environment. Expression of pknG in MS was decreased in acidic medium. Recombinant MS ectopically expressing pknG (MS-G) showed higher growth in acidic medium compared to wild type counterpart. MS-G also showed higher resistance upon exposure to 3.0 pH and better adaptability to acidic pH. Western blot analysis showed differential threonine but not serine phosphorylation of cellular proteins in MS at acidic pH which was restored by ectopic expression of pknG in MS. In Mtb H37Ra (Mtb-Ra), expression of pknG was increased at acidic pH. We also observed decreased expression of pknG in MS during infection in macrophages while the expression of pknG in Mtb-Ra was increased in similar conditions. Taken together, our data strongly suggests that pknG regulates growth of mycobacteria in acidic environment and is differentially transcribed in MS and Mtb under these conditions.

In-vivo studies on Transitmycin, a potent Mycobacterium tuberculosis inhibitor.

This study involves the in-vitro and in-vivo anti-TB potency and in-vivo safety of Transitmycin (TR) (PubChem CID:90659753)- identified to be a novel secondary metabolite derived from Streptomyces sp (R2). TR was tested in-vitro against drug resistant TB clinical isolates (n = 49). 94% of DR-TB strains (n = 49) were inhibited by TR at 10µg ml-1. In-vivo safety and efficacy studies showed that 0.005mg kg-1 of TR is toxic to mice, rats and guinea pigs, while 0.001mg kg-1 is safe, infection load did not reduce. TR is a potent DNA intercalator and also targets RecA and methionine aminopeptidases of Mycobacterium. Analogue 47 of TR was designed using in-silico based molecule detoxification approaches and SAR analysis. The multiple targeting nature of the TR brightens the chances of the analogues of TR to be a potent TB therapeutic molecule even though the parental compound is toxic. Analog 47 of TR is proposed to have non-DNA intercalating property and lesser in-vivo toxicity with high functional potency. This study attempts to develop a novel anti-TB molecule from microbial sources. Though the parental compound is toxic, its analogs are designed to be safe through in-silico approaches. However, further laboratory validations on this claim need to be carried out before labelling it as a promising anti-TB molecule.

Nontuberculous mycobacterium: An emerging pathogen: Indian perspective.

Nontuberculous mycobacteria (NTM), considered as mere contaminants, are off late, being reported as potential pathogens through various studies. The infections due to NTM range from pulmonary to extra pulmonary including skin and soft-tissue infections, traumatic and surgical wound infections, and catheter and implant-associated infections. Although extrapulmonary infections are extensively explored, pulmonary infections are scarcely reported due to their misdiagnosis as tuberculosis caused by M. tuberculosis (MTB). Appropriate detection methods are essential in order to facilitate the differential diagnosis of NTM from MTB infections. We aimed to collate the data available on NTM diagnosis and its epidemiology in India in this review. While diagnosis of MTB itself is more challenging in India, for appropriate treatment of NTM, special training and attention is needed for differential diagnosis of the former from latter. Currently, in India, in addition to the available techniques for identification of NTM, line probe assay (Hains life sciences) is proving to be a promising tool for the detection of NTM (common mycobacteria/additional species kit) and their antimicrobial resistance (Genotype NTM-DR VER 1.0). In future, with the price of sequencing steadily coming down, with proper monitoring, whole-genome sequencing could be the test of choice to predict the species, drug resistance, outbreaks in hospitals, and transmission dynamics.

Estimating TB diagnostic costs incurred under the National Tuberculosis Elimination Programme: a costing study from Tamil Nadu, South India.

BACKGROUND: The National Tuberculosis Elimination Programme (NTEP) of India is aiming to eliminate TB by 2025. The programme has increased its services and resources to strengthen the accurate and early detection of TB. It is important to estimate the cost of TB diagnosis in India considering the advancement and implementation of new diagnostic tools under the NTEP. The objective of this study was to estimate the unit costs of providing TB diagnostic services at different levels of public health facilities with different algorithms implemented under the NTEP in Chennai, Tamil Nadu, South India. METHODS: This costing study was conducted from the perspective of the health system. This study used only secondary data and information that were available in the public domain. Data were collected with the approval of health authorities. The patient's diagnostic path from the point of registration until the final diagnosis was considered in the costing exercise. The unit costs of different diagnostic tools used in the NTEP implemented by Chennai Corporation were calculated. RESULTS: We estimated the unit cost of the eight laboratory tests (Ziehl-Neelsen [ZN], fluorescence microscopy [FM], x-ray, digital x-ray, gene Xpert MTB/RIF (cartridge-based nucleic acid amplification test [NAAT] that identifies rifampicin resistant Mycobacterium Tuberculosis) Mycobacterium Tuberculosis/Rifampicin [MTB/RIF], mycobacteria growth indicator tube [MGIT], line probe assay [LPA] and Lowenstein Jensen [LJ] culture) for diagnosis of drug-sensitive and drug-resistant TB. The unit costs included fixed and variable costs for smear examination by ZN microscopy (₹ [Indian Rupee] 326 [US${\$}$4.72], FM (₹104 [US${\$}$1.5]), x-ray (₹218 [US${\$}$3.15]), digital X-ray (₹281 [US${\$}$4.07]), gene Xpert MTB/RIF (₹1137 [US${\$}$16.47]), MGIT (₹7038 [US${\$}$102]), LPA (₹6448 [US${\$}$93.44]) and LJ culture (₹4850 [US${\$}$70.28]). Out of 10 diagnostic algorithms used for TB diagnosis, algorithms using only smear microscopy had the lowest cost, followed by smear microscopy with x-ray for drug-sensitive TB (₹104 [US${\$}$1.5] to ₹544 [US${\$}$7.88]). Diagnostic algorithms for drug-resistant TB involving LPA and gene Xpert MTB/RIF were the most expensive. CONCLUSIONS: Understanding the various costs contributing to TB diagnosis in India provides crucial evidence for policymakers, programme managers and researchers to optimise programme spending and efficiently use resources.

Antibacterial Properties of Organosulfur Compounds of Garlic (Allium sativum).

Garlic (Allium sativum), a popular food spice and flavoring agent, has also been used traditionally to treat various ailments especially bacterial infections for centuries in various cultures around the world. The principal phytochemicals that exhibit antibacterial activity are oil-soluble organosulfur compounds that include allicin, ajoenes, and allyl sulfides. The organosulfur compounds of garlic exhibit a range of antibacterial properties such as bactericidal, antibiofilm, antitoxin, and anti-quorum sensing activity against a wide range of bacteria including multi-drug resistant (MDR) strains. The reactive organosulfur compounds form disulfide bonds with free sulfhydryl groups of enzymes and compromise the integrity of the bacterial membrane. The World Health Organization (WHO) has recognized the development of antibiotic resistance as a global health concern and emphasizes antibiotic stewardship along with the urgent need to develop novel antibiotics. Multiple antibacterial effects of organosulfur compounds provide an excellent framework to develop them into novel antibiotics. The review provides a focused and comprehensive portrait of the status of garlic and its compounds as antibacterial agents. In addition, the emerging role of new technologies to harness the potential of garlic as a novel antibacterial agent is discussed.

Prevalence of bacteriologically confirmed pulmonary tuberculosis and associated risk factors: A community survey in Thirvallur District, south India.

BACKGROUND: Tuberculosis (TB) prevalence surveys add to the active case detection in the community level burden of TB both national and regional levels. The aim of this study was to assess the prevalence of bacteriologically confirmed pulmonary tuberculosis (PTB) in the community. METHODS: Household community-based tuberculosis disease survey was conducted targeting 69054 population from 43 villages of 5 blocks in Tiruvallure district adopting cluster sampling methodology of ≥15 years old adult rural population of South India during 2015-2018. All eligible individuals with suspected symptoms of PTB were screened with chest X-ray. Two sputum specimens (one spot and the other early morning sample) were collected for M.tb smear and culture examination. Conversely demographical, smoking and alcohol drinking habits information were also collected to explore the risk factor. Stepwise logistic regression was employed to associate risk factors for PTB. RESULTS: A total of 62494 were screened among 69054 eligible population, of whom 6340 were eligible for sputum specimen collection. Sputum for M.tb smear and culture examination were collected in 93% of participants. The derived prevalence of PTB was 307/100000 population (smear-positive 130; culture positive 277). As expected that PTB has decreased substantially compared to preceding surveys and it showed that older age, male, low BMI, diabetes, earlier history of TB and alcohol users were significantly associated (p < .0001) with an increased risk of developing PTB. CONCLUSION: Upshot of the active survey has established a reduction in the prevalence of PTB in the rural area which can be accredited to better programmatic implementation and success of the National TB Control Programme in this district. It also has highlighted the need for risk reduction interventions accelerate faster elimination of TB.

A multi-targeting pre-clinical candidate against drug-resistant tuberculosis.

FNDR-20081 [4-{4-[5-(4-Isopropyl-phenyl)- [1,2,4]oxadiazol-3-ylmethyl]-piperazin-1-yl}-7-pyridin-3-yl-quinoline] is a novel, first in class anti-tubercular pre-clinical candidate against sensitive and drug-resistant Mycobacterium tuberculosis (Mtb). In-vitro combination studies of FNDR-20081 with first- and second-line drugs exhibited no antagonism, suggesting its compatibility for developing new combination-regimens. FNDR-20081, which is non-toxic with no CYP3A4 liability, demonstrated exposure-dependent killing of replicating-Mtb, as well as the non-replicating-Mtb, and efficacy in a mouse model of infection. Whole genome sequencing (WGS) of FNDR-20081 resistant mutants revealed the identification of pleotropic targets: marR (Rv0678), a regulator of MmpL5, a transporter/efflux pump mechanism for drug resistance; and Rv3683, a putative metalloprotease potentially involved in peptidoglycan biosynthesis. In summary, FNDR-20081 is a promising first in class compound with the potential to form a new combination regimen for MDR-TB treatment.

Reprocessing of Contaminated MGIT 960 Cultures to Improve Availability of Valid Results for Mycobacteria.

Optimal recovery of mycobacteria from the contaminated liquid culture is a challenge. While alternative methods have been suggested to reduce the rate of contamination in the BACTEC MGIT 960 system, reprocessing the contaminated liquid culture improves recovery of Mycobacterium tuberculosis. Among 793 MGIT cultures raised from as many sputum specimens after primary decontamination by the standard NaLC-NaOH method, valid results were available for 687 (86.6%) as 106 (13.4%) were contaminated. Reprocessing and reculturing of the contaminated cultures increased valid results to 739 (93.2%) and reduced the contamination rate to 6.8%. Both values were statistically significant. Recovery of the Mycobacterium tuberculosis complex increased from 45.6% to 48.4%. Valid negative results were available for an additional 3.4%. The method may be adopted to reduce the rate of contamination and to improve the valid culture results for mycobacteria.

Spoligotype Diversity of Mycobacterium tuberculosis over Two Decades from Tiruvallur, South India.

Geographically, most tuberculosis (TB) cases in 2018 were reported from India. This TB burden is compounded by MDR-TB and XDR-TB. The strategies for the management and control of TB in the community depend on an understanding of the mode of spread of the different strains of TB isolates in the community. To determine the distribution and trends of M. tb strains over the time period in the community due to treatment, we carried out the present study on changes over two decades. Design/Methods. A total of 1218 M. tb isolates (year: 2001-2018) from Tiruvallur, India, were genotyped by spoligotyping after DNA extraction and subjected to anti-TB drug susceptibility testing for the first-line anti-TB drugs. Results. On analysis with the SpolDB4 database, majority (2001-2003: 53.32% and 2015-2018: 46.3%) of the isolates belonged to East African Indian (EAI) lineage, and the orphans designated in comparison to SpolDB4 stood 33% among 2001-2003 strain collection and 46.3% among 2015-2018 strain collection. 10.2% (2001-2003) and 9.26% (2015 to 2018) of isolates were monoresistant to isoniazid (H). MDR strains were less common among EAI strains (3.2%) compared to non-EAI strains (10.32%). Conclusions. EAI is the most predominant lineage in Tiruvallur, despite the presence of highly transmissible lineages like Beijing for the last two decades. The prevalence of MDR-TB is below the national average of 2-3% among the new TB cases in the last two decades. The reason can be attributed to the well-established nature of the locally circulating strains in this region which are not associated with drug resistance.

Wild-Type MIC Distribution for Re-evaluating the Critical Concentration of Anti-TB Drugs and Pharmacodynamics Among Tuberculosis Patients From South India.

The World Health Organization (WHO) has developed specific guidelines for critical concentrations (CCs) of antibiotics used for tuberculosis (TB) treatment, which is universally followed for drug susceptibility testing (DST) of clinical specimens. However, the CC of drugs can differ significantly among the mycobacterial species based on the population, geographic location, and the prevalence of the infecting strain in a particular area. The association between CC and the minimal inhibitory concentration (MIC) of anti-TB drugs is poorly understood. In this study, we assessed the MICs of anti-TB drugs, including isoniazid (INH), rifampicin (RMP), moxifloxacin (MXF), ethambutol (ETH), and p-aminosalicylic acid (PAS) on drug-sensitive Mtb isolates from pulmonary TB patients in South India. The MIC assays performed using solid- and liquid-growth media showed changes in the CC of a few of the tested antibiotics compared with the WHO-recommended levels. Our observation suggests that the WHO guidelines could potentially lead to overdiagnosis of drug-resistant cases, which can result in inappropriate therapeutic decisions. To evaluate the correlation between drug-resistance and CC, we performed the whole-genome sequencing for 16 mycobacterial isolates, including two wild-type and 14 resistant isolates. Our results showed that two of the isolates belonged to the W-Beijing lineage, while the rest were of the East-African-Indian type. We identified a total of 74 mutations, including five novel mutations, which are known to be associated with resistance to anti-TB drugs in these isolates. In our previous study, we determined the serum levels of INH and RMP among the same patients recruited in the current study and estimated the MICs of the corresponding infected isolates in these cases. Using these data and the CCs for INH and RMP from the present study, we performed pharmacodynamics (PD) evaluation. The results show that the PD of RMP was subtherapeutic. Together, these observations emphasize the need for optimizing the drug dosage based on the PD of large-scale studies conducted in different geographical settings.

Validation of bedaquiline drug-susceptibility testing by BACTEC MGIT 960 system for Mycobacterium tuberculosis.

Background: Bedaquiline (BDQ) is a new antituberculosis (TB) drug effectively used for the treatment of multidrug-resistant and extensively drug-resistant TB. However, the reports on drug-susceptibility testing (DST) for BDQ are scarce. The study aimed to validate and standardize BDQ DST by BACTEC MGIT 960 system for Mycobacterium tuberculosis. Methods: A panel of ten M. tuberculosis isolates comprising 8 BDQ sensitive and 2 BDQ resistant strains were used to test accuracy, repeatability, and reproducibility of BDQDST by MGIT 960. BDQ DST by Middlebrook 7H11 agar method using polystyrene tubes was used as a standard method to calculate the accuracy of the validation. Results: DST by MGIT for BDQ showed 100% accuracy, repeatability, and reproducibility, although variations were observed in the growth units of the "test" MGIT tubes between technologist and drug stocks while testing for reproducibility. Conclusion: BDQ DST by MGIT 960 system is accurate, repeatable, and reproducible and hence can be implemented in certified laboratories routinely performing DST by MGIT 960 system.

Validation of environmental disinfection efficiency of traditional Ayurvedic fumigation practices.

Environmental disinfection greatly reduces the occurrence of nosocomial or healthcare associated infections (HCAIs) which are the major healthcare problems worldwide. In India, Ayurvedic traditional fumigation with natural plant products is used to disinfect environment. In the present study, environmental disinfection efficiency of traditional fumigation practice has been evaluated by using natural plant products such as garlic (Allium sativum) peel, turmeric (Curcuma longa) powder, Carom (Trachyspermum ammi) seeds (Ajwain) and Loban (resin of Styrax benzoin and Boswellia species). The efficiency of traditional fumigation using these natural products to disinfect air and surface was evaluated. The effect of traditional fumigation on the microbiological quality of air was revealed by active air sampling. In addition, the ability of the traditional fumigation using garlic peel to disinfect inanimate surface was evaluated using three strains of methicillin resistant Staphylococcus aureus (MRSA). Glass slide was artificially contaminated with the bacteria and fumigated whereas non-fumigated slide served as control. The control and fumigated slides were analyzed for surviving bacteria and subjected to scanning electron microscopy (SEM) analysis. Traditional fumigation performed separately with three grams of garlic peel, turmeric, carom seeds and loban powder reduced the average air borne bacterial colony forming units (cfu)/m3 compared to non-fumigated control. The SEM analysis showed reduced number of bacteria in garlic peel fumigated surface samples. The results of the study strongly suggested that the traditional Ayurvedic fumigation with natural plant products is effective in reducing air-borne bacteria and in disinfecting inanimate surfaces. The traditional fumigation with herbal products has huge potential to address the problem of nosocomial infections.

Differential Culturability of Mycobacterium tuberculosis in Culture-Negative Sputum of Patients With Pulmonary Tuberculosis and in a Simulated Model of Dormancy.

Tuberculosis (TB) remains a leading killer among infectious diseases of humans worldwide. Delayed diagnosis is a crucial problem in global TB control programs. Bacteriological methods currently used to diagnose TB in endemic countries take up to 8 weeks, which poses a significant delay in starting antibiotic therapy. The presence of a heterogeneous population of Mycobacterium tuberculosis, the causative agent of TB, is among the reasons for delayed diagnosis by bacteriological methods. Previously, it has been shown that mycobacterial resuscitation-promoting factors (RPFs), a family of proteins secreted by actively growing bacteria into the media, are capable of activating the growth of dormant bacteria, thus enhancing the detection of bacilli in the sputum of confirmed TB cases. However, the variability in bacterial resuscitation by RPF in the sputum of suspected pulmonary TB cases that showed differential smear and/or culture positivity during diagnosis has not been fully explored. Here, we report the presence of non-replicating bacteria in the sputum of suspected TB cases that show differential growth response to RPF treatment. Using crude and recombinant RPF treatment, we show improved sensitivity and reduced time to detect bacilli in the sputum samples of smear-positive/culture-negative or smear-negative/culture-negative cases. We also report the phenotypic heterogeneity in the RPF responsiveness among Mtb strains using an in vitro dormancy model. Our findings have implications for improving the bacteriological diagnostic modalities currently used to diagnose TB in endemic countries.

Extensively drug-resistant tuberculosis: current challenges and threats.

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.