Evaluation of Antimicrobial Effect of Silver Nanoparticle Based Whey Emulsions and Edible Films for the Extension of Shelf Life of Fruits and Vegetables.

The purpose of this study was to develop and assess the antimicrobial properties of silver nanoparticles (AgNPs)-based whey emulsions and edible films for extending the shelf life of fruits and vegetables. The AgNPs were synthesized using a biological method, and their morphological and topographical characteristics were evaluated using scanning electron microscopy (SEM). The AgNPs were incorporated into the emulsions and films to increase their antimicrobial efficacy. Bacterial and fungal strains were identified by DNA regions, including 16S and 18S rRNA, TEF-1α, and RPB2 to evaluate antimicrobial activity. AgNPs-based emulsions and films were used to extend the shelf life of fruits and vegetables for up to 15 days. The results showed that the use of AgNPs in the coated samples significantly increased their effectiveness against bacterial and fungal strains. SEM analysis revealed the presence of AgNPs of varying sizes, ranging from 21 to 62 nm. The zones of inhibition were measured against Staphylococcus aureus, Escherichia coli, Salmonella enterica, Aspergillus flavus, Aspergillus tamari, and Aspergillus niger. The total viable count (log cfu/ml) decreased from 6.423 in the control group to 3.301 in the treated samples. The antioxidant activity of the treated fruits and vegetables was also significantly improved, with values of 56.12, 23.36, 26.10, 7.6, 36.04, and 33.81% for strawberry, taro root, guava, peas, green chili, and carrot, respectively (p < 0.05). The AgNPs-based whey protein emulsions were found to exhibit the highest antimicrobial activity and are therefore a promising approach to extend the shelf life of fruits and vegetables.

Designing a Novel Peptide-Based Multi-Epitope Vaccine to Evoke a Robust Immune Response against Pathogenic Multidrug-Resistant Providencia heimbachae.

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.

In vitro and in silico Studies Reveal Bacillus cereus AA-18 as a Potential Candidate for Bioremediation of Mercury-Contaminated Wastewater.

Mercury (Hg) pollution is a worldwide problem and increasing day by day due to natural and anthropogenic sources. In this study, mercury-resistant (HgR) bacterial isolates were isolated from industrial wastewater of Ittehad Chemicals Ltd., Kala Shah Kaku, Lahore, Pakistan. Out of 65 bacterial isolates, five isolates were screened out based on showing resistance at 30-40 µg/ml against HgCl2. Selected Hg-resistant bacterial isolates were characterized as Bacillus subtilis AA-16 (OK562835), Bacillus cereus AA-18 (OK562834), Bacillus sp. AA-20 (OK562833), Bacillus paramycoides AA-30 (OK562836), and Bacillus thuringiensis AA-35 (OK562837). B. cereus AA-18 showed promising results in the resistance of HgCl2 (40 µg/ml) due to the presence of merA gene. Scanning electron microscopy (SEM) analysis of immobilized B. cereus AA-18 showed the accumulation Hg on the cell surface. The inoculation of immobilized B. cereus AA-18 remediated 86% Hg of industrial wastewater up to 72 h at large scale (p < 0.05). In silico analysis showed structural determination of MerA protein encoded by merA gene of B. cereus AA-18 (OK562598) using ProtParam, Pfam, ConSurf Server, InterPro, STRING, Jpred4, PSIPRED, I-TASSER, COACH server, TrRosetta, ERRAT, VERIFY3D, Ramachandran plot, and AutoDock Vina (PyRx 8.0). These bioinformatics tools predicted the structural-based functional homology of MerA protein (mercuric reductase) associated with mer operon harboring bacteria involved in Hg-bioremediation system.

Antibiotyping and genotyping of extensively drug-resistant (XDR) Salmonella sp. isolated from clinical samples of Lahore, Pakistan.

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.

Characterization of Mercury-Resistant Rhizobacteria for Plant Growth Promotion: An In Vitro and In Silico Approach.

In this study, a total 30 rhizobacterial isolates were screened out based on resistance against different concentrations of mercuric chloride (HgCl2), growth on nitrogen-free mannitol (NFM) and production of indole-3-acetic acid (IAA). The biochemical and plant growth promoting characterization of selected isolates was performed by different biochemical tests. Out of 30, six isolates, UM-3, AZ-5, UM-7, UM-11, UM-26, and UM-28 showed resistance at 30 µg/ml HgCl2, pronounced growth on NFM and high production of IAA as 18.6, 16.7, 16, 18.7, 14, and 16 µg/ml, respectively (P < 0.05). The 16S rDNA ribotyping and phylogenetic analysis of selected bacterial isolates were performed and characterized as Exiguobacterium sp. UM-3 (KJ736011), Bacillus thuringiensis AZ-5 (KJ675627), Bacillus subtilis UM-7 (KJ736013), Enterobacter cloacae UM-11 (KJ736014), Pseudomonas aeruginosa UM-26 (KJ736016), P. aeruginosa UM-28 (KJ736017) and Bacillus pumilus UM-16 (KJ736015) used as negative control. B. thuringiensis AZ-5 showed high resistance against 30 µg/ml of HgCl2 due to the presence of merB gene. The structural determination of MerB protein was carried out using bioinformatics tools, i.e., Protparam, Pfam, InterProScan, STRING, Jpred4, PSIPRED, I-TASSER, COACH server and ERRAT. These tools predicted the structural based functional homology of MerB protein (organomercuric lyase) in association with MerA (mercuric reductase) in bacterial Hg-detoxification system.

Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus.

Mercury-resistant (HgR) 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 (HgCl2). 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 (Hg0) 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 HgCl2 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).

Cloning, Expression, Isotope Labeling, and Purification of Transmembrane Protein MerF from Mercury Resistant Enterobacter sp. AZ-15 for NMR Studies.

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.

Screening of mercury-resistant and indole-3-acetic acid producing bacterial-consortium for growth promotion of Cicer arietinum L.

Mercury resistant (HgR ) 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-1 of HgCl2 and indole-3-acetic acid (IAA) production as 8-38 µg ml-1 . 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 (Hg0 ) 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+2 ) 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+2 ) 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-1 HgCl2 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).