Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

Our results indicated that a small fraction of people knew the exact definition and treatment duration of MDR TB and XDR TB in our society. There is a need to increase the awareness and knowledge status of university students about MDR and XDR TB.

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Antibiotic-resistant bacterial infections are of global concern nowadays. Environmental sources like water and soil are playing a key role in spreading antibiotic-resistance genes to humans, animals, and other environments. Objective: The purpose of this study was to identify and report the presence of multidrug-resistant bacteria (MDRs) in environmental water sources that can direct the spread of resistant genes to other bacteria/environments. Methodology: Environmental water samples were collected from 2 livestock farms and a fish pond. Bacterial isolation and identification were carried out by following Burgey’s manual of systematic bacteriology. Antibiotic susceptibility testing was done using the disk diffusion method and CLSI guidelines. Multiple antibiotic-resistant indexes were calculated. Whole genome sequences of previously reported bacteria were downloaded from NCBI to detect the resistance genes associated with phenotypic drug resistance and compared using the bioinformatics approach. Results: Microbial load was significantly high in all water sources. Following Genera were the most common: Klebsiella, Escherichia, Proteus, Serratia, Acinetobacter, Enterobacter, Pseudomonas, Bacillus, Lactobacillus, and Staphylococcus. Out of 10 classes of antibiotics, resistance against 8 classes were identified. Multiple Antibiotic Resistance (MAR) index range of isolated strains was between 0.4 and 0.9. Key Findings: Resistance against beta-lactam antibiotics was highest in our isolated strains with a MAR index of greater than 0.4 altogether. Conclusion: High burden of multidrug-resistant bacteria were detected in all water samples which can trigger the silent pandemic of antibacterial resistance.