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.

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 traditionalfumigation 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 naturalplant 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 efficiencyof traditional fumigation using these natural products to disinfect air and surface was evaluated. Theeffect 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 wasevaluated using three strains of methicillin resistant Staphylococcus aureus (MRSA). Glass slide wasartificially 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 electronmicroscopy (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 ingarlic peel fumigated surface samples. The results of the study strongly suggested that the traditionalAyurvedic fumigation with natural plant products is effective in reducing air-borne bacteria and indisinfecting inanimate surfaces. The traditional fumigation with herbal products has huge potential toaddress the problem of nosocomial infections.© 2019 The Authors. Published by Elsevier B.V. on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

TEM & SHV genes in extended spectrum beta-lactamase producing Klebsiella species beta their antimicrobial resistance pattern.

BACKGROUND & OBJECTIVES: Extended spectrum beta-lactamases (ESBLs) are often plasmid mediated derived from mutations in the classic TEM and SHV genes by one or more amino acid substitution around the active site. Detection of TEM and SHV genes by molecular methods in ESBL producing bacteria and their pattern of antimicrobial resistance can provide useful information about its epidemiology and risk factors associated with these infections. We investigated the presence of TEM and SHV genes in ESBL producing Klebsiella spp. and their antimicrobial resistance pattern in cases of neonatal septicaemia in a tertiary care hospital. METHODS: A total of 130 clinical isolates of Klebsiella spp. isolated from septicaemic neonates of a neonatal intensive care unit (NICU) from a tertiary care hospital in north India, were screened for ESBL production by combined disk diffusion method. PCR was used to detect TEM and SHV genes in ESBL positive isolates. Isoelectric points of ESBL enzymes from a few isolates (n = 6) were noted for typing of ESBL by isoelectric focusing. RESULTS: Of the 64 ESBL producing Klebsiella spp. isolates, 17 (26.5%) had both TEM and SHV genes, 31 (48.4%) had TEM alone and 13 (20.3%) had SHV gene alone. Three (4.6%) ESBL positive isolates were negative for both TEM and SHV. Isolates with both TEM and SHV genes were highly resistant to antibiotics used. Degree of resistance for 3(rd) generation cephalosporins was also high in these isolates. Six randomly selected isolates were subjected to isoelectric focussing. Results of isoelectric focussing were comparable with PCR. INTERPRETATION & CONCLUSION: Presence of TEM gene in ESBL producing Klebsiella spp. was more common than SHV gene. Frequency of antibiotic resistance was high in isolates having both TEM and SHV genes.

Novel mutations in emb B gene of ethambutol resistant isolates of Mycobacterium tuberculosis: a preliminary report.

BACKGROUND & OBJECTIVE: Ethambutol (EMB) resistance, thought to be occurring due to mutations in embB gene of Mycobacterium tuberculosis on the rise is a cause of grave concern. The present study was planned to investigate the presence of EMB resistance in M. tuberculosis isolates and to look for prevalent mutations in embB gene. METHODS: A total of 591(283 from new and 308 from previously treated cases) sputum samples from the same number of pulmonary tuberculosis cases were cultured. Isolates were tested by 1 per cent proportion method for resistance to isoniazid, rifampicin streptomycin and ethambutol. Minimum inhibitory concentration (MIC) of EMB was measured by absolute concentration method. Ten randomly selected isolates were subjected to single strand conformational polymorphism (SSCP) and direct DNA sequencing to look for mutation in 364 bp segments of embB gene. RESULTS: Of 353 isolates of M. tuberculosis from 591 sputum samples, 62 (17.58%) were resistant to EMB, of which, 16 (25.8%) showed initial resistance and 46 (74.2%) acquired. Mono resistance to EMB was rare. Only two isolates showed resistance to EMB alone. From 62 EMB resistant isolates, 88.7 per cent (55) were resistant to INH, 82.2 per cent (51) to rifampicin and 61.2 per cent (38) were resistant to streptomycin. Co-resistance to isoniazid and rifampicin (multidrug resistant, MDR-TB) with EMB resistance was seen in 41(66.1%) isolates. High level of EMB resistance was seen in 16.5 per cent isolates. SSCP showed altered mobility in 8 of 10 isolates tested. Among the 8 mutants, 4 had known mutations at codon Met 306 being replaced by Val/ Leu. The second most frequent mutation encountered was at codon Phe 287 being replaced by Val, Cys or Leu (novel mutations). Sequence analysis revealed 10 novel mutations in codon 221, 225, 227, 271, 272, 281, 282, 287, 293 and 294 within embB gene. INTERPRETATION & CONCLUSION: Presence of high frequency of EMB resistance, occurrence of high level EMB resistance, co-existence of MDR-TB with EMB resistance and novel mutations in emb B gene of M. tuberculosis clinical isolates reported highlight the need to work on larger samples to identify the diagnostic marker of EMB resistance in mycobacteria.

Novel mutations in emb B gene of ethambutol resistant isolates of Mycobacterium tuberculosis: A preliminary report.

Background & objectives: Ethambutol (EMB) resistance, thought to be occurring due to mutations in embB gene of Mycobacterium tuberculosis on the rise is a cause of grave concern. The present study was planned to investigate the presence of EMB resistance in M. tuberculosis isolates and to look for prevalent mutations in embB gene. Methods: A total of 591(283 from new and 308 from previously treated cases) sputum samples from the same number of pulmonary tuberculosis cases were cultured. Isolates were tested by 1 per cent proportion method for resistance to isoniazid, rifampicin streptomycin and ethambutol. Minimum inhibitory concentration (MIC) of EMB was measured by absolute concentration method. Ten randomly selected isolates were subjected to single strand conformational polymorphism (SSCP) and direct DNA sequencing to look for mutation in 364 bp segments of embB gene. Results: Of 353 isolates of M. tuberculosis from 591 sputum samples, 62 (17.58%) were resistant to EMB, of which, 16 (25.8%) showed initial resistance and 46 (74.2%) acquired. Mono resistance to EMB was rare. Only two isolates showed resistance to EMB alone. From 62 EMB resistant isolates, 88.7 per cent (55) were resistant to INH, 82.2 per cent (51) to rifampicin and 61.2 per cent (38) were resistant to streptomycin. Co-resistance to isoniazid and rifampicin (multidrug resistant, MDR-TB) with EMB resistance was seen in 41(66.1%) isolates. High level of EMB resistance was seen in 16.5 per cent isolates. SSCP showed altered mobility in 8 of 10 isolates tested. Among the 8 mutants, 4 had known mutations at codon Met 306 being replaced by Val/ Leu. The second most frequent mutation encountered was at codon Phe 287 being replaced by Val, Cys or Leu (novel mutations). Sequence analysis revealed 10 novel mutations in codon 221, 225, 227, 271, 272, 281, 282, 287, 293 and 294 within embB gene. Interpretation & conclusions: Presence of high frequency of EMB resistance, occurrence of high level EMB resistance, co-existence of MDR-TB with EMB resistance and novel mutations in emb B gene of M. tuberculosis clinical isolates reported highlight the need to work on larger samples to identify the diagnostic marker of EMB resistance in mycobacteria.

Prevalence of multidrug resistant Mycobacterium tuberculosis in Lucknow, Uttar Pradesh.

BACKGROUND & OBJECTIVE: Multi-drug resistant (MDR) Mycobacterium tuberculosis isolates may be transmitted within communities due to dense population and poor hygienic conditions. For proper management and control of MDR-TB, understanding drug susceptibility pattern of M. tuberculosis isolates and their transmission pattern in every health care setting are essential. In the present study, we attempted to describe the current prevalence of MDR-TB in Lucknow district, Uttar Pradesh, and our observations on transmission of MDR isolates among populations in and around this area. METHODS: Patients diagnosed as that of pulmonary tuberculosis (PTB) were enrolled from primary level (PLH), secondary level (SLH) and tertiary level (TLH) healthcare centres from Lucknow district. Detailed history of intake of antitubercular drug in the past was taken to decipher initial/ acquired drug resistance. Sputum samples were cultured on Lowenstein-Jensen media to isolate mycobacteria. Drug susceptibility patterns of isolated M. tuberculosis isolates were recorded using 1 per cent proportion method. Transmission of MDR isolates in community was accessed by random amplified polymorphic DNA (RAPD). Isolates showing same band pattern on RAPD were retyped using different primers targeted to the inverted repeat sequence of IS6110 copies in M. tuberculosis genome. RESULTS: A total of 686 M. tuberculosis isolates were obtained from 1162 patients, of which 318 were from untreated subjects and 368 were from patients who were treated for tuberculosis in the past. Prevalence of MDR was 19.8 per cent, initial and acquired being 13.2 and 25.5 per cent respectively. Prevalence of resistance to any drug, MDR and individual drug resistance to isoniazid, streptomycin, ethambutol and rifampicin was significantly higher in patients who were treated in the past. Drug resistance was significantly higher at tertiary level health care compared to primary level health care. Genotypically similar clusters were seen at all levels of health care. It was not always possible to establish geographic connections within clusters. INTERPRETATION & CONCLUSION: High prevalence of both initial and acquired MDR was noted in M. tuberculosis isolates collected from pulmonary tuberculosis patients. Presence of small clusters of MDR isolates at all health care levels suggests transmission within the studied community.

Detection of extended spectrum beta-lactamase production in clinical isolates of Klebsiella spp.

BACKGROUND & OBJECTIVE: Clinical laboratories need to develop quick screening methods for detection of extended spectrum beta-lactamase (ESBL) producing strains, so that the appropriate medication can be started without delay. In this study, we report the screening sensitivity of four representative antimicrobial agents i.e., cefpodoxime, cefotaxime, ceftazidime and aztreonam, commonly used for ESBL detection in Klebsiella spp. METHODS: A total of 100 clinical isolates of Klebsiella spp. from the cases of neonatal septicaemia at a tertiary care hospital from north India, were screened for ESBL production by Kirby- Bauer's disc diffusion (cefpodoxime, cefotaxime, ceftazidime and aztreonam) and minimum inhibitory concentration (MIC) test by agar dilution methods. Confirmation was done by double disc method. RESULTS: Results showed that 58 of the 100 isolates tested were ESBL positive by confirmatory test and cefpodoxime was more efficient ESBL screening antimicrobial agent than ceftazidime, cefotaxime and aztreonam. INTERPRETATION & CONCLUSION: Using the standard disk diffusion as screening test for identifying ESBL producers, cefpodoxime was found to be the most efficient antimicrobial agent in screening isolates as potential ESBL producers followed by ceftazidime in Klebsiella spp. isolates.

Prevalence & antimicrobial resistance pattern of extended spectrum beta-lactamase producing Klebsiella spp isolated from cases of neonatal septicaemia.

BACKGROUND & OBJECTIVE: Extended spectrum beta-lactamase (ESBL) producing Klebsiella spp led to serious concern about septicaemic neonates in neonatal intensive care units (NICU) due to high resistance against commonly used antimicrobial agents. Knowledge of disease burden and information on resistance to antimicrobials are required for proper management of such cases in NICUs. Here we report the prevalence and resistance pattern of ESBL producing Klebsiella spp isolated from cases of neonatal septicaemia at a tertiary care hospital from north India. METHODS: A total of 100 clinical isolates of Klebsiella spp isolated from 2995 blood samples of suspected cases of neonatal septicaemia were studied. Antimicrobial susceptibility was determined by Kirby- Bauer's disc diffusion method. All isolates were screened for ESBL production on the basis of inhibition zone against cephotaxime (<27 mm) and ceftazidime (<22 mm) and a breakpoint of minimum inhibitory concentration (MIC) (<2 microg/ml for cephotaxime and <8 microg/ml for cefpodoxime) by agar dilution method. Resistance pattern of ESBL producers and non-ESBL producers was compared. RESULTS: Of the 100 Klebsiella isolates, 58 were positive for ESBL production, which was much lower than 86.6 per cent reported in 2003. Almost all the isolates were sensitive to imipenam and meropenam. Drug resistance was found to be significantly more common in ESBL producing isolates than in non-ESBL producers. INTERPRETATION & CONCLUSION: We found that 56 per cent of Klebsiella spp isolates were ESBL producers. There is a need to carefully formulate therapeutic strategies to control infections in NICUs. The high percentage of drug resistance in ESBL producing Klebsiella spp suggests that routine detection of ESBL is required by reliable laboratory methods.