Characterization of proteome wide antigenic epitopes to design proteins specific and proteome-wide ensemble vaccines against heartland virus using structural vaccinology and immune simulation approaches.

Heartland virus is a single-stranded negative-sense RNA virus that infects humans and causes lethargy, myalgia, headaches, nausea, diarrhea, weight loss, arthralgia, loss of appetite, leukopenia, and easy bruising due thrombocytopenia. The unavailability of antiviral drugs for HRTV infection is a major obstacle to treat this infection, therefore supportive care management is adopted in the case of a severe ailment. In this scientific study, proteins specific and proteome-wide Helper T-cell (HTL), linear B cell, and cytotoxic T-cell (CTL) epitopes mapping joined together with suitable linkers to design multi-epitopes subunit vaccine (MEVC). The constructed four vaccines from nucleocapsid protein, replicase, glycoprotein and finally whole proteome-wide constructs demonstrated stronger antigenic and non-allergenic behavior. Physiochemical properties evaluation also reported easy and efficient expression and downstream analysis of the constructs. Molecular docking of these constructs with toll-like receptor 7 (TLR7) revealed good binding and further validation based on MM/GBSA also demonstrated stronger interaction between the vaccine constructs and TLR7. Moreover, in silico cloning reported CAI value of 0.96 for each construct and excellent GC contents percentage required for experimental analysis. Furthermore, immune simulation-based immune response surveillance revealed that upon the injection of antigen the primary and secondary antibodies were produced between 5 and 15 days, and a more robust neutralization of the antigen by the proteome-wide vaccine construct was observed. This research could pave the way for the development of dynamic and efficient vaccines that contain a unique mix of numerous HRTV derived antigenic peptides to control HRTV infection.

Antibiogram and molecular characterization of multi-drug resistant microorganisms isolated from urinary tract infections.

Bacteria are the commonest etiological factor among the microbes that cause UTIs. The most prevalent bacteria identified in the lab are Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. Antibiotics are the empiric therapy for such infections but the reoccurrence rate is becoming high owing to the development of resistance due to their irrational and indiscriminate use across the globe. This study was designed on UTI cases of OPD, Medical, Nephrology, Surgical, Main OT, Urology and ICU wards of Allied hospital Faisalabad. 11 antibiotics were used which showed that E. coli is sensitive to Amikacin, Gentamicin, Imipenem, Piperacillin tazobactam, and Polymyxin B. Klebsiella pneumonia showed sensitivity for Amikacin, Gentamicin, Nitrofurantoin, Imipenem, Polymyxin B, Piperacillin tazobactam and Trimethoprim-sulfamethoxazole. While Pseudomonas aurignosa showed resistance to Amikacin, Ciprofloxacin, Gentamicin, Piperacillin tazobactam, Imipenem, and Polymyxin B. E. coli exhibited the highest sensitivity for Piperacillin tazobactam, Klebsiella pneumonia for Imipenem and Pseudomonas aurignosa for Ciprofloxacin. Further, the isolated DNA samples of these microorganisms were confirmed by gel electrophoresis and subjected to molecular characterization by performing trace file and phylogenetic tree analysis.

Structural basis for the mechanism of interaction of SARS-CoV-2 B.1.640.2 variant RBD with the host receptors hACE2 and GRP78.

The inflicted chaos instigated by the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) globally continues with the emergence of novel variants. The current global outbreak is aggravated by the manifestation of novel variants, which affect the effectiveness of the vaccine, attachment with hACE2 (human Angiotensin-converting enzyme 2) and immune evasion. Recently, a new variant named University Hospital Institute (IHU) (B.1.640.2) was reported in France in November 2021 and is spreading globally affecting public healthcare. The B.1.640.2 SARS-CoV-2 strain revealed 14 mutations and 9 deletions in spike protein. Thus, it is important to understand how these variations in the spike protein impact the communication with the host. A protein coupling approach along with molecular simulation protocols was used to interpret the variation in the binding of the wild type (WT) and B.1.640.2 variant with hACE2 and Glucose-regulating protein 78 (GRP78) receptors. The initial docking scores revealed a stronger binding of the B.1.640.2-RBD with both the hACE2 and GRP78. To further understand the crucial dynamic changes, we looked at the structural and dynamic characteristics and also explored the variations in the bonding networks between the WT and B.1.640.2-RBD (receptor-binding domain) in association with hACE2 and GRP78, respectively. Our findings revealed that the variant complex demonstrated distinct dynamic properties in contrast to the wild type due to the acquired mutations. Finally, to provide conclusive evidence on the higher binding by the B.1.640.2 variant the TBE was computed for each complex. For the WT with hACE2 the TBE was quantified to be-61.38 ± 0.96 kcal/mol and for B.1.640.2 variant the TBE was estimated to be -70.47 ± 1.00 kcal/mol. For the WT-RBD-GRP78 the TBE -was computed to be 32.32 ± 0.56 kcal/mol and for the B.1.640.2-RBD a TBE of -50.39 ± 0.88 kcal/mol was reported. This show that these mutations are the basis for higher binding and infectivity produced by B.1.640.2 variant and can be targeted for drug designing against it.Communicated by Ramaswamy H. Sarma.

Antibacterial efficacy of silver nanoparticles (AgNPs) against metallo-ß-lactamase and extended spectrum ß-lactamase producing clinically procured isolates of Pseudomonas aeruginosa.

Resistance to carbapenems is a global threat, especially in developing countries with limited health resources. Prevalence, antibiogram, PCR detection of antibiotic resistance genes, and potency of Silver Nanoparticles (AgNPs) against multidrug-resistant (MDR) Pseudomonas aeruginosa were studied. Kirby-Bauer disc method and PCR were used to study antibiogram and drug resistance genes respectively in 255 isolates of Pseudomonas aeruginosa obtained from a tertiary care hospital. Silver nitrate (AgNO3) precursor salts were reacted with Aspergillus flavus culture filtrate to trigger the extracellular mycosynthesis of AgNPs. Mycosynthesis was first monitored regularly by visible ultraviolet spectroscopy that recorded AgNP peaks of approximately 400-470 nm. Confirmation by Transmission electron micrographs provided confirmation of AgNPs formed within a range of 5-30 nm. Individual and combined antibacterial activity of ten antibiotics and AgNPs was analyzed. Pearson correlation coefficients (r) were calculated for phenotypic and genotypic multidrug resistance. Data were evaluated using SPSS version 20. p-value < 0.05 was considered statistically significant. 61.5% were carbapenemase producers (p < 0.01). The recorded frequency of blaIMP-1, blaSHV, blaVIM, blaOXA, and blaTEM were 13%, 32%, 15%, 21%, and 43%, respectively. The reducing order of antimicrobial activity of antibiotics and AgNPs was piperacillin/tazobactam + AgNPs (31 mm), cefoxitin + AgNPs (30 mm) > amikacin + AgNPs (25 mm) > aztreonam + AgNPs (23 mm) > meropenem + AgNPs (22 mm) > imipenem + AgNPs (20 mm) > gentamycin + AgNPs (17 mm) > ciprofloxacin + AgNPs (16 mm) > cefoperazone/sulbactam + AgNPs (14 mm) ≥ ceftazidime + AgNPs (14 mm). The conjugated effect of AgNPs plus antibiotics showed a 0.15-3.51 (average of 2.09) fold-area augmentation of antimicrobial activity. AgNPs conjugated with antibiotics effectively inhibited MDR Pseudomonas aeruginosa. To the best of our understanding, this is an inaugural report from Punjab Pakistan enlisting co-expression of Metallo-ß-lactamases, extended-spectrum ß-lactamases, and AmpC-ß-lactamase plus activity of antibiotic-AgNPs.

Epidemiology and high incidence of metallo-ß-lactamase and AmpC-ß-lactamases in nosocomial Pseudomonas aeruginosa.

OBJECTIVES: Isolates producing metallo-ß-lactamase (MBL) have a significant impact on therapeutic and diagnostic layouts, plus their increased frequency has been reported globally. Determination of incidence of clinical isolates of Pseudomonas aeruginosa that are capable of producing MBL and AmpC-ß-lactamases making them resistant to imipenem and cefoxitin. MATERIALS AND METHODS: Out of 1159 collected samples of urine, wound swabs, blood, tissue, and pus, the isolation rate of P. aeruginosa in the period of March 2020 to February 2021 was 22.0% (255/1159). Bacterial strains that were resistant towards imipenem were further processed for detecting the ß-lactamase group of genes followed by statistical analysis of risk factors done based on clinical sample, gender, plus department of sample collection. RESULTS: The percentage of resistance against imipenem was found to be 53%. Out of 135 strains, phenotypic tests revealed MBLs incidence to be 61.5% by combination disc test and 81.5% by Modified Hodge test (MHT). Frequencies of blaIMP-1, blaVIM, blaSHV, blaTEM, and blaOXA genes were calculated to be 13%, 15%, 32%, 43%, and 21%, respectively. Co-expressions of blaMBLs (blaVIM and blaIMP-1) plus blaESBL (blaSHV, blaOXA, blaTEM) were detected using simplex and multiplex PCR. blaTEM, blaSHV, and blaOXA co-existed in 7.5% of clinical isolates. 5.5% of the isolates exhibited simultaneous expression of MBL/ESBL genes. 15% of the isolates resistant to cefoxitin were positive for the blaAmpC gene (17/114). CONCLUSION: This is a pioneer report from Pakistan that concomitantly presents expression of blaVIM and blaIMP-1 with blaTEM, blaOXA, blaSHV, and blaAmpC in isolates of P. aeruginosa.