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.

Targeting inflammation for the treatment of endometritis in bovines.

The uterine endometrial surface of bovines is in constant exposureconstantly exposed with to a multitude ofmany microbial populations that changes throughout the post-partum phase in terms of complexity and dynamics. These microbes contribute to the host pathology, leading to severe economic losses along withnd reproductive capabilities. The basic primary interface that occurs between the internal tissues of the body of the hostbetween the host body's internal tissues and the microbes is the endometrial surface of the uterus. As a result of the infinite pathogenic population, there is always a danger for the opportunistic organisms to attack. Therefore, it is paramount that any interactions, especially microbial microbes with the endometrial surface, are regulated by the host cells. However, the inflammatory response as the defense mechanism contributes a pivotal roleis pivotal in host immunity and pathology. The inflammatory cascade and pathways are important essential to eliminate this clinical problem. In this review, we will discuss and explain how the inflammation and the various components of the immune system play their role in host pathology and therapeutic strategies, taking into account the interface between the host and the microbes on the surface of the endometrium. This review is also instrumental in further explanation of inflammatory uterine disease by discussing the response of inflammation to external insult.

Patterns of Common Dermatological Conditions among Children and Adolescents in Pakistan.

Background and Objectives: Dermatological disorders are highly prevalent among children in Pakistan. The present cross-sectional study aims to identify the spectrum of dermatological conditions among children and adolescents in Pakistan. Materials and Methods: A total of 582 patients (50.9% males; 49.1% females) were included in the study based on their age (5.7 ± 4.1 years), dermatological condition, and epidemiology. The youngest patient was aged ten days, whereas the eldest was seventeen. Age criteria were further stratified into three categories: infants and toddlers (≤5 years), children (≥5 to <12 years), and adolescents (≥12 to <18 years). Amongst them, the majority was from Punjab (81.6%), while the other regions included were Azad Jammu and Kashmir (14.4%), Islamabad (3.3%), and Khyber Pakhtunkhwa (0.7%). Results: Scabies was the highest reported skin condition with 281 (45.55%) patients, followed by 114 (19.6%) with eczema, 60 (10.3%) with dermatitis, 33 (5.7%) with tinea capitis, 17 (2.9%) with tinea corporis, 16 (2.7%) with impetigo, and 15 (2.6%) with folliculitis. Other conditions include urticaria, burns, infections, pediculosis, tinea inguinalis, tinea faciei, nappy rashes, alopecia, warts, tinea incognito, tinea cruris, and acne vulgaris. The chi-squared test showed a high prevalence of tinea corporis and acne among adolescents (12-17 years), whereas eczema, dermatitis, and impetigo were more prevalent among infants and toddlers. Conclusions: Pets or livestock and poor hygiene were found to be highly reported risk factors for many dermatological conditions like scabies and fungal infections. Dermatological conditions are common in younger individuals, but unfortunately, many children do not receive the desired medical assistance.

Seroprevalence of Newcastle disease virus and avian influenza virus in poultry and captive wild birds in poultry-dense regions of Pakistan.

Newcastle disease virus (NDV) and avian influenza virus (AIV) are causing contagious diseases in chickens and wild birds worldwide; however, there is a paucity of information on the current status of seropositivity of Newcastle and avian influenza diseases in chickens and wild birds of Pakistan. Therefore, the present study aimed to investigate the serological evidence of both diseases in commercial poultry (broiler, layer chickens), backyard poultry, and captive wild birds in poultry­dense regions of Punjab, Pakistan. Enzyme­linked immunosorbent (ELISA) and haemagglutination inhibition (HI) assays were performed for the determination of antibodies against NDV and AIV and their genotyping and subtyping, respectively. Overall, 47.5% and 67.4% seroprevalence of NDV and AIV, respectively, was observed in both poultry and wild birds. Based on bird's category, layer chickens had the highest seroprevalence of NDV (60.8%, 95% CI: 52.95­68.22, OR: 0.71) followed by backyard poultry (56.8%, 95% CI: 47.92­65.32, OR: 0.82), broilers (52.7%, 95% CI: 46.84­58.64), pigeons (41.3%, 95% CI: 30.53­52.81, OR: 1.59), peafowls (26.1%, 95% CI: 11.09­48.69, OR: 3.16), ducks (23.8%, 95% CI: 12.59­39.8, OR: 3.57), turkeys (16.7%, 95% CI: 4.41­42.27, OR: 5.58), parrots (14.3%, 95% CI: 2.52­43.85, OR: 6.70) and quails (2.3%, 95% CI: 0.2­13.51, OR: 4.8). Comparatively, backyard chickens had the highest seroprevalence of AIV (78.8%, 95% CI: 70.64­85.22, OR: 0.63) followed by ducks (73.8%, 95% CI: 57.68­85.6, OR: 0.83), layers (73.5%, 95% CI: 65.98­79.89, OR: 0.84), pigeons (72.5%, 95% CI: 61.2­81.61, OR: 0.89), broilers (70.1%, 95% CI: 64.44­75.29), turkeys (55.5%, 95% CI: 31.35­77.6, OR: 1.87), peafowls (47.8%, 95% CI: 27.42­68.9, OR: 2.56) and parrots (42.8%, 95% CI: 18.8­70.3, OR: 3.1). Overall, 40.1%, 34.2%, 31.3%, and 25.1% sera were positive for H9 AIV, G­VII NDV, H7 AIV, and G­VI NDV, respectively. The current study revealed a widespread exposure to NDV and AIV in poultry and captive wild birds. Therefore, it is crucial to include captive wild birds in NDV and AIV surveillance programs to further strengthen disease control measures, particularly in endemic regions.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Insights into the Prognostic Role of Serum Interleukin-6 and Hematobiochemical Alterations in Cattle during Recent Outbreaks of Lumpy Skin Disease in Lodhran District, Pakistan.

Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus of the Poxviridae family, genus Capripoxvirus. The present study was designed to determine the prognostic ability of serum IL-6 in LSD using a binary logistic regression model at baseline sampling. A 17-day cohort study was conducted on a recent outbreak of LSD among cattle in the Lodhran District of Punjab, Pakistan. Infected cattle were divided into two categories based on their clinical status on day 17 as recovered (n = 33) or unrecovered (n = 17). Nodular lesions and scab specimens (n = 50) were used for the isolation of the lumpy skin disease virus and were confirmed by PCR. In recovered animals, hematological results showed marked leukocytosis, eosinophilia, lymphocytosis, neutrophilia, and monocytopenia. However, marked erythrocytosis, leukopenia, and thrombocytopenia were observed in the unrecovered animals at the final sampling point of the study. Serum levels of total protein, albumin, and glucose were significantly higher in the recovered animals. Meanwhile, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatinine phosphokinase, total bilirubin, and direct bilirubin were found considerably higher in the unrecovered group. Receiver-operating characteristic curve analysis for serum IL-6 at baseline predicts the extended clinical conditions at the cut-off value of 85.16 pg/mL (55% specificity, 94% sensitivity, area under the curve 0.8039, respectively). In conclusion, the disease-induced hematological and biochemical alterations were significantly ameliorated in the recovered animals. In addition, the study revealed that serum IL-6 can be used as a valid marker for predicting the clinical worsening of LSD in cattle.

Inter-Relationship Between a Transcriptional Regulator of Flagella Genes cj0440c and Thiamine Metabolic Pathway in Campylobacter jejuni.

Campylobacter jejuni is a major cause of gastroenteritis in humans. It has been reported that the pathogenesis of C. jejuni is closely related to the formation, adhesion, and invasion of flagella toxin in host epithelial cells. A putative transcriptional regulator, known as cj0440c, is thought to be involved in the regulation of flagellar synthesis. However, confirmation of this hypothesis requires deep insight into the regulation mechanism of cj0440c and its possible relationship with different antibiotics. Therefore, the study explained here was designed to determine the relationship and function (phenotypically and genotypically) of cj0440c in the flagellar synthesis of C. jejuni NCTC11168. The study determined the mode of expression of cj0440c and flagella-related genes under exposure to various drugs. To verify the involvement of cj0440c protein in the metabolic pathway of thiamine, an enzymatic hydrolysis experiment was performed and analyzed through the application of mass spectrometry. The overexpression vector of C. jejuni NCTC11168 was also constructed to find out whether or not target genes were regulated by cj0440c. The findings of the study showed that cj0440c and other flagella-related genes were expressed differentially under the influence of various antibiotics including erythromycin, tylosin, azithromycin, gentamicin, etimicin, enrofloxacin, gatifloxacin, tetracycline, and tigecycline. The analysis showed that the cj0440c protein did not catalyze the degradation of thiamine. In conclusion, the study aids in the understanding of the inter-relationship between the regulatory mechanism of flagella genes and the thiamine metabolic pathway.

Renessans Helps in Early Clearance of SARS-CoV-2: In-Vivo Activity of the Iodine Complex in Rhesus macaque.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

Epidemiology, Environmental Risks, Virulence, and Resistance Determinants of Klebsiella pneumoniae From Dairy Cows in Hubei, China.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen, which causes serious infections in humans and animals. To investigate the antimicrobial resistance pattern and virulence profile of K. pneumoniae, a total of 887 samples were collected from both the healthy and mastitis cows and the bedding, feed, feces, air, drinking water, spraying water, washing water, and milk cup swabs from five dairy farms in Hubei, China, during 2019 and 2020. K. pneumoniae was isolated and identified using PCR of the khe and 16S rDNA sequencing. A genotypic characterization was performed for K. pneumoniae isolates using wzi typing and multilocus sequence typing (MLST). Antimicrobial resistances were confirmed using broth microdilution against 17 antimicrobial agents and resistance and virulence genes were determined by PCR. The prevalence of K. pneumoniae was 26.94% (239/887) distributed in 101 wzi allele types (199/239, 83.26%) and 100 sequence types (STs) (209/239, 87.45%), including 5 new wzi allele type and 25 new STs. Phylogenetic analysis showed that K. pneumoniae isolated from milk, nipple swab, feed, and feces is classified in the same clone complex. By comparing with the PubMLST database, at least 67 STs have the risk of spreading in different species and regions. Interestingly, 60 STs have been isolated from humans. The isolates were highly sensitive to meropenem and colistin, but resistant to ampicillin (100%), sulfisoxazole (94.56%), cephalothin (47.28%), streptomycin (30.13%), and so on. Noteworthy, multidrug-resistant (MDR) rate was found to be 43.93% in this study. By PCR, 30 of 68 antimicrobial resistance (AMR) genes were identified; the prevalence rate of blaTEM, blaSHV, strA, strB, aadA1, and aac(6')-Ib-cr was more than 50%. Eleven CTX-M-producing K. pneumoniae were found. The detection rate of fimH, mrkD, uge, wabG, entB, iutA, iroN, and ureA was over 85%. This study reinforces the epidemiological importance of K. pneumoniae in food-producing animals in Hubei. The emergence and spread of environmental MDR K. pneumoniae may pose a potential threat to food safety and public health.

Molecular Characterization of Toxoplasma gondii in Cats and Its Zoonotic Potential for Public Health Significance.

Toxoplasmosis is a globally distributed disease of warm-blooded animals. It is caused by the opportunistic parasite Toxoplasma gondii (T. gondii). One-third of the global human population is believed to be infected with T. gondii. Cats serve as final host of T. gondii and are the main source of contamination of soil and water. This study aimed to detect genotypes of T. gondii in cats. Fecal samples (n = 400) were collected from districts of South Punjab (Khanewal and Sahiwal), and were processed by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. The obtained oligonucleotide sequences (T. gondii) were submitted to the GenBank database, and the evolutionary tree was constructed using MEGA-X software. Seven fecal samples (3.5%) from cats were positive. Five out of thirteen fecal samples (38.46%) found to be positive for T. gondii with microscopy were confirmed by PCR. After phylogenetic analysis with 3 clonal types and atypical strains, isolates of T. gondii in current study were more closely linked to a typical strain (AF249696). Besides genotyping from cats, seroprevalence from humans and ruminants is still considered to be the best and easiest way to identify the Toxoplasma. Blood samples were collected from sheep and goats (n = 2000 each), and human blood samples (n = 400) were collected from the same vicinity. Seroprevalence was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. In Khanewal, the blood samples of 292 goats (29.2%) and 265 sheep (26.5%), and 6 fecal samples from cats (3%) were positive. Out of 200 human blood samples, 52 were positive, with a seroprevalence of 26%. In the Sahiwal district, the blood samples from 49 humans, 235 sheep and 348 goats were positive, with seroprevalence of 24.5%, 23.5% and 34.8%, respectively. The present study revealed the current circulating genotype of T. gondii from cats in the districts Khanewal and Sahiwal and the seroprevalence of the organism in small ruminants and humans living in the same vicinity. Further genotype analyses of the organism from ruminants and humans are needed.

CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.

Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.

Dose Optimization of Aditoprim-Sulfamethoxazole Combinations Against Trueperella pyogenes From Patients With Clinical Endometritis by Using Semi-mechanistic PK/PD Model.

Combinations of two and more drugs with different target sites are being used as a new treatment regimen for resistant clones of bacteria. Though, achieving the right combination of the drugs for optimal dosage regimen is challenging. In our study, we studied the antimicrobial effect of aditoprim, a novel dihydrofolate reductase inhibitor, and its synergistic effect with sulfamethoxazole. Synergy testing was performed by checkerboard micro dilution method and validation of different checkerboard ratios by static and dynamic time-kill analysis and in vitro pharmacokinetic/pharmacodynamics (PK/PD) model, and semi mechanistic PK/PD modeling was used to calculate and validate the synergistic effect of drug combination. Both checkerboard and static time-kill assays demonstrated the greater synergistic effect [fractional inhibitory concentration index (FICI) = 0.37] of the aditoprim [minimum inhibitory concentration (MIC) = 0.25 µg/ml]-sulfamethoxazole (MIC=>64 µg/ml) combination against all T. Pyogenes isolates. In the in vitro PK/PD model, the dosage proportion of sulfamethoxazole 4 mg/ml twice a day in combination with steady-state aditoprim 1 mg/ml efficiently repressed the growth of bacteria in 24 h with the ratio of 2-log10 decrease, related to the early inoculum against three T. Pyogenes isolates. The semi mechanistic PK/PD model projected that a combination of a high dose of aditoprim (2 mg/ml) with sulfamethoxazole (2 mg/day) was necessary to attain the killing of bacteria below the detection limit (limit of detection (LOD); i.e., 1 log10 CFU/ml) at 24 h with an MIC sulfamethoxazole (SMZ) of 64 µg/ml. However, it is anticipated that a combination of high dose of aditoprim with sulfamethoxazole is critical to attain the suppressed bacterial growth to < LOD. This study represents essential PK/PD modeling for optimization of combination of aditoprim and sulfamethoxazole to suppress growth of T. Pyogenens.

A Cross-Sectional Survey of Knowledge, Attitude, and Practices of University Students in Pakistan Regarding COVID-19.

The COVID-19 pandemic is striking the world with serious public health and socioeconomic complications. The pandemic has influenced all forms of daily life, including educational institutions. Therefore, this cross-sectional survey was conducted to understand knowledge, attitudes, and practices related to COVID-19 among the students of the University of Veterinary and Animal Sciences, Lahore. The data was collected using an online self-directed questionnaire. The survey form includes six items about sociodemographic characteristics, 14 knowledge-based questions, seven questions on attitude, and eight questions on practices. The sample number was calculated using the Raosoft sample size calculator. A total number of 3,854 students, including 1,823 men and 2,031 women, were engaged in this survey, having student representation from all the provinces in the country. The data were analyzed using a chi-square test. A total of 97% of the students knew that the etiological agent of COVID-19 is a virus and that it is a disease of the respiratory system (94%). Many students kept visiting their relatives during the lockdown (45%), and their relatives kept visiting them at home (59%). The responses from the students varied a lot on specific questions about the transmission of the virus. Women tended to have less information regarding precautionary travel measures (p < 0.01), but supplemental knowledge of prevention of disease transmission from positive patients (p < 0.01). Conclusively, the majority of the university students surveyed had imperative knowledge, a good attitude, and active practice in response to the COVID-19 outbreak. Moreover, the KAP scores have varied by demography, gender, and the number of family members. Therefore, continuous awareness of preventative behaviors should be disseminated regularly in emergencies.

RNA-seq-based transcriptome analysis of a cefquinome-treated, highly resistant, and virulent MRSA strain.

The emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains of animal origin that are resistant to several antibiotics is of great concern. Cefquinome is a fourth-generation cephalosporin developed specifically for veterinary use. The mechanism of MRSA resistance to cefquinome is still not established. Therefore, we designed this study to evaluate the effect of cefquinome on the transcriptome of MRSA1679a, a strain that was isolated from a chicken. The transcriptome analysis indicated that multiple efflux pumps (QacA, NorB, Bcr, and ABCb) were upregulated in MRSA1679a as a resistance mechanism to expel cefquinome. Additionally, penicillin-binding protein 1A was overexpressed, which conferred resistance to cefquinome, a ß-lactam antibiotic. Adhesion and the biofilm-forming capacity of the MRSA strain was also enhanced in addition to overexpression of many stress-related genes. Genes related to carbohydrate metabolism, secretion systems, and transport activity were also significantly upregulated in MRSA1679a. In conclusion, global transcription was triggered to overcome the stress induced by cefquinome, and the MRSA1679a showed a great genetic potential to survive in this challenging environment. This study provides a profound understanding of MRSA1679a as a potentially important pathogen and identifies key resistance characteristics of MRSA against cefquinome. Studies should be aimed to demonstrate multidrug resistance mechanisms of virulent strains by exposing to different antibiotic combinations.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

Microbiological inhibition-based method for screening and identifying of antibiotic residues in milk, chicken egg and honey.

This present study was designed to develop a novel microbiological inhibition-based method for the rapid screening and identification of antibiotic residues in milk, chicken egg and honey. Geobacillus stearothermophilus C953 was used as test bacterium in the detection system of this study. The optimization of nutrients and other supplements were performed to promote the growth of test bacterium and thus shorten the detection time. Furthermore, the synergetic agents were added to improve the sensitivity of test bacterium to more antibiotics. Additionally, confirmatory solutions such as ß-lactamase, p-aminobenzoic acid, MgSO4 and cysteine were added to classify and identify different kinds of antibiotics. We observed that the LOD of this detection system was at or close to maximum residue limits established by EU for ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides and quinolones in milk. The LOD of different kinds of antibiotics in chicken egg was less than or similar to the MRL and the LOD of Premi®test (except sulfonamides). For honey, there are no MRL, the LOD was less than or similar to the recommended concentration and the LOD of Premi®test. Noteworthy, the detection system also can identify these six kinds of antibiotics in milk, chicken egg and honey, and there were satisfactory results of specificity experiments and confirmation experiments by LC-MS/MS. Accordingly, the present study provides a reliable preliminary characterization of antibiotic residues in animal foods and improves the detection efficiency for the following chemical confirmation experiments by HPLC, LC-MS/MS, immunological and receptor-based tests.

The search for a microbiological inhibition method for the rapid, broad-spectrum and high-throughput screening of six kinds of antibiotic residues in swine urine.

In this study, a microbiological inhibition method for rapidly screening antibiotics in swine urine was established with an easy sample pre-treatment. The microbiological system consisted of an agar medium mixed with nutrients, sensitizers, a test bacterium (Geobacillus stearothermophilus ATCC12980) and pH indicator (bromocresol purple). It was observed that the detection limits of the test kit for twenty-eight common antimicrobial residues in urine, including ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides, and lincosamides, were less than or equal to the maximum residue limits of the kidney, as determined by the EU and China. Moreover, the false negative rate and the false positive rate, along with other performance indexes such as interassay coefficients of variation and shelf life of the kit, all met the standard requirements of the ISO13969:2003 guidelines. Additionally, our results were consistent with those using the gold-standard physical chemistry method, which suggest the proposed method is suitable for screening antibiotic residues.

A comparative phylogenomic analysis of SARS-CoV-2 strains reported from non-human mammalian species and environmental samples.

Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV (hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis.

A comparative evaluation of serum biochemistry profile and antigenic relatedness among velogenic and mesogenic Avian avulavirus 1 infection in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10-6.51 EID50/0.1 mL for sub-genotype VIIi and 10-6.87 EID50/0.1 mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (p < 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (p < 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (p < 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (R-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.