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Mercury resistant (HgR) Enterobacter sp. AZ-15 was isolated from heavy metal polluted industrial wastewater samples near to districts Kasur and Sheikhupura, Pakistan. 16S rDNA ribotyping and phylogentic analysis showed 98% homology with already reported Enterobacter species. The merF gene encoding transmembrane protein-MerF was amplified from genomic DNA and ligated into pET31b+ vector using restriction endonucleases, SphI and XhoI. The genetic codons of merF gene encoding cysteine residues were mutated into codons, translating into serine residues by site-directed mutagenesis. Ketosteroid isomerase (KSI), a fusion tag which is present in pET31b+ vector, was used in the expression of merFm gene. KSI was used to drive the target peptide (MerFm) into inclusion bodies so that the peptide yield and purity were increased. The stable plasmid pET31b+:merFm was transformed into C43(DE3) E.coli cells. The high expression of uniformly 15N isotopically labeled-MerFm protein was induced with 1 mM IPTG. The purification of 15N-MerFm recombinant protein by Ni-NTA and size exclusion chromatography involved an unfolding/refolding procedure. The two-dimensional HSQC NMR spectra of MerFm protein showed the purity and correct number of resonances for each amide. 1H–15N HSQC NMR experiment also confirmed that no modification of the tryptophan residue occurred during cyanogen bromide cleavage. A small scale reservoir of Luria Bertani (LB) medium supplemented with 20 μg/ml of HgCl2 showed 90% detoxification of Hg by Enterobacter sp. AZ-15. The accumulation of Hg on the cell surface of this strain was visualized by scanning electron microscopy (SEM) which confirmed its potential use in Hg-bioremediation.

Mercury resistant (Hg ) bacteria were screened from industrial effluents and effluents-polluted rhizosphere soils near to districts Kasur and Sheikhupura, Pakistan. Out of 60 isolates, three bacterial strains, Bacillus sp. AZ-1, Bacillus cereus AZ-2, and Enterobacter cloacae AZ-3 showed Hg-resistance as 20 μg ml of HgCl and indole-3-acetic acid (IAA) production as 8-38 μg ml . Biochemical and molecular characterization of selected bacteria was confirmed by 16S ribotyping. Mercury resistant genes merA, merB, and merE of mer operon in Bacillus spp. were checked by PCR amplification. The merE gene involved in the transportation of elemental mercury (Hg ) via cell membrane was first time cloned into pHLV vector and transformed in C43(DE3) Escherichia coli cells. The recombinant plasmid (pHLMerE) was expressed and purified by nickel (Ni ) affinity chromatography. Chromatographic techniques viz. thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and Gas chromatography-mass spectrometry (GC-MS) confirmed the presence of Indole-3-acetic acid (IAA) in supernatant of selected bacteria. The strain E. cloacae AZ-3 detoxified 88% of mercury (Hg ) from industrial effluent (p < 0.05) after immobilization in Na-alginate beads. Finally, Hg-resistant and IAA producing bacterial consortium of two strains, Bacillus sp. AZ-1 and E. cloacae AZ-3, inoculated in mercury amended soil with 20 μg ml HgCl resulted 80, 22, 64, 116, 50, 75, 30, and 100% increase as compared to control plants in seed germination, shoot and root length, shoot and root fresh weight, number of pods per plant, number of seeds and weight of seeds, respectively, of chickpea (Cicer arietinum L.) in pot experiments (p < 0.05).

Mercury resistant Pseudomonas strains were isolated from polluted water, rhizospheric soil and root nodules of different plant species. Selected bacterial isolates showed potential to produce florescence under UV light and to tolerate heavy metal stress of 10-20 µg/ml HgCl2. Biochemical characterization revealed all selected bacterial isolates belong to genus Pseudomonas. A total of 27 bacterial isolates were evaluated for bioremediation of mercury due to their ability to produce H2S and fix nitrogen. Genomic DNA of 12 Pseudomonas strains was isolated and their genetic diversity was evaluated using SSR (GACA)4 microsatellite finger printing. On the basis of banding pattern of PCR amplified products, a phylogenetic tree was constructed using Minitab 13 software which showed 71%, 46% and 44% homology of SZ-30, SZ-6 and SZ-8 with strains of cluster 1 and SZ-16 respectively. Results exhibited significant diversity among selected Pseudomonas strains.

Aims This study was designed to analyse the prevalence of class 1 and class 2 integron integrase genes among antibiotic‐resistant coliform bacteria isolated from paper currency circulating in Pakistan. Methods and Results A total of 500 individual currency notes were collected from different food vending sites at Lahore, Pakistan. Bacterial population were identified by biochemical and PCR techniques. Antimicrobial susceptibility testing was performed by disc diffusion assay. The highest bacterial population on currency was found from street vendors and butcher shops. Escherichia coli was found to be the most prevalent coliform bacteria followed by Klebsiella sp. and Enterobacter sp. PCR amplification of antimicrobial resistance gene showed the presence of ampC, blaTEM, blaNDM‐1, qnrA, tet(A) and tet(B) genes among coliform isolates. A total of 47 integron integrase bearing strains of coliform bacteria were analysed. Sequence analysis showed the presence of dfrA1‐aadA1, dfrA1, dfrA5, dfrA7, aadA1, aadA4 cassette arrays in class 1 integron and dfrA1‐sat2‐aadA1 in class 2 integrase genes. Conclusion Circulating currency was heavily contaminated with antimicrobial‐resistant coliform bacteria bearing class 1 and class 2 integron integrase genes. Significance and Impact of the Study This study describes a potential threat of severe bacterial infections due to improper hand hygiene and community sanitation when dealing with the currency notes.

Mercury-resistant (Hg R) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 µg/ml against mercuric chloride (HgCl 2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercuryresistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury (Hg 0) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing 30 µg/ml of HgCl 2 was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

Aims Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi. The objective of this study was to evaluate the prevalence of XDR Salmonella among local population of Lahore and genotyping of isolates for antibiotic‐resistant genes. Methods and Results A total of 200 blood samples from suspected typhoid fever patients were collected. One hundred and fifty‐seven bacterial samples were confirmed as Salmonella Typhi and 23 samples were confirmed as Salmonella Paratyphi after biochemical, serological and PCR based molecular characterization. Antibiogram analysis classified 121 (67·2%) Salmonella isolates as MDR and 62 isolates (34·4%) as XDR. The predominant resistance gene was ampC with 47·7% prevalence, followed by gyrA, catA1, tet(A), aac (3)‐la, qnrS, blaNDM‐1 and blaCTX‐M‐15 genes in 45·5, 40, 21·6, 18·3, 11·6, 2·2 and 0·5% isolates respectively. Sequence analysis showed the presence of sul1 and dfrA7 gene cassette arrays in 12 class 1 integron integrase positive isolates. Conclusion Large number of clinical XDR S. Typhi‐resistant against third generation cephalosporins have been reported. Significance and Impact of the Study The current study highlights the possible emergence of clinical XDR S. Typhi cases in Lahore, Pakistan. Potential attribution of phenotypic and genotypic XDR cases may help to contribute targeted therapy.

Providencia heimbachae, a Gram -ve, rod-shaped, and opportunistic bacteria isolated from the urine, feces, and skin of humans engage in a wide range of infectious diseases such as urinary tract infection (UTI), gastroenteritis, and bacteremia. This bacterium belongs to the Enterobacteriaceae family and can resist antibiotics known as multidrug-resistant (MDR), and as such can be life-threatening to humans. After retrieving the whole proteomic sequence of P. heimbachae ATCC 35613, a total of 6 non-homologous and pathogenic proteins were separated. These shortlisted proteins were further analyzed for epitope prediction and found to be highly non-toxic, non-allergenic, and antigenic. From these sequences, T-cell and B-cell (major histocompatibility complex class 1 and 2) epitopes were extracted that provided vaccine constructs, which were then analyzed for population coverage to find its reliability worldwide. The population coverage for MHC-1 and MHC-2 was 98.29% and 81.81%, respectively. Structural prediction was confirmed by validation through physiochemical molecular and immunological characteristics to design a stable and effective vaccine that could give positive results when injected into the body of the organism. Due to this approach, computational vaccines could be an effective alternative against pathogenic microbe since they cover a large population with positive results. In the end, the given findings may help the experimental vaccinologists to develop a very potent and effective peptide-based vaccine.