Repositioning of anti-infective compounds against monkeypox virus core cysteine proteinase: a molecular dynamics study.

Monkeypox virus (MPXV) core cysteine proteinase (CCP) is one of the major drug targets used to examine the inhibitory action of chemical moieties. In this study, an in silico technique was applied to screen 1395 anti-infective compounds to find out the potential molecules against the MPXV-CCP. The top five hits were selected after screening and processed for exhaustive docking based on the docked score of ≤ -9.5 kcal/mol. Later, the top three hits based on the exhaustive-docking score and interaction profile were selected to perform MD simulations. The overall RMSD suggested that two compounds, SC75741 and ammonium glycyrrhizinate, showed a highly stable complex with a standard deviation of 0.18 and 0.23 nm, respectively. Later, the MM/GBSA binding free energies of complexes showed significant binding strength with ΔGTOTAL from -21.59 to -15 kcal/mol. This report reported the potential inhibitory activity of SC75741 and ammonium glycyrrhizinate against MPXV-CCP by competitively inhibiting the binding of the native substrate.

Designing Novel Multi-Epitope Vaccine Construct against Prevotella intermedia-Interpain A: An Immunoinformatics Approach.

Background and Objectives: Periodontitis is a chronic multifactorial inflammatory infectious disease marked by continuous degradation of teeth and surrounding parts. One of the most important periodontal pathogens is P. intermedia, and with its interpain A proteinase, it leads to an increase in lethal infection. Materials and Methods: The current study was designed to create a multi-epitope vaccine using an immunoinformatics method that targets the interpain A of P. intermedia. For the development of vaccines, P. intermedia peptides InpA were found appropriate. To create a multi-epitope vaccination design, interpain A, B, and T-cell epitopes were found and assessed depending on the essential variables. The vaccine construct was evaluated based on its stability, antigenicity, and allergenicity. Results: The vaccine construct reached a more significant population and was able to bind to both the binding epitopes of major histocompatibility complex (MHC)-I and MHC-II. Through the C3 receptor complex route, P. intermedia InpA promotes an immunological subunit. Utilizing InpA-C3 and vaccination epitopes as the receptor and ligand, the molecular docking and dynamics were performed using the ClusPro 2.0 server. Conclusion: The developed vaccine had shown good antigenicity, solubility, and stability. Molecular docking indicated the vaccine's 3D structure interacts strongly with the complement C3. The current study describes the design for vaccine, and steady interaction with the C3 immunological receptor to induce a good memory and an adaptive immune response against Interpain A of P. intermedia.

Insight into the Interaction Mechanism of HSA with Aztreonam: A Multispectroscopic and Computational Approach.

Aztreonam is a Gram-negative bacteria-targeting synthetic monobactam antibiotic. Human serum albumin (HSA) plays an important role in the transference of pharmaceuticals, hormones, and fatty acids, along with other compounds, determining their biodistribution and physiological fate. Using several biophysical and in silico approaches, we studied the interaction of aztreonam with HSA under physiological environments in this study. Results confirm the formation of HSA-aztreonam complex where aztreonam showed moderate affinity towards HSA. A static mode of quenching was confirmed from the steady state fluorescence data. FRET findings also showed that there was a significant feasibility of energy transfer between HSA and aztreonam. Site marker displacement experimental conclusion suggested the binding site of aztreonam was the sub-domain IB of HSA. Circular dichroic spectroscopic analysis suggested that aztreonam interaction decreases the α-helical content of HSA. Changes in microenvironment were studied through synchronous fluorescence data. According to molecular docking results, the HSA-aztreonam complex is mostly maintained by non-covalent forces, with a binding energy of 7.7 kcal mol-1. The presence of a hydrogen bond, van der Waal interaction, and pi-anion interaction in the binding process, as well as conformational changes in HSA after binding with aztreonam, are all confirmed by molecular dynamic simulation.

An Overview on Phenotypic and Genotypic Characterisation of Carbapenem-Resistant Enterobacterales.

Improper use of antimicrobials has resulted in the emergence of antimicrobial resistance (AMR), including multi-drug resistance (MDR) among bacteria. Recently, a sudden increase in Carbapenem-resistant Enterobacterales (CRE) has been observed. This presents a substantial challenge in the treatment of CRE-infected individuals. Bacterial plasmids include the genes for carbapenem resistance, which can also spread to other bacteria to make them resistant. The incidence of CRE is rising significantly despite the efforts of health authorities, clinicians, and scientists. Many genotypic and phenotypic techniques are available to identify CRE. However, effective identification requires the integration of two or more methods. Whole genome sequencing (WGS), an advanced molecular approach, helps identify new strains of CRE and screening of the patient population; however, WGS is challenging to apply in clinical settings due to the complexity and high expense involved with this technique. The current review highlights the molecular mechanism of development of Carbapenem resistance, the epidemiology of CRE infections, spread of CRE, treatment options, and the phenotypic/genotypic characterisation of CRE. The potential of microorganisms to acquire resistance against Carbapenems remains high, which can lead to even more susceptible drugs such as colistin and polymyxins. Hence, the current study recommends running the antibiotic stewardship programs at an institutional level to control the use of antibiotics and to reduce the spread of CRE worldwide.

Heavy Metal (Arsenic) Induced Antibiotic Resistance among Extended-Spectrum ß-Lactamase (ESBL) Producing Bacteria of Nosocomial Origin.

Antimicrobial resistance (AMR) is a leading cause of treatment failure for many infectious diseases worldwide. Improper overdosing and the misuse of antibiotics contributes significantly to the emergence of drug-resistant bacteria. The co-contamination of heavy metals and antibiotic compounds existing in the environment might also be involved in the spread of AMR. The current study was designed to test the efficacy of heavy metals (arsenic) induced AMR patterns in clinically isolated extended-spectrum ß-lactamase (ESBL) producing bacteria. A total of 300 clinically isolated ESBL-producing bacteria were collected from a tertiary care hospital in Lahore, Pakistan, with the demographic characteristics of patients. After the collection of bacterial isolates, these were reinoculated on agar media for reidentification purposes. Direct antimicrobial sensitivity testing (AST) for bacterial isolates by disk diffusion methods was used to determine the AST patterns with and without heavy metal. The heavy metal was concentrated in dilutions of 1.25 g/mL. The collected bacterial isolates were isolated from wounds (n = 63, 21%), urine (n = 112, 37.3%), blood (n = 43, 14.3%), pus (n = 49, 16.3%), and aspirate (n = 33, 11%) samples. From the total 300 bacterial isolates, n = 172 were Escherichia coli (57.3%), 57 were Klebsiella spp. (19%), 32 were Pseudomonas aeruginosa (10.6%), 21 were Proteus mirabilis (7%) and 18 were Enterobacter spp. (6%). Most of the antibiotic drugs were found resistant to tested bacteria. Colistin and Polymyxin-B showed the highest sensitivity against all tested bacteria, but when tested with heavy metals, these antibiotics were also found to be significantly resistant. We found that heavy metals induced the resistance capability in bacterial isolates, which leads to higher AMR patterns as compared to without heavy metal tested isolates. The results of the current study explored the heavy metal as an inducer of AMR and may contribute to the formation and spread of AMR in settings that are contaminated with heavy metals.

Designing mRNA- and Peptide-Based Vaccine Construct against Emerging Multidrug-Resistant Citrobacter freundii: A Computational-Based Subtractive Proteomics Approach.

Background and Objectives: Citrobacter freundii (C. freundii) is an emerging and opportunistic Gram-negative bacteria of the human gastrointestinal tract associated with nosocomial and severe respiratory tract infections. It has also been associated with pneumonia, bloodstream, and urinary tract infections. Intrinsic and adaptive virulence characteristics of C. freundii have become a significant source of diarrheal infections and food poisoning among immune-compromised patients and newborns. Impulsive usage of antibiotics and these adaptive virulence characteristics has modulated the C. freundii into multidrug-resistant (MDR) bacteria. Conventional approaches are futile against MDR C. freundii. Materials and Methods: The current study exploits the modern computational-based vaccine design approach to treat infections related to MDR C. freundii. A whole proteome of C. freundii (strain: CWH001) was retrieved to screen pathogenic and nonhomologous proteins. Six proteins were shortlisted for the selection of putative epitopes for vaccine construct. Highly antigenic, nonallergen, and nontoxic eleven B-cell, HTL, and TCL epitopes were selected for mRNA- and peptide-based multi-epitope vaccine construct. Secondary and tertiary structures of the multi-epitope vaccine (MEVC) were designed, refined, and validated. Results: Evaluation of population coverage of MHC-I and MHC-II alleles were 72% and 90%, respectively. Docking MEVC with TLR-3 receptor with the binding affinity of 21.46 (kcal/mol) occurred through the mmGBSA process. Further validations include codon optimization with an enhanced CAI value of 0.95 and GC content of about 51%. Immune stimulation and molecular dynamic simulation ensure the antibody production upon antigen interaction with the host and stability of the MEVC construct, respectively. Conclusions: These interpretations propose a new strategy to combat MDR C. freundii. Further, in vivo and in vitro trials of this vaccine will be valuable in combating MDR pathogens.

Prevalence of Bacterial Pathogens among Symptomatic-SARS-CoV-2 PCR-Negative Patients.

The epidemiological and clinical aspects of coronavirus disease-2019 (COVID-19) have been subjected to several investigations, but little is known about symptomatic patients with negative SARS-CoV-2 PCR results. The current study investigated patients who presented to the hospital with respiratory symptoms (but negative SARS-CoV-2 RT-PCR results) to determine the prevalence of bacterial pathogens among these patients. A total of 1246 different samples were collected and 453 species of bacterial pathogens were identified by culture. Antibiotic susceptibility testing was performed via the Kirby Bauer disc diffusion test. Patients showed symptoms, such as fever (100%), cough (83%), tiredness (77%), loss of taste and smell (23%), rigors (93%), sweating (62%), and nausea (81%), but all tested negative for COVID-19 by PCR tests. Further examinations revealed additional and severe symptoms, such as sore throats (27%), body aches and pain (83%), diarrhea (11%), skin rashes (5%), eye irritation (21%), vomiting (42%), difficulty breathing (32%), and chest pain (67%). The sum of n = 1246 included the following: males, 289 were between 5 and 14 years, 183 (15-24 years), 157 (25-34 years), 113 (35-49 years), and 43 were 50+ years. Females: 138 were between 5 and 14 years, 93 (15-24 years), 72 (25-34 years), 89 (35-49 years), and 68 were 50+ years. The Gram-positive organisms isolated were Staphylococcus aureus (n = 111, 80.43%, MRSA 16.6%), E. faecalis (n = 20, 14.49%, VRE: 9.4%), and Streptococcus agalactiae (n = 7, 5.07%), while, Gram-negative organisms, such as E. coli (n = 135, 42.85%, CRE: 3.49%), K. pneumoniae (n = 93, 29.52%, CRE: 1.58%), P. aeruginosa (n = 43, 13.65%), C. freundii (n = 21, 6.66%), Serratia spp. (n = 8, 2.53%), and Proteus spp. (n = 15, 4.76%) were identified.

Regulation of Host Immune Response against Enterobacter cloacae Proteins via Computational mRNA Vaccine Design through Transcriptional Modification.

Enterobacter cloacae is mainly responsible for sepsis, urethritis, and respiratory tract infections. These bacteria may affect the transcription of the host and particularly their immune system by producing changes in their epigenetics. In the present study, four proteins of Enterobacter cloacae were used to predict the epitopes for the construction of an mRNA vaccine against Enterobacter cloacae infections. In order to generate cellular and humoral responses, various immunoinformatic-based approaches were used for developing the vaccine. The molecular docking analysis was performed for predicting the interaction among the chosen epitopes and corresponding MHC alleles. The vaccine was developed by combining epitopes (thirty-three total), which include the adjuvant Toll-like receptor-4 (TLR4). The constructed vaccine was analyzed and predicted to cover 99.2% of the global population. Additionally, in silico immunological modeling of the vaccination was also carried out. When it enters the cytoplasm of the human (host), the codon is optimized to generate the translated mRNA efficiently. Moreover, the peptide structures were analyzed and docked with TLR-3 and TLR-4. A dynamic simulation predicted the stability of the binding complex. The assumed construct was considered to be a potential candidate for a vaccine against Enterobacter cloacae infections. Hence, the proposed construct is suitable for in vitro analyses to validate its effectiveness.

Immune and Oxidative Response against Sonicated Antigen of Mycoplasma capricolum subspecies capripneumonia-A Causative Agent of Contagious Caprine Pleuropneumonia.

Vaccines are vital for prevention and control of mycoplasma diseases. The exploration of a vaccine candidate for the development of a vaccine is imperative. The present study envisages the evaluation of immune and oxidative response against an adjuvanted, sonicated antigen of Mycoplasma capricolum subsp. capripneumonia in male Angora rabbits (1 year old, 2 kg) divided in four groups, each having six animals. Group 1 was the healthy control and received 1 mL PBS via subcutaneous route. Group 2 was administered 1 mL of saponin-adjuvanted and -sonicated antigen, Group 3 was given 1 mL of montanide ISA 50-adjuvanted and-sonicated antigen, and Group 4 was given 1 mL of standard vaccine via subcutaneous route. Animals were evaluated for cellular and humoral immune response and oxidative parameters at 0, 7, 14, 21, and 28 days of the study. Total leukocytic, neutrophilic, and basophilic counts showed a significant (p < 0.05) increase in vaccinated groups compared to the healthy group on most of the intervals. TNF-α levels were significantly (p < 0.05) higher in the Group 2 than the Group 1 at all the time intervals and more comparable to Group 4 than Group 3. IL-10 levels were significantly (p < 0.05) higher in vaccinated groups compared to the healthy group on days 14, 21, and 28, but were lower in Group 3 than in Group 2 and Group 4. More hypersensitivity as inflammation and histopathological cellular infiltration in the ear was produced in Group 2 and Group 4 than in Group 3. IgG levels were significantly (p < 0.05) higher in Group 2 and Group 4 than in Group 3 on days 14 and 21. Antibody titers were comparatively higher in Group 4, followed by Group 2 and 3, than Group 1. Significantly (p < 0.05) higher oxidant and lower antioxidant values were noted in Group 2 and 4 compared to Group 3 and Group 1 on most of the intervals. The TLC and antibody titer showed increasing trend throughout the trial, whereas TNF-α, IgG, L, M and E started decreasing from day 14, and IL-10, N and B started decreasing from day 21. This study concludes that the saponin-adjuvanted and-sonicated antigen induces comparatively higher immune response than montanide but is associated with oxidative and inflammatory reactions.

Genotypic and Phenotypic Characterization of Erythromycin-Resistant Staphylococcus aureus Isolated from Bovine Mastitis and Humans in Close Contact.

Staphylococcus aureus (S. aureus) is a major causative agent of mastitis and is resistant to many antibiotics. Thus, there is a need to characterize the genetic determinants of S. aureus erythromycin resistance, such as ermA, ermB and ermC. The current study aimed to determine the phenotypic and genotypic erythromycin resistance profile and relatedness of S. aureus recovered from bovine mastitis and humans in close contact. A total of 14 mastitis-infected buffalo milk samples and 16 samples from their respective milkers were collected from different farms of Lahore, Pakistan. The antibiotic resistance profile was determined through the disk diffusion test. The overall prevalence of S. aureus in mastitis-affected buffaloes was found to be 75%, of which 52.1% were resistant to erythromycin and 42.8% to clindamycin. S. aureus isolates recovered from milker nasal samples showed 56.25% resistance to erythromycin and 44% resistance to clindamycin. Genotypic antibiotic resistance profiles were determined from 14 milk samples through PCR. Overall, eight (52.1%), three (21.4%) and five (35.7%) S. aureus isolates were positive for the ermA, ermB and ermC genes, respectively. Moreover, 16 milker nasal S. aureus isolates were also tested for the presence of ermA, ermB and ermC genes. The ermA, ermB and ermC genes were observed in nine(56.7%), five (31.3%) and seven (43.7%) isolates, respectively. A significant association was shown between phenotypic and genotypic erythromycin resistance. The results indicate both that there are sufficient genetic similarities, and the actual transmission of erythromycin resistance genes between these two hosts of S. aureus.