Search

Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health issue in Pakistan. Isoniazid is a first-line pro-drug that requires activation through an enzyme called catalase peroxidase, but is subject to widespread resistance, driven by mutations in katG and inhA genes and other loci with compensatory effects (e.g., ahpC). Here, we used whole genome sequencing data from 51 M. tuberculosis isolates collected from Khyber Pakhtunkhwa province (years 2016-2019; all isoniazid phenotypically resistant) to investigate the genetic diversity of mutations in isoniazid candidate genes. The most common mutations underlying resistance were katG S315T (37/51), fabG1 -15CT (13/51; inhA promoter), and inhA -154GA (7/51). Other less common mutations (n 5) were also identified in katG (R128Q, V1A, W505*, A109T, D311G) and candidate compensatory genes ahpC (-54CT, -51GA) and oxyS (M249T). Using DynaMut2 software, the mutants exhibited various degrees of stability and flexibility on protein structures, with some katG mutations leading to a decrease in KatG protein flexibility. Overall, the characterisation of circulating isoniazid resistant-linked mutations will assist in drug resistant TB management and control activities in a highly endemic area of Pakistan.

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries.

Fluoroquinolones (FQs) are broad-spectrum second-line antimicrobial drugs commonly used in the treatment of tuberculosis (TB). Data on FQ resistance in the Khyber Pakhtunkhwa (KP) province of Pakistan, a high-burden country, are scarce. This study aimed to analyze the resistance to FQs in this specific geographic area. Samples were collected from 25 districts of KP from 2014 to 2019. Data regarding suspected TB patients were collected from their guardians or secondary caregivers. All the samples were subjected to decontamination and digestion processing. Drug susceptibility testing (DST) was performed according to the standard minimum inhibitory concentration for ofloxacin (OFX), levofloxacin (LEV), and moxifloxacin (MOX), taken as 2, 1, and 1 μg/mL, respectively. For the 5,759 clinical samples collected from 25 districts, DST was conducted for a total of 3,158 samples. Out of the total DSTs, the OFX profile was available for 2,983, MOX profile for 2,290, and LEV profile for 544 samples. OFX and LEV resistance was found to be evenly distributed and has remained the same for the past few years, whereas MOX resistance increased from 1% in 2017 to 4% in 2019. Among a total of 807 OFX-resistant isolates, 218 (27%) were observed to be monoresistant to OFX, whereas 589 (73%) isolates were resistant to OFX and at least one other anti-TB drug. Drug resistance to OFX was higher in multidrug-resistant TB (MDR-TB), that is, 428 (53%). It was concluded that resistance to MOX has been increasing, whereas OFX resistance is much higher in MDR cases. FQ resistance needs to be continuously monitored to avoid further side effects. This study provides useful information for better management of FQ resistance with reference to the global TB control program 2030.

Regardless of a plethora of advanced diagnostics, TB and drug resistance remains a principal killer. We proposed gold nanoparticles (AuNPs) attached with probes to enhance the efficiency of GeneXpert MTB/RIF assay instead of conventional dye probes for molecular detection. A total of 15,000 samples were collected from TB suspects and subjected to Xpert MTB/RIF assay, where 6800 (45.3%) were detected as MTB positive, 280 (4.3%) were detected to harbor mutations in the RRDR, while invalid /errors were found in 690 (4.6%) cases. The mutations were detected by probe E, 199 (71.1%), while probes B and D, 30 and 26 (10% and 9%), respectively. In the Xpert MTB/RIF Assay were found mutations picked by probes E and B codons 529-533 (71%) and 512-518 (10%), respectively. The fast-rising works of TB nano-diagnostics, of Xpert probes, may improve by the applications of gold nanoparticle probes.

Background Tuberculosis (TB) is a global public health issue, getting worse due to emergence of resistance. Pyrazinamide (PZA) is first-line antimicrobial drugs used against non-replicated Mycobacterium tuberculosis (MTB). Data is scarce about whole genome sequencing of PZA resistance (PZA-R) in Khyber Pakhtunkhwa (KP) province of high burden country, Pakistan. In the current study we aimed to find the most common mutations in PZA-R MTB isolates in association with other candidate genes in a whole genome sequence (WGS). Samples were collected from TB suspects and drug susceptibility testing (DST) was performed according according to the WHO standards. The resistant samples were subjected for whole genome sequencing (WGS). The sequence data was through MTBseq and Total Genotyping Solution for Mycobacterium tuberculosis (TGS-TB). Metabolic model was analyzed, using RAST server. Results Among the three whole genome sequences, (NCBI BioProject Accession: PRJNA629298, PRJNA629388) 1997, 1162, and 2053 mutations including indel, was detected. Diverse variability has been detected in the membrane proteins PE and PPE, modulating the host immune response. Nine mutations in coding and promotor region have been detected in pncA with one novel (T-4C) variants. Mutations in the other drug candidate genes, KatG, rpoB have also been detected. Conclusion The metabolic model shows a distinct property. Diversity of variants has been detected in majority of MTB essential genes, functions from cell growth to cell signaling. The current study provides useful information, associated with geographic specific strains for biomarkers development and better management of drug resistance isolates.

Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.

Anti-tuberculosis therapy involves the combination of drugs to hamper the growth of Mycobacterium tuberculosis (MTB). The emergence of multidrug-resistant tuberculosis (MDR-TB) is a global concern. Pakistan has been ranked 5th position in terms of a high burden of MDR-TB in the world. The aim of the current study was to investigate the prevalence of drug resistance in MTB in Khyber Pakhtunkhwa. Random samples were collected from 25 districts using the simple random sampling formula. All samples were processed in a biosafety level 3 laboratory for culture and drug susceptibility testing. Among 5759 presumptive tuberculosis (TB) cases, 1969 (34%) were positive. The proportion of TB was higher in females (39%) than males (29%), thus it represents a significant association between gender and tuberculosis (p < 0.05). People ages between 25 to 34 years were more likely to be infected with MTB (40%). Drug-resistant profile showed 97 (4.9%) patients were infected with MDR-TB. Streptomycin resistance was the highest and was observed in 173 (9%) isolates followed by isoniazid in 119 (6%) isolates. The lowest resistance was observed to pyrazinamide (3%). The prevalence of MDR-TB (10.4%) among patients that previously received anti-tuberculosis treatment is seemingly high. A large-scale drug resistance survey is required to evaluate the drug resistance for better management of tuberculosis.

Sajid Ali,1 Muhammad Tahir Khan,2 Khan Anwar Sheed,3 Muhammad Mumtaz Khan,4 Fariha Hasan11Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan; 2Department of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan; 3Provincial TB Reference Laboratory, Provincial TB Control Program, Khyber Pakhtunkhwa, Pakistan; 4Department of Microbiology, University of Haripur, Haripur, PakistanBackground: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.Keywords: spoligotyping, VNTR, L3/CAS, genotyping, molecular characterization, genetic diversity