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.

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.

Comparative Yolk Proteomic Analysis of Fertilized Low and High Cholesterol Eggs during Embryonic Development.

The yolk is the principal part of the egg that contains vitamins, minerals, lipids, and proteins which are essential for embryo development and hatching. The egg yolk contains significant amounts of lipoproteins, triacylglycerides, and cholesterol, whose dynamics are indistinct during embryogenesis. The effects of cholesterol on the yolk protein abundance, intensity, and function are ill-defined during embryonic development. Using two-dimensional gel electrophoresis, eggs with respective high and low cholesterol protein abundance were investigated after 0, 2, 6, and 13 days of embryogenesis and further analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results revealed that the vitellogenin proteins are the most abundant egg yolk protein that showed proximity and a high degree of variation in isoelectric point and molecular weight. The results demonstrated increased expression of vitellogenin-1 and vitellogenin-3 at two days and vitellogenin-2 protein at 13 days of embryogenesis in both egg types. The ovoinhibitor, immunoglobulin lambda light chain precursor, Ig-gamma (clone-36 chicken), and beta-2-glycoprotein-1 precursor proteins were significantly expressed in high cholesterol eggs while haptoglobin protein PIT-54 and vitelline membrane outer layer proteins intensities were significant in low cholesterol eggs at two days of embryogenesis. The high cholesterol eggs showed a modest increase in egg weight, yolk weight, albumen height, yolk color, and egg strength relative to the low cholesterol eggs. The gene ontology enrichment analysis revealed that the differentially expressed proteins such as vitellogenin proteins were involved in lipid transport and lipid localization biological processes and showed nutrient reservoir activity function. The ovotransferrin regulated the biological processes of plasminogen activation and extracellular matrix disassembly and characterized the anchored component of the plasma membrane. The ovoinhibitor protein was involved in response to mineralocorticoid and corticosterone biological processes whereas the vitellin membrane outer layer protein constituted the extracellular exosome, extracellular organelle, and membrane-bounded vesicle cellular components. Collectively, our study revealed yolk protein abundance, molecular function, cellular components, and biological processes and concluded that yolk protein intensities were significantly altered by cholesterol concentration.

Path-based extensions of local link prediction methods for complex networks.

Link prediction in a complex network is a problem of fundamental interest in network science and has attracted increasing attention in recent years. It aims to predict missing (or future) links between two entities in a complex system that are not already connected. Among existing methods, local similarity indices are most popular that take into account the information of common neighbours to estimate the likelihood of existence of a connection between two nodes. In this paper, we propose global and quasi-local extensions of some commonly used local similarity indices. We have performed extensive numerical simulations on publicly available datasets from diverse domains demonstrating that the proposed extensions not only give superior performance, when compared to their respective local indices, but also outperform some of the current, state-of-the-art, local and global link-prediction methods.

Computation of solution to fractional order partial reaction diffusion equations.

In this article, the considered problem of Cauchy reaction diffusion equation of fractional order is solved by using integral transform of Laplace coupled with decomposition technique due to Adomian scheme. This combination led us to a hybrid method which has been properly used to handle nonlinear and linear problems. The considered problem is used in modeling spatial effects in engineering, biology and ecology. The fractional derivative is considered in Caputo sense. The results are obtained in series form corresponding to the proposed problem of fractional order. To present the analytical procedure of the proposed method, some test examples are provided. An approximate solution of a fractional order diffusion equation were obtained. This solution was rapidly convergent to the exact solution with less computational cost. For the computation purposes, we used MATLAB.