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Microorganisms inhabiting cold environments have an incredible potential to produce secondary metabolites including antibacterial, antifungal and antiviral compounds. The aim of the study was to evaluate the possibility of using cold adapted bacteria isolated from Passu glacier for the production of antimicrobial metabolites. Agar well diffusion assay was used to select four best antimicrobial compound producers coded as HTP12, HTP13, HTP36 and LTP10. These strains were identified as Alcaligenes faecalis, Pseudochrobactrum saccharolyticum, Alcaligenes pakistanensis and Alcaligenes pakistanensis, respectively, by their phylogenetic analysis. The media, temperature, incubation time and pH were optimized for antimicrobial compound production. The effect of nitrogen sources, carbon sources and salts on antibiotic production was also determined. Alcaligenes pakistanensis (LTP10) showed the best antimicrobial activity in Luria Bertani broth, at 25°C, pH 7 and incubation time of 96 hrs against various bacterial ATCC strains and clinical isolates. Yeast extract increased antibiotic activity of Alcaligenes pakistanensis (LTP10) while glucose, starch, tryptophan, threonine, L-leucine, arginine, NaCl and KCl decreased the activity. This study concludes that the psychrophilic bacteria are abundant, undiscovered, and good producers of antimicrobial metabolites under optimum conditions. This will lead to the discovery of potent and novel metabolites which could be of medical and industrial importance. [ABSTRACT FROM AUTHOR]

Lipases are fat splitting enzymes. Psychrotrophic enzymes including lipases possess huge potential for many industries due to their high activity at low temperatures. Lipase produced by the psychrophilic bacteria HTB2 identified as Pseudomonas mandelii, by using different molecular techniques, isolated from Batura glacier, Hunza Valley, Pakistan, was partially purified through acetone precipitation and gel permeation chromatography. During purification studies two types of peaks were observed in spectrophotometric analysis which indicated the presence of two different sizes of lipases produced by Pseudomonas mandelii HTB2. The purified enzyme from P. mandelii HTB2 showed best activity at 45°C, however, maximum lipase activity was attained at pH 10. It is a unique finding that an enzyme from a psychrophilic bacterium was active at thermophilic range and extreme alkaline condition. This is the first report of purification of lipase from psychrophilic bacteria from Batura glacier, and it holds promise for potential industrial applications. [ABSTRACT FROM AUTHOR]

In this study, the bacterial isolate HI6 was recovered from the ice of the Hopar (Bualtar) glacier, Karakoram Mountain Range, Pakistan. Isolate HI6 was identified as Serratia marcescens through microscopic and molecular analysis (16S rRNA sequencing). Serratia marcescens HI6 was analyzed for different physiological characteristics including temperature, pH and culture media. Serratia marcescens HI6 showed best growth at 25°C, on LB medium and pH 7. In addition, the Fatty acid methyl ester (FAME) was extracted and subjected to Fourier Transform infrared (FTIR) and Gas chromatography/mass spectroscopy (GC/MS) analysis. FTIR and GC/MS analysis has revealed that cell membrane of Serratia marcescens HI6 contains various types of fatty acids (FA). i–C16:1 (35.3% out of total cell membrane fatty acids) was found most predominantly followed by i–C15:1 (18.5%) and n-C16 (13.2%). Moreover, branched fatty acids (62%) including monounsaturated fatty acids (MUFA) (e.g. i–C15:1 and i–C16:1) and saturated fatty acids (SFA) were the dominant types of fatty acids in the cell membrane. A polyunsaturated fatty acid (PUFA), C18:2(cis–9) (known as linoleic acid) was also detected among analyzed fatty acids. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria (MTB) have ability to accumulate biominerals intracellularly as iron nanoparticles in the form of ferric oxide (Magnetite) and ferric sulphide (Greigite), known as magnetosomes. Most of them belong to the α-proteobacteria, β-proteobacteria and γ-proteobacteria class. In Pakistan, the magnetotactic bacterial strains are not yet isolated. In current study, the Magnetotactic bacterial strains are isolated from different environmental samples and a modified specific growth medium was used to culture the isolated MTB strains. The technique used for accumulation of bacteria in water samples was the magnetic enrichment technique. The isolation of magnetotactic bacteria is difficult and identified number of strains of magnetotactic bacteria is less than a hundred. So, there is a need to focus more on the identification and classification of MTB. The selected strains were cultured on scale up production and extraction of magnetosomes were carried out by Boiling method. The extracted magnetosomes were characterized by Scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy and X-ray powder diffraction (XRD). [ABSTRACT FROM AUTHOR]

Among our collection of psychrophilic bacteria from various non-polar glaciers, the present study involves the study of the ability of the psychrophilic bacterial strains, isolated from Passu Glacier, Pakistan, to produce thermolabile alkaline phosphatase extracellularly as well as intracellularly. The biophysio-chemical characteristics and 16S rRNA signatures of the isolate MRLBA1 were similar to the members of genus Pseudomonas. This rod shaped MRLBA1 isolated from glacial ice exhibited aerobic growth cycle with cardinal limits of pH; 4-11 and temperature; 2-30°C. The strain showed highest extracellular alkaline phosphatase activity at pH 8.0 and 18°C when inoculated with 24 h old inoculum (5%), after 48 h of incubation in shake flask at 150 rpm. The strain’s targeting mechanism of alkaline phosphatase at cell membrane as well as in the extracellular medium is interesting. The results demonstrated that alkaline phosphatase being thermolabile, acts as a stress protein also in addition to harvesting the energy directly in carbon deficient ice. [ABSTRACT FROM AUTHOR]

Abstract: Human group A rotaviruses (RVAs) possess a large genetic diversity and new RVA strains and G/P genotype combinations are been identified frequently. Only a few studies reporting the distribution and co-circulation of RVA G and P genotypes are available for Pakistan. This hospital based study showed a RVA prevalence rate of 23.8%, which is similar to RVA detection rates estimated in other Eastern Mediterranean countries. During 2010, the following RVA strains were found to co-circulate: G1P[8] and G2P[4] (both 24.3%), G1P[6] (12.1%), G9P[8] (10.8%), G9P[6] (5.4%), G12P[6] (6.7%), G6P[1] (2.7%) and mixed infections (6.7%). Sequence analyses of selected G1, G2, G9 and G12 RVA strains revealed a close evolutionary relationship with typical human RVA strains. Sequence identities among the Pakistani VP7 RVA genes encoding G1, G2, G9 and G12 ranged between 91.5–98.7%, 99.6–98.9%, 97.7–99.5% and 99.2–99.9%, respectively. Analysis of the VP4 genes revealed co-prevalence of distinct lineages of the P[8] genotype. P[6] and P[4] showed a close relationship with typical human RVA strains detected in several Asian countries. The two G6P[1] RVA strains were closely related to typical bovine RVA strains, suggesting one or multiple interspecies transmission events. Our data provide important baseline data on the burden of RVA disease and genotype distribution in Rawalpindi, Pakistan, which is important with respect to vaccine introduction in national immunization programs. [Copyright &y& Elsevier]

Poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases. [ABSTRACT FROM AUTHOR]

Abstract: In the earlier part of the year 2003, various pipeline ruptures were caused by sabotage activities in the southern part of Pakistan. The event being presented in this paper was fascinating because a pipe, which was blasted with the help of explosives, subsequently caused another pipe (buried 20ft away) to rupture through slow and gradual consequential erosion. On 8 April 2003, two parallel-running gas pipelines (30 and 24-in. diameters), in the main gas transmission pipelines-network of Pakistan, were ruptured. The 30-in. line was the first to rupture (at about 3.30AM), while after a time interval of about 80min the 24-in. line also developed a rupture. At the site of the rupture, the 30-in. line passed over an irrigation canal, while the 24-in. line was buried a few feet below the canal-bed at a horizontal distance of about 20ft from the 30-in. line. The above-ground 30-in. line was ruptured with the help of an explosive device that had been placed in the space between the pipe and the top of the canal-bank. The blast created an opening of about 4×10 in. at about 5 o′clock position. The leaking gas (a gas-jet, to be exact), which was co-incidentally directed towards the nearby-buried 24-in. line, caused a ‘sand-blasting’ action on the surface of the 24-in. pipe. The slow but persistent abrasion of the 24-in. pipe by the ‘sand-blasting’ caused a gradual reduction in the wall-thickness of the pipe with the result that when the wall-thickness had been reduced to a level that it was unable to support the inside gas-pressure of 1000psi, the pipe developed a longitudinal rupture. [Copyright &y& Elsevier]

At currently, approximately 70 species of magnetotactic bacteria have been identified; thus, there is an urgent need to identify more magnetotactic bacteria from diverse environmental sources with potential applications in industry and biotechnology. To the best of our knowledge, this is the first magnetotactic bacterial strain discovered in Pakistan. The first magnetotactic bacteria, Magnetospirillum moscoviense MS-24, was isolated from Banjosa Lake (Rawalakot), Pakistan, in the current investigation. Magnetospirillum moscoviense MS-24 was screened using the Racetrack method. The Magnetospirillum moscoviense MS-24 were physically characterised using Atomic Force Microscopy, High-Resolution Scanning Electron Microscopy, and Transmission Electron Microscopy. The current study used microscopy to illustrate the shape of bacteria and to find a very obvious chain of magnetosomes within the bacterial cell. The Magnetospirillum moscoviense MS-24 measured about 4 ± 0.04 µm in length and 600 ± 0.02 nm in diameter. The microfluidic chip experiments were also used to detect magnetotaxis behaviour in bacteria., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; bla AmpC 6 (100%), bla VIM-1, bla SHV and bla DHA 5 (100%) each, bla OXA-1 1 (100%), bla CMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for bla KPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in bla NDM-1, bla OXA-1, bla SHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The participation of numerous physicochemical and biological functions maintains the evolution and expansion of the remarkable nature. Due to its vast applicability in several engineering disciplines, naturally occurring bio-mineralization or microbially induced calcium carbonate (MICP) precipitation is attracting more interest. Cave bacteria contribute to the precipitation of calcium carbonate (CaCO 3 ). In the present study, soil sediments were collected from Kashmir cave, KPK, Pakistan, and plated on B4 specific nutrients limited medium for bacterial isolation and the viable bacterial count was calculated. Three bacterial strains named GSN-11, TFSN-14, and TFSN-15 were capable of precipitating CaCO 3 . These bacterial isolates were identified through 16S rRNA gene sequencing and strain GSN-11 was identified as Bacillus toyonensis, TFSN-14 as Paracoccus limosus and TFSN-15 as Brevundimonas diminuta. Enhanced CaCO 3 precipitation potential of these bacteria strains was observed at 25°C and pH 5. The precipitated CaCO 3 was confirmed by scanning electron microscopy, X-ray powder diffraction, and Fourier transform infra-red spectroscopy. The findings showed that the precipitates were dominated by calcite, aragonite, and nanosize vaterite. Current research suggests that precipitation of CaCO 3 by proteolytic cave bacteria is widespread in Kashmir cave and these bacterial communities can actively contribute to the formation of CaCO 3 by enhancing the pH of the microenvironment. RESEARCH HIGHLIGHTS: Kashmir cave inhabit potentially active bacteria in terms of biogeochemical processes. Cave bacteria significantly precipitated CaCO 3 . Calcite, aragonite, and nanosize vaterite were dominant in precipitates., (© 2022 Wiley Periodicals LLC.)

Rock microbes are capable to solubilize phosphate present in the rocks.. In this study, we focused on the isolation of phosphate solubilizing bacteria from rocks of Murree, Pakistan. Both endolithic and epilithic bacteria were screened for phosphate solubilization. Three bacterial strains were selected based on halozone formation inNational Botanical Research Institute for phosphate) medium supplemented with TCP (tribasic calcium phosphate). The solubilization index for these bacteria was recorded as 4.29, 4.03 and 3.99. The pH of the medium dropped from 7.0 to 4.0 after 5 days with continuous shaking at 150 rpm, which facilitate the phosphate solubilization. The strains P26, P4 and N27 were identified as Pseudomonas putida strain (KT004381), Pseudomonas grimontii (KT223621) and Alcaligenes faecalis (KT004385). Strain P26 showed maximum phosphate solubilization (367.54 µg/ml), while P4 and N27 showed 321.88 and 291.36 µg/ml after 3 days of incubation. Such inorganic phosphate solubilization could be attributed to the organic acids production by bacteria. The presence of organic acids is determined by high-performance liquid chromatography. Three different types of acids, gluconic, oxalic and malic acid were the dominant acids found in the culture medium. It may be assumed that these bacteria can play a role in weathering of rocks as well. PSB is likely to serve as an efficient biofertilizer, especially in areas deficient in P to increase the overall performance of crops., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study was conducted to explore the frequency of positivity of cutaneous leishmaniasis (CL) in the tribal district Bajaur located near the Pak-Afghan border. The present study was conducted at the Leishmaniasis Center of Headquarter Hospital Khar District Bajaur, Pakistan. In total, 646 patients were recruited and included in the study after ethical approval and consent from the patients. CL was confirmed by taking blood samples from the sides of the lesion and observing them under a microscope using Giemsa staining. Information about demographic factors was collected from the study participants with a questionnaire and analyzed by SPSS. It was found that 73.8% of suspected patients were positive and 26.2% were negative for CL. There were 51.9% male and 48.1% female patients. The most frequently affected site was the face (42.6%), and most of the patients (85.8%) had only one lesion. The positivity of CL was higher among those under age 15 years. The area of most positivity, with 45.2% of the cases, was Tehsil Mamund. Most of the patients (46.6%) lived in stone houses, with 98.6% of patients having domestic animals in their houses. Approximately 198 patients were treated with intramuscular and intralesional injections of meglumine antimoniate, and their weekly follow-up revealed that 48% of patients recovered, while the remaining patients left the course of treatment at different stages of therapy. The positivity of CL is high in this area and is confirmed by the detection of Leishmania amastigotes in the blood collected from their lesions. Socioeconomic factors are the main underlying causes of the rapid spread of this disease and meglumine antimoniate is an effective drug., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Biogeographic variations in the gut microbiota are pivotal to understanding the global pattern of host-microbiota interactions in prevalent lifestyle-related diseases. Pakistani adults, having an exceptionally high prevalence of type 2 diabetes mellitus (T2D), are one of the most understudied populations in microbiota research to date. The aim of the present study is to examine the gut microbiota across individuals from Pakistan and other populations of non-industrialized and industrialized lifestyles with a focus on T2D. The fecal samples from 94 urban-dwelling Pakistani adults with and without T2D were profiled by bacterial 16S ribosomal RNA gene and fungal internal transcribed spacer (ITS) region amplicon sequencing and eubacterial qPCR, and plasma samples quantified for circulating levels of lipopolysaccharide-binding protein (LBP) and the activation ability of Toll-like receptor (TLR)-signaling. Publicly available datasets generated with comparable molecular methods were retrieved for comparative analysis of the bacterial microbiota. Overall, urbanized Pakistanis' gut microbiota was similar to that of transitional or non-industrialized populations, depleted in Akkermansiaceae and enriched in Prevotellaceae (dominated by the non-Westernized clades of Prevotella copri ). The relatively high proportion of Atopobiaceae appeared to be a unique characteristic of the Pakistani gut microbiota. The Pakistanis with T2D had elevated levels of LBP and TLR-signaling in circulation as well as gut microbial signatures atypical of other populations, e.g., increased relative abundance of Libanicoccus / Parolsenella , limiting the inter-population extrapolation of gut microbiota-based classifiers for T2D. Taken together, our findings call for a more global representation of understudied populations to extend the applicability of microbiota-based diagnostics and therapeutics.

Multiple Rotavirus A (RVA) strains are linked with gastrointestinal infections in children that fall in age bracket of 0 to 60 months. However, the problem is augmented with emergence of unique strains that reassort with RVA strains of animal origin. The study describes the sequence analysis of a rare G6P[1] rotavirus strain isolated from a less than 1 year old child, during rotavirus surveillance in Rawalpindi district, Pakistan in 2010. Extracted RNA from fecal specimen was subjected to high throughput RT-PCR for structural and nonstructural gene segments. The complete rotavirus genome of one isolate RVA/Human-wt/PAK/PAK99/2010/G6P[1] was sequenced for phylogenetic analysis to elucidate the evolutionary linkages and origin. Full genome examination of novel strain RVA/Human-wt/PAK/PAK99/2010/G6P[1] revealed the unique genotype assemblage: G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H1. The evolutionary analyses of VP7, VP4, NSP1 and NSP3 gene segments revealed that PAK99 clustered with bovine, or cattle-like rotavirus strains from other closely related species, in the genotypes G6, P[1], A3 and T6 respectively. Gene segments VP6, VP1, VP2, VP3, NSP2 and NSP4 all possessed the DS-1-like bovine genotype 2 and bovine (-like) RVA strains instead of RVA strains having human origin. However, the NSP5 gene was found to cluster closely with contemporary human Wa-like rotavirus strains of H1 genotype. This is the first report on bovine-human (Wa-like reassortant) genotype constellation of G6P[1] strain from a human case in Pakistan (and the second description worldwide). Our results emphasize the significance of incessant monitoring of circulating RVA strains in humans and animals for better understanding of RV evolution., (© 2019 Wiley Periodicals, Inc.)

The gold standard therapy for treating the periodontal infections is the removal of bacterial plaque and deposits of calculus from tooth surfaces by scaling and root planning. In order to eliminate these bacterial reservoirs, beside conventional treatment, chemo therapeutic agents are commonly prescribed by periodontologists. To avoid the systemic side effects and development of antibiotics resistance, local drug delivery methods has gained the attention of dentists to treat periodontal infections, along with scaling and root planning. The aim of this study was to evaluate the effectiveness of local drug delivery system in combination with scaling and root planning, by using 1% metronidazole gel and mouthwash. The patients were divided into 3 groups. Group I: conventional treatment group. Group II: patients received treatment with gel. Group III: patients received treatment with mouthwash. All groups received treatment for 30 days. Clinical parameters and salivary concentration of TNF-α, PGE2 and nitric oxide were measured before and after treatment in both groups. All clinical parameters and inflammatory biomarkers significantly reduced in gel and mouthwash group patients (p≤0.001) as compared to patients received conventional treatment. The gel is found to be more efficacious than mouthwash especially in reducing clinical attachment loss (p< 0.05) and in reducing inflammatory biomarkers (p≤0.001). We strongly suggest the use of metronidazole via local drug delivery system combined with scaling and root planning to treat periodontal diseases.

The Hindu Kush mountains spread over Northern areas of Pakistan having hundreds of glaciers representing a unique ecosystem driven by the specific geochemistry and climate. The current study measured the distribution of culturable fungi in Tirich Mir glacier, Hindu Kush range, and the potential of these isolates to show antimicrobial activity and produce biotechnologically important enzymes. Samples of glacial ice, sediments, and meltwater were collected from Tirich Mir glacier, and 46 fungal strains were isolated and characterized for identity and biotechnological applications. The findings revealed Penicillium (10) as the most common genus, followed by Alternaria (9), Cladosporium (7), Coprinopsis, two isolates each belonging to genus Phoma, Ulocladium, Epicoccum, Onygenales, and Didymella, and one isolate of genus Davidiella, Aspergillus, Geomyces, Dothideomycetes, Pseudogymnoascus, Irpex, Scopulariopsis, Ascochyta, Tomicus, and Davidiellaceae. Davidiella tassiana HTF9 showed growth in the presence of 18% NaCl and pH 2, 3, 5, 7, 9, and 11. The isolates Ulocladium sp. and Onygenales sp. inhibited the growth of test fungi, Gram-negative and positive bacteria. Fungal strains were capable of producing cold-active enzymes, including cellulase, lipase, amylase, and deoxyribonuclease. The isolate Penicillium chrysogenum HTF24 was an efficient producer of amylase, deoxyribonuclease, and cellulase. The fungi of high-altitude glaciers are potent candidates for biotechnological applications; however, studies using more sensitive techniques are needed for further exploration., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, cyanobacterium isolate CHS1 isolated from Hopar glacier, Pakistan, was analyzed for the first time for cell membrane fatty acids and production of pigments. Sequencing of the 16-23S intergenetic region confirmed identification of the isolate CHS1 as Nodularia spumigena. All chlorophyll and carotenoid pigments were quantified using high-performance liquid chromatography and experiments to test tolerance against a range of physico-chemical conditions were conducted. Likewise, the fatty acid profile of the cell membrane CHS1 was analyzed using gas chromatography and mass spectroscopy. The cyanobacterium isolate CHS1 demonstrated tolerance to 8 g/L% NaCl, 35°C and pH 5-9. The characteristic polyunsaturated fatty acid (PUFA) of isolate CHS1, C18:4, was observed in fatty acid methyl esters (FAMEs) extracted from the cell membrane. CHS1 was capable of producing saturated fatty acids (SFA) (e.g., C16:0), monounsaturated fatty acids (MUFA) (e.g., C18:1) and polyunsaturated fatty acids (e.g., C20:5) in the cell membrane. In this study, we hypothesize that one mechanism of cold adaptation displayed by isolate CHS1 is the accumulation of high amounts of PUFA in the cell membrane.

Introduction of animal group A rotavirus (RVA) gene segments into the human RVA population is a major factor shaping the genetic landscape of human RVA strains. The VP6 and NSP4 genes of 74 G/P-genotyped RVA isolates collected in Rawalpindi during 2010 were analyzed, revealing the presence of VP6 genotypes I1 (60.8%) and I2 (39.2%) and NSP4 genotypes E1 (60.8%), E2 (28.3%) and E-untypable (10.8%) among the circulating human RVA strains. The typical human RVA combinations I1E1 and I2E2 were found in 59.4% and 24.3% of the cases, respectively, whereas 5.4% of the RVA strains were reassortants, i.e., either I1E2 or I2E1. The phylogeny of the NSP4 gene showed that one G2P[4] and two G1P[6] RVA strains clustered with porcine E1 RVA strains or RVA strains that were considered to be (partially) of porcine origin. In addition, the NSP4 gene segment of the unusual human G6P[1] RVA strains clustered closely with bovine E2 RVA strains, further strengthening the hypothesis of an interspecies transmission event. The study further demonstrates the role of genomic re-assortment and the involvement of interspecies transmission in the evolution of human RVA strains. The VP6 and NSP4 nucleotide sequences analyzed in the study received the GenBank accession numbers KC846908- KC846971 and KC846972-KC847037, respectively.

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan., Objective: The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan., Methodology: Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18., Results: Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10-25 years) and previously treated TB patients., Conclusion: DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.

Background: The association between dyspepsia, H. pylori and psychological distress has remained a topic of intense debate over the past several decades. In Pakistan, where depression is highly prevalent and dyspepsia is possibly present in a high percentage of population, little data exist about these common health problems. This study was conducted to determine the frequency and predictors of depression among patients presenting with dyspeptic symptoms in the Gastrointestinal (GI) Clinic of a tertiary care hospital in Pakistan., Methods: Two hundred and sixty-nine consecutive patients were enrolled in the study based on their presenting symptoms in the GI clinic at Shifa International Hospital, Islamabad. Subjects with prior history of peptic ulcer disease (PUD), gallstones and HCV infections were excluded from the study. Demographic and socioeconomic variables as well as dyspeptic symptoms and important causes of dyspeptic symptoms were recorded. Depression was analysed based on the Urdu version of Beck's Depression Inventory-II (BDI-II). The data were analysed using SPSS-10 for univariate and multivariate analyses., Results: Mild depression was associated with lower education status (p < 0.001), lesser income (p < 0.018), and lower socioeconomic status (0 < 0.009) as well as rural residence (p < 0.026). Smoking, alcohol-use, H. pylori infection, gender and dyspepsia were not found to have any association with depression. On multivariate analysis, education and income group remained significantly associated with mild depression. Clinically significant depression was found to be associated with lower education and rural residence., Conclusion: Depression among dyspeptic patients was found to be associated with socioeconomic status rather than dyspeptic symptoms or important risk factors associated with dyspeptic symptoms.

Objectives: To determine whether phylogenetic type is associated with the development of multidrug resistance to antibiotics., Methods: Urinary tract infection (UTI) isolates from three hospitals in Pakistan were collected over a period of 10 months, and analysed in terms of causative bacterial species and drug susceptibility., Results: Multidrug resistance was widespread and resistance frequencies were >50% for several of the most commonly used antibiotics, including ciprofloxacin and third-generation cephalosporins for Escherichia coli isolates. The great majority of E. coli isolates remained susceptible to meropenem and fosfomycin. Sixty E. coli isolates were analysed in detail to determine correlations between resistance phenotypes and genotypes, mutation rates and phylogenetic group. Most isolates had elevated mutation rates, suggesting this was being selected. The majority of ciprofloxacin-resistant isolates carried a specific set of mutations in the quinolone resistance-determining region of gyrA and parC (S83L, D87N, S80I and E84V). In addition, 67% of the ciprofloxacin-resistant E. coli isolates carried one or more horizontally transmissible determinants of resistance to ciprofloxacin, including aac(6')-Ib-cr, qepA, qnrA and qnrB. There was a significant correlation between resistance to third-generation cephalosporins, being an extended-spectrum β-lactamase producer, being resistant to ciprofloxacin and belonging to phylogenetic group B2., Conclusions: The data suggest that features of the bacterial genotype might facilitate the development of multidrug resistance in particular lineages. Better understanding of the mechanistic basis for correlations between drug resistance and genotype could potentially be exploited to develop molecular tools for the prediction of resistance development.

In this study, 121 Escherichia coli samples isolated from clinical specimens obtained from Pakistan Institute of Medical Science, Islamabad, Pakistan, were analyzed for extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases using disk-diffusion assay and polymerase chain reaction. Of the isolates, 78 and 43 were identified as ESBL and AmpC producers, respectively. The highest resistance (89%) was observed against cefotaxime, followed by ciprofloxacin (87.6%) and cefepime (87%). Genetic analysis showed the presence of different class A and class C β-lactamase genes, either alone (44.7%) or in combination (53.6%). CTX-M (57.7%) was the most prevalent among class A, followed by TEM (20.3%) and SHV (15.4%). CIT (including LAT-1 to LAT-4, CMY-2 to CMY-7, and BIL-1) and MOX (including MOX-1, MOX-2, CMY-1, and CMY-8 to CMY-11) family-specific plasmid-mediated AmpC β-lactamases were the most prevalent among these isolates. Our study showed that both class A and class C β-lactamases contributed to cephalosporin resistance in the E. coli isolates, thereby limiting therapeutic options. Co-expression of these enzymes may further hinder the identification of ESBLs, which is a critical step for designing a successful treatment for multidrug-resistant E. coli.

Common alpha-thalassemia (thal) rearrangements were studied in a normal random population and in six ethnic groups of Pakistan. Analyses of 204 individuals from the normal population revealed the presence of only the -alpha(3.7) allele with an overall frequency of 8.3%. Ethnic differences were statistically significant for Pashtoon vs. Balochi (p < 0.0005) and Pashtoon vs. Sindhi (p < 0.002). Two hundred and eighty-five thalassemia patients were also studied to identify rare alpha-thal alleles. In this group, 24.6% of the patients had one or two alpha genes deleted. Two rare alleles in the Pakistani population, -alpha(4.2) (0.2%) and alphaalphaalpha(anti3.7) (0.9%), were identified in these patients. The -alpha(4.2) allele was found only in Sindhis, while alphaalphaalpha(anti3.7) was present in Punjabis, Sindhis and Balochis. Five patients with triplicated alpha genes were homozygous for either the beta+ or the beta(0) genotype.

The prevalence of extended-spectrum beta-lactamases producing Enterobacteriaceae (ESBLPE) among nosocomial and community acquired isolates was studied (September, 1999 - August, 2000). Nosocomial and OPD isolates (200 each) were collected from OPD and different wards of the Pakistan Institute of Medical Sciences (PIMS) Islamabad, Pakistan, and tested for the production of ESBLs using the double disc diffusion technique. The prevalence of ESBL was highest in nosocomial isolates among the patients of age group III (50-60 years) (48%) with Escherichia coli as the most prevalent organism. ESBL positive isolates were mostly reported in males (65.33%) compared to females (34.67%).