Exploring the Interplay of the CRISPR-CAS System with Antibiotic Resistance in Staphylococcus aureus: A Poultry Meat Study from Lahore, Pakistan.

Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus.

Design and Assessment of a Novel In Silico Approach for Developing a Next-Generation Multi-Epitope Universal Vaccine Targeting Coronaviruses.

In the past two decades, there have been three coronavirus outbreaks that have caused significant economic and health crises. Biologists predict that more coronaviruses may emerge in the near future. Therefore, it is crucial to develop preventive vaccines that can effectively combat multiple coronaviruses. In this study, we employed computational approaches to analyze genetically related coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, focusing on the spike glycoprotein as a potential vaccine candidate. By predicting common epitopes, we identified the top epitopes and combined them to create a multi-epitope candidate vaccine. The overall quality of the candidate vaccine was validated through in silico analyses, confirming its antigenicity, immunogenicity, and stability. In silico docking and simulation studies suggested a stable interaction between the multi-epitope candidate vaccine and human toll-like receptor 2 (TLR2). In silico codon optimization and cloning were used to further explore the successful expression of the designed candidate vaccine in a prokaryotic expression system. Based on computational analysis, the designed candidate vaccine was found to be stable and non-allergenic in the human body. The efficiency of the multi-epitope vaccine in triggering effective cellular and humoral immune responses was assessed through immune stimulation, demonstrating that the designed candidate vaccine can elicit specific immune responses against multiple coronaviruses. Therefore, it holds promise as a potential candidate vaccine against existing and future coronaviruses.

Evidence of Salmonella enterica in Pakistani backyard poultry breeds: isolation, molecular characterization, and pathology.

The present study evaluated the presence of Salmonella enterica in Pakistani backyard poultry. A total 48 chickens from 4 backyard poultry breeds with the clinical presentation of S. enterica infection were randomly selected from villages in the Punjab Province. Cloacal swabs from live poultry and liver samples from the dead birds were collected for bacterial culture and biochemical identification. Liver and spleen samples from dead birds were evaluated for gross and histopathological changes. Bacterial isolates were subjected to PCR and sequencing of ratA gene. Biochemical identification revealed 5/48 (10.42%) chickens positive for S. enterica. Gross pathology included enlarged, discoloured and congested liver and congested spleen. Histopathology demonstrated congestion of sinusoidal capillaries, cellular swelling and cellular/ballooning degeneration, congestion of central hepatic vein, granular hepatocytic cytoplasm and the presence of variable-sized vacuoles in hepatocytes. The PCR yielded a S. enterica specific amplicon (1047 bp). All liver samples that were positive for S. enterica by biochemical tests, were also positive by PCR. The ratA gene sequencing revealed a close resemblance with S. enteritidis isolates from humans. The present study highlights zoonotic risk from backyard poultry and suggests that PCR can be used as an alternate method for rapid detection of Salmonella serovars.

Post-harvest biocontrol of Salmonella Enteritidis on Chicken breast meat and Shell eggs using multiphage cocktail.

The study aimed to evaluate the efficacy of a phage cocktail to reduce Salmonella Enteritidis contamination on perishable food items viz. chicken breast meat and shell eggs using different concentrations. Initially, four bacteriophages €P54, €P59, €P66, and €P72 were isolated from sewage water using Salmonella Enteritidis as a target strain. €P54 and €P66 were found to be Myoviruses while €P59 and €P72 belonged to the Siphoviridae family. A phage cocktail was applied at a concentration of 100 and 10,000 multiplicity of infection (MOI) after artificially contaminating both food items with Salmonella Enteritidis. Results showed that, phage cocktail significantly (p ≤ 0.05) reduced Salmonella Enteritidis count at both concentrations. However, the increased reduction was witnessed at 10,000 MOI. In comparison to untreated control, on chicken breast meat bacterial count was reduced to 1.94 and 3.17 Log10 cfu/g at 100 and 10,000 MOI respectively at 4oC. Similarly, on shell eggs, the bacterial count was reduced to 3.09 and 2.81 Log10 cfu/mL at 10,000 MOI at 4°C and 25°C respectively, while at 100 MOI there was less drop in bacterial count at both 4°C and 25°C. The results showed a better reduction at 4°C as compared to 25°C. Our data showed that the phage cocktail is an effective alternative and additional measure compared to conventional bacterial control methods for meat and eggs.

Current methods for the detection of Plasmodium parasite species infecting humans.

Malaria is the world's fatal parasitic disease. The ability to quickly and accurately identify malaria infection in challenging environments is crucial to allow efficient administration of the best treatment regime for human patients. If those techniques are accessible and efficient, global detection of Plasmodium species will become more sensitive, allowing faster and more precise action to be taken for disease control strategies. Recent advances in technology have enhanced our ability to diagnose different species of Plasmodium parasites with greater sensitivity and specificity. This literature review provides a summary and discussion of the current methods for the diagnosis and identification of Plasmodium spp. in human blood samples. So far not a single method is precise, but advanced technologies give consistent identification of a Plasmodium infection in endemic regions. By using the power of the recent methods, we can provide a broader understanding of the multiplicity of infection and or transmission dynamics of Plasmodium spp. This will result in improved disease control strategies, better-informed policy, and effective treatment for malaria-positive patients.

Physiological properties of indigenous lytic bacteriophages as monophage suspension and cocktail against poultry-adapted typhoidal Salmonella variants.

The emergence and spread of multidrug resistance among pathogens of the agro-food sector is increasing at an alarming rate, which has directed attention to the search for alternative to antibiotic therapy. The present work studied the physiological and population dynamics of lytic bacteriophages against avian-adapted Salmonella. Out of 28 positive samples, four bacteriophage isolates (SalØ-ABF37, SalØ-RCMPF12, SalØ-MCOH26, SalØ-DNLS42) were selected based on their ability to clearly lyse bacterial test strains. The isolates propagated were active against closely related D1 serotypes, i.e., S. Enteritidis and S. Typhimurium, with no heterologous activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 23235. Each of the monophage suspension and cocktail efficiently suppressed the bacterial count from exponential increase in comparison to the untreated bacterial control. The bacterial turbidity was recorded as 0.244 at λ600 during 400 min of co-incubation, in contrast to bacterial control showing λ600 = 0.669. The latent period was recorded to be 25, 35, 25 and 30 for SalØ-ABF37, SalØ-RCMPF12, SalØ-MCOH26 and SalØ-DNLS42, with 73.00, 97.00, 132 and 75.00 PFU cell-1, respectively. The highest lytic activity was seen at 37.00 ˚C - 42.00 ˚C, with phage particle count being fairly stable at pH 3.00 - 9.00. Each of the isolates possessed dsDNA by being resistant to RNase A. The current study concludes that lytic phages are promising alternative to combat multidrug resistant superbugs. The physiological characterization and bacterial growth inhibition are important parameters in standardization of phage therapy.

Effect of carboxylated poly l-Lysine as a cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull semen.

Buffalo bull sperm are more prone to cryo-injuries. Glycerol being the most common permeable cryoprotectant exerts cytotoxic effects on sperm which cause a reduction in fertility. Thus, the exploration of new cryoprotectant is needed. For this purpose, we investigated the effect of carboxylated poly l-Lysine (CPLL) as cryoprotectant used with different concentrations of glycerol on post-thaw sperm motility, kinematics, plasma membrane integrity, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), catalase concentration and in vivo fertility of Nili Ravi buffalo bull semen. In experiment 1, semen samples (n = 15, bulls = 3) were diluted with Tris-citrate-egg yolk extender containing different concentration of CPLL [0% (C0), 0.25% (C0.25), 0.5% (C0.5), 0.75% (C0.75), 1% (C1)]. Each concentration of CPLL was added in extender containing either 7% (G7) or 5% (G5) glycerol. Diluted semen samples were cooled and cryopreserved using standard procedures. Post-thaw total and progressive motility, plasma membrane integrity, acrosome integrity, and MMP were found higher (P < 0.05) in group (G5C0.75) containing 0.75% CPLL and 5% glycerol as compared to the control group (G7C0) and other groups while LPO was recorded lower (P < 0.05) in the same group (G5C0.75). In experiment 2, in vivo fertility was compared between G5C0.75 (5% Glycerol+ 0.75% CPLL; depicted better post-thaw quality) and control group G7C0. Buffaloes were inseminated after 24 h of onset of estrus. Pregnancy diagnosis was performed per rectum at least 60 days post insemination. The fertility rates [56% (58/102) vs. 36% (37/103)] were higher (P < 0.05) in G5C0.75 as compared to the control group G7C0. Based upon these results, this study concludes that the addition of 0.75% CPLL in combination with 5% glycerol in freezing extender improves the post-thaw structure, function and in vivo fertility of Nili Ravi buffalo bull semen.