Molecular characterization of human adenoviruses associated with pediatric respiratory infections in Karachi, Pakistan.

Human adenoviruses (HAdVs) are a diverse group of viruses associated with respiratory infections in humans worldwide. However, there is a lack of research on the genetic diversity and epidemiology of HAdVs in Pakistan. This study characterized HAdVs in pediatric patients with respiratory tract infections in Karachi, Pakistan, between 2022 and 2023. We analyzed 762 nasopharyngeal samples of children ≤ 5 years. DNA extraction, followed by PCR targeting E2B and hexon genes, was carried out. Data analysis was performed on SPSS 25.0, and phylogenetic analysis of hexon gene was performed on MEGA 11. HAdV was detected in 7.34% (56/762) of patients round the year, but at a significantly higher rate during the winter season. Age was insignificantly associated with HAdV incidence (p = 0.662), but more than 62.5% (35/56) of positive cases were younger than 10 months. The circulating HAdVs were identified as six different types from species B (78.57%) and C (21.42%), with the majority of isolates found to be like B3. HAdV was found to be co-infected with bocavirus (5.4%) and measles (7.14%). These findings revealed a high frequency and genetic diversity of respiratory HAdVs in Karachi, Pakistan. We conclude that periodic and continuous surveillance of adenoviruses and other respiratory pathogens is necessary to improve the prognosis and management of respiratory diseases, thereby reducing the child mortality rate in Pakistan.

Effect of ellagic acid and mesocarp extract of Punica granatum on productive and reproductive performances of laying hens.

In the present study, we determined the potential effects of ellagic acid and mesocarp extract of Punica granatum on the productive and reproduction performance of laying hens. Five treatment groups were setup: (1) control group (without ellagic acid), (2) 50 mg of ellagic acid, (3) 100 mg of ellagic acid, (4) 200 mg of ellagic acid, and (5) mesocarp extract of P. granatum. All the groups were investigated for feed intake, body weight, egg production, egg quality, fertility, hatchability, antioxidant status of serum and liver, lipid peroxidation, and antibacterial activities. Egg production, feed intake, and bodyweight were significantly increased (p < 0.05) with 100 mg of ellagic acid and P. granatum extract while no significant effect was observed on albumen and yolk weight, yolk index, yolk color, egg-shape index, and Haugh unit. Both ellagic acid and P. granatum extract significantly improved hatchability while 100 and 200 mg/kg of ellagic acid numerically decreased fertility. Besides, ellagic acid (100 mg/kg) and P. granatum extract significantly decreased malondialdehyde concentration and increased total antioxidant capacity, glutathione peroxidase, and total superoxide dismutase in serum and liver samples of laying hens (p < 0.05). The lipid peroxidation was decreased among the treatment groups, with 100 mg of ellagic acid and P. granatum extract showed the best activity. Moreover, ellagic acid demonstrated strong killing activity against Escherichia coli and Staphylococcus aureus while it was ineffective against methicillin-resistant S. aureus. Our results conclude that ellagic acid and P. granatum promoted egg production, hatchability, and antioxidant enzyme activities of the laying hens.

A novel type I toxin-antitoxin system modulates persister cell formation in Staphylococcus aureus.

A plethora of toxin-antitoxin systems exist in bacteria and has multilateral roles in bacterial pathogenesis and virulence. Toxin-antitoxin systems have been involved in persister cell formation in Escherichia coli and Mycobacterium but have not been reported to be associated with Staphylococcus aureus persistence. Persistence is the ability of bacterial cells to tolerate unfavorable conditions and multiple stresses. There are less known and more unknown factors that either alleviate or aggravate bacterial persistence phenomenon. For the first time, we reported a new chromosomally encoded tripartite toxin-antitoxin system and its role in S. aureus persister cell formation. The toxin gene is bacteriostatic in action and counterbalanced by antitoxin RNA that could basepair with the toxin mRNA and formed a duplex. The transcriptional regulator positively regulates the toxin expression under certain stress conditions. The toxin ectopic induction increased S. aureus susceptibility to norfloxacin, ciprofloxacin, and ofloxacin. Whole-genome RNA sequencing revealed that MDR efflux pump norA is significantly down-regulated by toxin ectopic induction. The deletion of norA from S. aureus genome reduced resistance toward ciprofloxacin, norfloxacin, and ofloxacin, as well as resulted in a decrease in minimal inhibitory concentration while complementation of norA successfully restored the phenotypes. The persistence assay of the norA mutant revealed that deletion of norA increased persister cell survival in S. aureus. Altogether, we have provided insight into the first tripartite type-I TA system and revealed the role of MDR NorA in the persister cell formation of S. aureus.

Contribution of the gyrA and waaG mutants to fluoroquinolones resistance, biofilm development, and persister cells formation in Salmonella enterica serovar Typhi.

Fluoroquinolone resistance in Salmonella has been reported worldwide and poses a serious public health threat in developing countries. Multiple factors contribute to fluoroquinolone resistance, including mutations in DNA gyrase and the acquisition of antimicrobial resistance genes. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans, which is highly prevalent in counties with poor sanitation and hygiene standards. Here, we reported S. Typhi clinical isolates that showed varying degrees of susceptibility to fluoroquinolones and were characterized by Analytical Profile Index 20E test kit and 16S rRNA sequencing. S. Typhi strain S27 was resistant to fluoroquinolones and had multiple mutations in the gyrA gene. The gyrA lies in the quinolone resistance determining region of S. Typhi and has mutations at codon 83 (Ser83Phe), codon 87 (Asp87Gly), codon 308 (Lys308Glu), and codon 328 (Val328Ile). S. Typhi strain S6 has no gyrA mutations and is sensitive to fluoroquinolones but forms a strong biofilm relative to S. Typhi S27. Transcriptional analysis of biofilm associated genes revealed that the waaG gene was significantly downregulated. The ΔwaaG mutant showed a significant decrease in persister cells and a strong biofilm formation relative to wild type and gyrA mutant. The gyrA tetra mutant persister assay revealed a significant increase in persister cells compared to wild type and ΔwaaG. Collectively, this is the first report of S. Typhi's two key genes and their roles in antibiotic tolerance, biofilm formation, and fluoroquinolone resistance that can help in understanding the mechanism of persister formation and eradication.

Clinical manifestations of active tuberculosis patients coinfected with severe acute respiratory syndrome coronavirus-2.

Summary background: The coronavirus 2019 pandemic was caused by a new single-strand RNA virus that originated from Wuhan, China, and infected more than 190 countries. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) coinfection with tuberculosis posed a serious public health concern and complicated the prognosis and treatment of patients. Since both are respiratory diseases, the sign and symptoms may overlap and could have synergistic effects on the host that can increase mortality during coinfection. The present investigation reported the clinical characteristics of patients having coinfection of COVID-19 and tuberculosis (COVID-TB). Methods: We performed a retrospective investigation on COVID-19 infection in tuberculosis patients between the years 2020 and 2021. The SARS-CoV-2 was confirmed by PCR and the COVID-TB epidemiological and clinical findings were recorded on the day of admission and followed up for 25 days. Results: The mean age of the COVID-19 patients was 50 ± 15 years, 76.36% were male and 23.64% were female. Weight loss, sore throat, whooping cough, chest pain, and vomiting were common symptoms, and asthma, diabetes, arthritis, and hypertension were found as co-morbidities in COVID-TB. The D-dimer, lactate dehydrogenase, C-reactive protein, erythrocyte sedimentation rate, and creatine kinase levels increased 14-fold, 12.5-fold, 11-fold, 10-fold, and 7-fold respectively during COVID-TB. The patients suffered from hyperferritinemia and lymphocytopenia which increased the likelihood of death. The levels of D-dimer, lactate dehydrogenase, C-reactive protein, erythrocyte sedimentation rate, and creatinine kinase were positively correlated with patient age. The chest radiograph showed the infectious agents have consolidated opacity and peripheral dissemination in the lungs. Conclusion: Tuberculosis coinfection augmented the severity of COVID-19 and the likelihood of death, and high vigilance is recommended for respiratory pathogens in COVID-19.

Teicoplanin associated gene tcaA inactivation increases persister cell formation in Staphylococcus aureus.

Staphylococcus aureus is part of normal human flora and is widely associated with hospital-acquired bacteremia. S. aureus has shown a diverse array of resistance to environmental stresses and antibiotics. Methicillin-resistant S. aureus (MRSA) is on the high priority list of new antibiotics discovery and glycopeptides are considered the last drug of choice against MRSA. S. aureus has developed resistance against glycopeptides and the emergence of vancomycin-intermediate-resistant, vancomycin-resistant, and teicoplanin-resistant strains is globally reported. Teicoplanin-associated genes tcaR-tcaA-tcaB (tcaRAB) is known as the S. aureus glycopeptide resistance operon that is associated with glycopeptide resistance. Here, for the first time, the role of tcaRAB in S. aureus persister cells formation, and ΔtcaA dependent persisters' ability to resuscitate the bacterial population was explored. We recovered a clinical strain of MRSA from a COVID-19 patient which showed a high level of resistance to teicoplanin, vancomycin, and methicillin. Whole genome RNA sequencing revealed that the tcaRAB operon expression was altered followed by high expression of glyS and sgtB. The RNA-seq data revealed a significant decrease in tcaA (p = 0.008) and tcaB (p = 0.04) expression while tcaR was not significantly altered. We knocked down tcaA, tcaB, and tcaR using CRISPR-dCas9 and the results showed that when tcaA was suppressed by dCas9, a significant increase was witnessed in persister cells while tcaB suppression did not induce persistence. The results were further evaluated by creating a tcaA mutant that showed ΔtcaA formed a significant increase in persisters in comparison to the wild type. Based on our findings, we concluded that tcaA is the gene that increases persister cells and glycopeptide resistance and could be a potential therapeutic target in S. aureus.

The functional repertoire of AmpR in the AmpC ß-lactamase high expression and decreasing ß-lactam and aminoglycosides resistance in ESBL Citrobacter freundii.

Citrobacter freundii is characterized by AmpC ß-lactamases that develop resistance to ß-lactam antibiotics. The production of extended-spectrum ß-lactamase (ESBL) is substantially high in Escherichia coli, C. freundii, Enterobacter cloacae, and Serratia marcescens, but infrequently explored in C. freundii. The present investigation characterized the ESBL C. freundii and delineated the genes involved in decrease in antibiotics resistance. We used the VITEK-2 system and Analytical Profile Index (API) kit to characterize and identify the Citrobacter isolates. The mRNA level of AmpC and AmpR was determined by RT-qPCR, and gel-shift assay was performed to evaluate protein-DNA binding. Here, a total of 26 Citrobacter strains were isolated from COVID-19 patients that showed varying degrees of antibiotic resistance. We examined and characterized the multidrug resistant C. freundii that showed ESBL production. The RT-qPCR analysis revealed that the AmpC mRNA expression is significantly high followed by a high level of AmpR. We sequenced the AmpC and AmpR genes that revealed the AmpR has four novel mutations in comparison to the reference genome namely; Thr64Ile, Arg86Ser, Asp135Val, and Ile183Leu while AmpC remained intact. The ΔAmpR mutant analysis revealed that the AmpR positively regulates oxidative stress response and decreases ß-lactam and aminoglycosides resistance. The AmpC and AmpR high expression was associated with resistance to tazobactam, ampicillin, gentamicin, nitrofurantoin, and cephalosporins whereas AmpR deletion reduced ß-lactam and aminoglycosides resistance. We conclude that AmpR is a positive regulator of AmpC that stimulates ß-lactamases which inactivate multiple antibiotics.

Pathophysiology of Methicillin-Resistant Staphylococcus aureus Superinfection in COVID-19 Patients.

The global spread of the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has infected humans in all age groups, deteriorated host immune responses, and caused millions of deaths. The reasons for individuals succumbing to COVID-19 were not only the SARS-CoV-2 infection but also associated bacterial infections. Antibiotics were largely used to prevent bacterial infections during COVID-19 illness, and many bacteria became resistant to conventional antibiotics. Although COVID-19 was considered the main culprit behind the millions of deaths, bacterial coinfections and superinfections were the major factors that increased the mortality rate in hospitalized patients. In the present study, we assessed the pathophysiology of methicillin-resistant Staphylococcus aureus (MRSA) superinfection in COVID-19 patients in Pakistan. A total of 3492 COVID-19 hospitalized patients were screened among which 224 strain were resistant to methicillin; 110 strains were tazobactam-resistant; 53 strains were ciprofloxacin-resistant; 23 strains were gentamicin-resistant; 11 strains were azithromycin-resistant; 3 strains were vancomycin-resistant. A high frequency of MRSA was detected in patients aged ≥50 with a prevalence of 7.33%, followed by patients aged >65 with a prevalence of 5.48% and a 5.10% prevalence in patients aged <50. In addition, pneumonia was detected in the COVID-19-associated MRSA (COVID-MRSA) that showed decreased levels of lymphocytes and albumin and increased the mortality rate from 2.3% to 25.23%. Collectively, an MRSA superinfection was associated with increased mortality in COVID-19 after 12 to 18 days of hospitalization. The study assessed the prevalence of MRSA, mortality rate, pneumonia, and the emergence of antibiotic resistance as the main outcomes. The study summarized that COVID-MRSA aggravated the treatment and recovery of patients and suggested testing MRSA as critical for hospitalized patients.

Genetic resilience in chickens against bacterial, viral and protozoal pathogens.

The genome contributes to the uniqueness of an individual breed, and enables distinctive characteristics to be passed from one generation to the next. The allelic heterogeneity of a certain breed results in a different response to a pathogen with different genomic expression. Disease resistance in chicken is a polygenic trait that involves different genes that confer resistance against pathogens. Such resistance also involves major histocompatibility (MHC) molecules, immunoglobulins, cytokines, interleukins, T and B cells, and CD4+ and CD8+ T lymphocytes, which are involved in host protection. The MHC is associated with antigen presentation, antibody production, and cytokine stimulation, which highlight its role in disease resistance. The natural resistance-associated macrophage protein 1 (Nramp-1), interferon (IFN), myxovirus-resistance gene, myeloid differentiation primary response 88 (MyD88), receptor-interacting serine/threonine kinase 2 (RIP2), and heterophile cells are involved in disease resistance and susceptibility of chicken. Studies related to disease resistance genetics, epigenetics, and quantitative trait loci would enable the identification of resistance markers and the development of disease resistance breeds. Microbial infections are responsible for significant outbreaks and have blighted the poultry industry. Breeding disease-resistant chicken strains may be helpful in tackling pathogens and increasing the current understanding on host genetics in the fight against communicable diseases. Advanced technologies, such as the CRISPR/Cas9 system, whole genome sequencing, RNA sequencing, and high-density single nucleotide polymorphism (SNP) genotyping, aid the development of resistant breeds, which would significantly decrease the use of antibiotics and vaccination in poultry. In this review, we aimed to reveal the recent genetic basis of infection and genomic modification that increase resistance against different pathogens in chickens.

Bioinformatics and Functional Assessment of Toxin-Antitoxin Systems in Staphylococcus aureus.

Staphylococcus aureus is a nosocomial pathogen that can cause chronic to persistent infections. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. These systems are frequently studied in Escherichia coli and Mycobacterial species but rarely explored in S. aureus. In the present study, we thoroughly analyzed the S. aureus genome and screened all possible TA systems using the Rasta bacteria and toxin-antitoxin database. We further searched E. coli and Mycobacterial TA homologs and selected 67 TA loci as putative TA systems in S. aureus. The host inhibition of growth (HigBA) TA family was predominantly detected in S. aureus. In addition, we detected seven pathogenicity islands in the S. aureus genome that are enriched with virulence genes and contain 26 out of 67 TA systems. We ectopically expressed multiple TA genes in E. coli and S. aureus that exhibited bacteriostatic and bactericidal effects on cell growth. The type I Fst toxin created holes in the cell wall while the TxpA toxin reduced cell size and induced cell wall septation. Besides, we identified a new TA system whose antitoxin functions as a transcriptional autoregulator while the toxin functions as an inhibitor of autoregulation. Altogether, this study provides a plethora of new as well as previously known TA systems that will revitalize the research on S. aureus TA systems.