Methicillin resistant Staphylococcus aureus: A brief review of virulence and resistance.

Staphylococcus aureus is a common gram-positive human pathogen involved in both community-acquired and nosocomial infections ranging from localised superficial lesions to food poisoning and fatal systemic infections owing to its impressive array of virulence factors responsible for attaching, colonising, invading, and avoiding host immune system. The discovery of antibiotics effectively checked the once deadly infections. However, resistance started soon after their discovery and the first methicillin-resistant strain of staphylococcus aureus was reported in the early 1960s. The most important attribute of methicillin-resistant staphylococcus aureus is its acquisition of mecA gene coding for penicillin-binding protein-2a that blocks inhibitory action on peptidoglycan cross-linking. Methicillin-resistant staphylococcus aureus presents a serious global healthcare concern being responsible for prolonged hospital stays and increased mortality. The precise information of virulence factors and resistant traits of methicillin-resistant staphylococcus aureus and their interplay in a community is key to minimize the intermixing of resistant and susceptible pathogens in the community.

Cognitive therapy for depression in tuberculosis treatment: protocol for multicentre pragmatic parallel arm randomised control trial with an internal pilot.

INTRODUCTION AND OBJECTIVES: There is an unmet need to develop high-quality evidence addressing tuberculosis (TB)-related mental health comorbidity, particularly in the context of lower-middle-income countries. This study aims to examine the effectiveness and cost-effectiveness of cognitive behavioural therapy (CBT) versus enhanced treatment as usual (ETAU) in improving depressive symptoms in people with TB and comorbid depression, enhancing adherence with anti-TB treatment (ATT) and its implementation in the real-world setting of Pakistan. METHODS: We will conduct a pragmatic parallel arm randomised control trial with an internal pilot. A brief psychological intervention based on CBT has been developed using a combination of qualitative and ethnographic studies. The inbuilt pilot trial will have a sample size of 80, while we plan to recruit 560 (280 per arm) participants in the definitive trial. Participants who started on ATT within 1 month of diagnosis for pulmonary and extrapulmonary TB or multidrug resistant TB (MDR-TB) and meeting the criteria for depression on Patient Health Questionnaire-9 (PHQ-9) will be randomised with 1:1 allocation to receive six sessions of CBT (delivered by TB healthcare workers) or ETAU. Data on the feasibility outcomes of the pilot will be considered to proceed with the definitive trial. Participants will be assessed (by a blinded assessor) for the following main trial primary outcomes: (1) severity of depression using PHQ-9 scale (interviewer-administered questionnaire) at baseline, weeks 8, 24 and 32 postrandomisation and (2) ATT at baseline and week 24 at the end of ATT therapy. ETHICS AND DISSEMINATION: Ethical approval has been obtained from Keele University Research Ethics Committee (ref: 2023-0599-792), Khyber Medical University Ethical Review Board (ref: DIR/KMU-EB/CT/000990) and National Bioethics Committee Pakistan (ref: No.4-87/NBC-998/23/587). The results of this study will be reported in peer-reviewed journals and academic conferences and disseminated to stakeholders and policymakers. TRIAL REGISTRATION NUMBER: ISRCTN10761003.

Co-occurrence of antibiotic resistance and virulence Genes in Methicillin Resistant Staphylococcus aureus (MRSA) Isolates from Pakistan.

Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is one of the major human pathogen that is associated with hospital as well as community acquired infections and is responsible for huge amount of life-threatening diseases. Objective: Objective of the study was to determine MRSA prevalence, their antibiotic sensitivity patterns, frequency of virulence genes (sea, seb, sed, tst, hla, hld) and their co-occurrence with resistance marker mecA among Rawalpindi and its nearby regions of Pakistani clinical isolates. Methodology: The present study was carried out to identify the virulence and antibiotic resistance genes that co-occur in MRSA through polymerase chain reaction. Antibiotic sensitivity, presence of virulence genes and their co-occurrence with resistance marker mecA were analyzed. Results: These isolates were found resistant to number of antibiotics i.e. Amoxicillin (16.1%), Cefixime (48.38%), Doxycycline (27.415), Trimethoprim/sulfamethoxazole (37.09%), Clindamycin (30.64%), Erythromycin (83.87%), Penicillin (100%), Vancomycin (4.83%), Ciprofloxacin (70.96%), Tetracycline (20%), Linezolid (3.22%) and Fusidic acid (11.295). The frequency of antibiotic resistant gene (mecA) was 69.35% and that of virulence genes hla, hld, sea, seb, sed and tst was 100, 100, 53.2, 30.6, 3.2 and 24.2% respectively. Amongst all examined genes, hla and hld genes had the highest and sed gene had the lowest frequency. The maximum coexistence of genes was observed for hla+hld+mecA gene combination (42 out of 62 isolates). Conclusion: This study reports the presence of multidrug resistant, vancomycin-resistant and mecA negative MRSA isolates in infected patients of Rawalpindi and nearby regions of Pakistan that may have attributed to treatment failures, adaptability of new virulence characteristics and spread of antibiotic resistance.

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. In the present study, we designed a B- and T-cell multi-epitope vaccine against P. aeruginosa using a subtractive proteomics and immunoinformatics approach. A total of five proteins were shortlisted based on essentiality, extracellular localization, virulence, antigenicity, pathway association, hydrophilicity, and low molecular weight. These include two outer membrane porins; OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These shortlisted proteins were further analyzed to identify immunogenic and antigenic B- and T-cell epitopes. The best scoring epitopes were then further subjected to the construction of a polypeptide multi-epitope vaccine and joined with cholera toxin B subunit adjuvant. The final chimeric construct was docked with TLR4 and confirmed by normal mode simulation studies. The designed B- and T-cell multi-epitope vaccine candidate is predicted immunogenic in nature and has shown strong interactions with TLR-4. Immune simulation predicted high-level production of B- and T-cell population and maximal expression was ensured in E. coli strain K12. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10255-3.

Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression.

Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent yet, not diagnosed and reported properly. Due to the simultaneous occurrence of both infections, diagnosis is delayed leading to inadequate treatments and mortality. With the rise of MDR Klebsiella and Mycobacterium, a prophylactic and an immunotherapeutic vaccine has to be entailed for preemptive and adroit therapeutic approach. In this study, we aim to implement reverse vaccinology approach that encompasses a comprehensive evaluation of vital aspects of the pathogens to explore immunogenic epitopes against Omp A of Klebsiella and Rv1698, Rv1973 of Mtb that may help in vaccine development. The designed multi-epitopic vaccine was assessed for antigenicity, allergenicity and various physiochemical parameters. Molecular docking and simulations were executed to assess the immunogenicity and complex stability of the vaccine. The final multi-epitopic vaccine is validated to be highly immunogenic and can serve as a valuable proactive remedy for subject pathogens.

Draft genome sequence of an extensively drug-resistant neonatal Klebsiella pneumoniae isolate harbouring multiple plasmids contributing to antibiotic resistance.

OBJECTIVES: Klebsiella pneumoniae is a notorious nosocomial pathogen that has become a significant cause of neonatal infections causing morbidity and mortality. A multidrug-resistant K. pneumoniae isolate (K184) was isolated from a 5-day-old infant admitted to the neonatal intensive care unit of a local hospital in Rawalpindi, Pakistan. Whole-genome analysis of the isolated strain was performed to gain a better understanding of the genetic basis of antimicrobial resistance and virulence determinants. METHODS: K. pneumoniae isolate K184 was sequenced on an Illumina HiSeq 2500 platform. The genome was assembled using SPAdes with 30× coverage and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.4.3. Characterisation of the strain was performed using MLST 2.0 server. Plasmids, antimicrobial resistance determinants and virulence factors were identified using PlasmidFinder v.2.0, the Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factors Database (VFDB), respectively. RESULTS: Neonatal K. pneumoniae isolate K184 has a considerably large genome with a size of 6,686,067 bp and a GC content of 55.6%. The isolate possesses three plasmids actively contributing to antimicrobial resistance, which classifies it as heavily loaded genome, along with three prophage regions. With 15 antimicrobial resistance determinants and various virulence factors, the neonatal isolate belongs to ST2096. CONCLUSION: The genome of neonatal isolate K184 studied here provides an insight into antibiotic resistance and virulence determinants. This draft genome can be used to compare antimicrobial-resistant K. pneumoniae strains isolated from the neonatal population.

Designing a multi-epitopic vaccine against the enterotoxigenic Bacteroides fragilis based on immunoinformatics approach.

Enterotoxigenic Bacteroides fragilis is an enteric pathogen which is described as a causative agent of various intestinal infections and inflammatory diseases. Moreover, various research studies have reported it to be a leading factor in the development of colorectal cancer. As a part of the normal human microbiome, its treatment has become quite a challenge due to the alarming resistance against the available antibiotics. Although, this particular strain of B. fragilis shows susceptibility to few antibiotics, it is pertinent to devise an effective vaccine strategy for its elimination. There is no vaccine available against this pathogen up to date; therefore, we systematically ventured the outer membrane toxin producing proteins found exclusively in the toxigenic B. fragilis through the in-silico approaches to predict a multi-epitopic chimeric vaccine construct. The designed protein constitutes of epitopes which are predicted for linear B cells, Helper and T cells of outer membrane proteins expected to be putative vaccine candidates. The finalized proteins are only expressed in the enterotoxigenic B. fragilis, thus proving them to be exclusive. The 3D structure of the protein was first predicted followed by its refinement and validation via utilizing the bioinformatic approaches. Docking of the designed protein with the TLR2 receptor forecasted apt binding. Upon immune simulation, notable levels were observed in the expression of the immune cells.