The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

BACKGROUND: Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. MATERIAL AND METHODS: The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. RESULT: The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions of breast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. CONCLUSION: In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan.

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408 bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Phytochemical composition and In-vitro activity of ethanolic extract of Eucalyptus globulus leaves against multidrug resistant poultry pathogens.

Aim of the present study was to determine the In-vitro antibacterial activity of ethanolic extract of E. globulus leaves against common multidrug resistant poultry pathogens. Phytochemical analysis through HPLC revealed that kaempeferol (7.315min) followed by querecetin (6.655min) and myrecetin (3.655min). Percent area of kaempeferol (6826.88%) was highest, followed by myrecetin (5516.22%) and querecetin (163.748%). Phytochemical investigation of ethanolic extract of E. globulus leaves through GCMS showed highest retention time (min) α-pinene (20.43) and α-terpineol (20.15) accompanied by spathulenol (11.97), piperitone (11.04). The ethanolic extracts of E. globulus leaves showed a highest zone of inhibition against S. pullorum SP6; 20.64± 2.08, E. coli SE 12; 19.75± 2.83, C. perfringens type A (CPM38-01); 19.46± 2.02. The highest level of MIC of E. globulus noted were against S. gallinarum S22; 133.37±53.294, S. gallinarum S1; 130.20±45.10, S. gallinarum S4; 129.47±24.182, S. gallinarum S3; 126.83±72.392. In conclusion, the study confirmed that the ethanolic extract of E. globulus is composed of active ingredients having antibacterial activity and can be referred as an alternate to antibiotics.

Activity of ethanolic extract of Eucalyptus globulus leaves against multi drug resistant poultry pathogens in broiler chicks.

The present study was designed to evaluate the antimicrobial activity of E. globulus leaves in broiler chicks. Total (n=255) day-old chicks were segregated into five groups i.e. Pathogenic E. coli, S. pullorum, S. gallinarum and C. perfringens type A and control negative group. Each bacterial challenged (1x 107 CFU) group was divided into control positive, antibiotic, probiotic and E. globulus group. Experimental birds were exposed to E. coli, S. pullorum, S. gallinarum and C. perfringens type A at different ages. At 35th day of experiment the log reduction for each group was determined. The highest log reduction in E. coli and C. perfringens Type A colonies count were found in E. globulus (3.26) (2.33) treated group followed by antibiotic (2.85) (1.59) and probiotic (2.84) (1.50) respectively. The log reduction in S. pullorum colonies count was highest in E. globulus (2.50) followed by probiotic (2.24) and antibiotic (2.16). The S. gallinarum colonies count log reduction was found highest for antibiotic (2.84) followed by probiotic (2.48) and E. globulus group. The results of in-vivo experiment revealed that ethanolic extract of E. globulus has antibacterial activity and it can be used as a replacement to low level of antibiotics added in poultry feed.

In silico analysis of four structural proteins of aphthovirus serotypes revealed significant B and T cell epitopes.

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.

Role of Rab GTPases in HSV-1 infection: Molecular understanding of viral maturation and egress.

Most enveloped viruses exploit complex cellular pathways for assembly and egress from the host cell, and the large DNA virus Herpes simplex virus 1 (HSV-1) makes no exception, hijacking several cellular transport pathways for its glycoprotein trafficking and maturation, as well as for viral morphogenesis and egress according to the envelopment, de-envelopment and re-envelopment model. Importantly Rab GTPases, widely distributed master regulators of intracellular membrane trafficking pathways, have recently being tightly implicated in such process. Indeed, siRNA-mediated genetic ablation of specific Rab proteins differently affected HSV-1 production, suggesting a complex role of different Rab proteins in HSV-1 life cycle. In this review, we discuss how different Rabs can regulate HSV-1 assembly/egress and the potential therapeutic applications of such findings for the management of HSV-1 infections.

Short Communication - Study on quality and efficacy of commercial tylosin and doxycycline products against local isolates of mycoplasma in broilers.

The present study was conducted to investigate the quality and efficacy of commercially available preparations of tylosin and doxycycline available in the local market at Peshawar for poultry. In vitro and in vivo, tests were conducted to check the quality of these antimicrobial drugs. In vitro quality control test was performed by High performance liquid chromatographic (HPLC) and micro dilution method. In vivo, efficacy of the test drugs was checked in broilers infected with Mycoplasma gallisepticum. Results of HPLC indicated that test drug-2 contains doxycycline hydrochloride within specified limits but contain high quantity of active ingredient (Tylosin tartrate 120%). Recovery percentage of test drugs (3, 4, 5) were below the pharmacopoeial limit, which contained low quantity of tylosin tartrate (85%, 87.5%, 85%) respectively however, percent recovery of doxycycline were in the appropriate limits. All the tested drugs were effective against Mycoplasma gallisepticum and showed minimum inhibitory concentration (MIC) at 1.9µg/ml. The in vivo result indicated that all tested drugs decreased morbidity and mortality in infected chicks. The birds treated with test drugs (3 and 5) showed mortality of 9.5%, which was slightly higher than the other test groups. The current study suggested that there are incidences of substandard drugs in Pakistan and the drug regularity authorities should take strict actions against the manufacturing companies.

Microbial communities present in the lower respiratory tract of clinically healthy birds in Pakistan.

Commercial poultry is an important agricultural industry worldwide. Although dense living conditions and large flocks increase meat and egg production, they also increase the risk of disease outbreaks and zoonoses. Current pathogen identification methods mostly rely on culture-dependent techniques and, therefore, are limited to a very small number of bacteria present in the environment. Next Generation Sequencing allows for culture-independent characterization of lower respiratory microbiome of birds including the identification of novel commensals and potentially emerging pathogens. In this study, we collected tracheo-bronchoalveolar lavage of 14 birds raised at 3 different farms in the Punjab province of Pakistan. To characterize the lower respiratory microbiome of these birds, we sequenced hyper-variable regions of the 16S ribosomal subunit gene. Although dominated by bacteria belonging to a small number of taxonomic classifications, the lower respiratory microbiome from each farm was far more diverse and novel than previously known. The differences in microbiome among farms suggest that inter-farm differences affect the microbiome of birds more than breed, geographic location, or management system. The presence of potential and known pathogens in genetically similar specialty breeds of chickens kept at unnaturally high densities and under variable conditions presents an extraordinary opportunity for the selection of highly pathogenic bacteria. In some instances, opportunistic respiratory pathogens were observed in apparently healthy birds. Understanding and monitoring the respiratory microbiome of such populations may allow the early detection of future disease threats.

Report-Isolation identification and control of vancomycin resistant Staphylococcus aureus.

Vancomycin resistant Staphylococcus aureus (VRSA) has been reported from many parts of the world including Asian countries. Hence, main objective of study was to evaluate the possible occurrence of VRSA in hospitals of Lahore city and to ensure the effectiveness of various substitute therapeutic options. A total of 150 samples of pus/wounds were collected from three hospitals of the city and VRSA were isolated and confirmed through recommended method of Clinical and Laboratory Standards Institute. Out of 51 (49.04%) methicillin resistant S. aureus (MRSA) isolates, 5 (9.8%) were found resistant to vancomycin. Minimum inhibitory concentration (MIC) of Linezolid (LZD), Moxifloxacin (MFX) and Clindamycin (CD) were calculated against VRSA isolates by broth microdilution test. All 5 (100%) isolates were susceptible to Linezolid and Clindamycin, while 4 (80%) were susceptible to Moxifloxacin. Ethanolic extracts of Turmeric, Mint, Coriander, Garlic, Kalonji, Cinnamon and Cloves illustrate average MIC values of 140.8 µg/mL, 563.2 µg/mL, 486.4 µg/mL, 614.4 µg/mL, 409.6 µg/mL, 281.6 µg/mL and 64 µg/mL, respectively against 5 VRSA strains. Concentration dependent increase in growth inhibition zones of ethanolic plant extract was recorded by agar well diffusion test. This study was helpful to find out the effective antibiotic against VRSA. Plant extracts encompass anti-staphylococcal activity and this finding demands necessity of further exploration of potential found in these natural herb.

Antibacterial activity of herbal extracts against multi-drug resistant Escherichia coli recovered from retail chicken meat.

Increasing incidence rate of multiple drug resistance in Escherichia coli (E. coli) due to extensive uses of antibiotics is a serious challenge to disease treatment. Contaminated retail chicken meat is one of the major sources of spread of multi drug resistant (MDR) E. coli. Current study has been conducted to study the prevalence of MDR E. coli in retail chicken meat samples from Lahore city of Pakistan and it was found that 73.86% of E. coli isolates have MDR pattern. In vitro evaluation of antibacterial activity of crude ethanolic extracts of six herbs against MDR E. coli phenotypes has revealed that clove and cinnamon have maximum zones of inhibition as compared to other herbal extracts. Mint and coriander gave the intermediate results while garlic and kalonji showed the least antibacterial activity against the MDR E. coli phenotypes using the agar well diffusion technique. Average Minimum Inhibitory Concentrations (MICs) for clove, mint, cinnamon, coriander, kalonji and garlic extracts were 1.15, 1.38, 0.5, 1.99, 2.41, 8.60 mg/mL respectively using the broth micro dilution method. The results obtained in present study were revealed that crude ethanol extracts of selected herbs have had significant antibacterial activity. Hence they can be used as promising alternatives of antimicrobials against MDR E. coli species and can be used for cooked food preservation.

Identifying key soil characteristics for Francisella tularensis classification with optimized Machine learning models.

Francisella tularensis (Ft) poses a significant threat to both animal and human populations, given its potential as a bioweapon. Current research on the classification of this pathogen and its relationship with soil physical-chemical characteristics often relies on traditional statistical methods. In this study, we leverage advanced machine learning models to enhance the prediction of epidemiological models for soil-based microbes. Our model employs a two-stage feature ranking process to identify crucial soil attributes and hyperparameter optimization for accurate pathogen classification using a unique soil attribute dataset. Optimization involves various classification algorithms, including Support Vector Machines (SVM), Ensemble Models (EM), and Neural Networks (NN), utilizing Bayesian and Random search techniques. Results indicate the significance of soil features such as clay, nitrogen, soluble salts, silt, organic matter, and zinc , while identifying the least significant ones as potassium, calcium, copper, sodium, iron, and phosphorus. Bayesian optimization yields the best results, achieving an accuracy of 86.5% for SVM, 81.8% for EM, and 83.8% for NN. Notably, SVM emerges as the top-performing classifier, with an accuracy of 86.5% for both Bayesian and Random Search optimizations. The insights gained from employing machine learning techniques enhance our understanding of the environmental factors influencing Ft's persistence in soil. This, in turn, reduces the risk of false classifications, contributing to better pandemic control and mitigating socio-economic impacts on communities.

In Vitro Characterization of Probiotic Potential of Limosilactobacillus fermentum against Salmonella Gallinarum Causing Fowl Typhoid.

Fowl typhoid, a septicaemic disease of poultry, is caused by Salmonella Gallinarum and leads to severe economic losses. The aim of the present study was to isolate, select and characterize indigenous probiotic lactobacilli with anti-Salmonella Gallinarum activity. A total 55 lactobacilli were isolated from the caeca and ileum parts of healthy chickens and identified to species level by 16S rDNA sequencing. All the isolates were initially screened for antimicrobial activity and selected isolates were further subjected to in vitro evaluation of probiotic properties. Lactobacilli isolates (n = 21) showed varying levels of activity (08-18 mm) against Salmonella Gallinarum. These selected isolates also showed tolerance to acidic conditions (pH 3 and 4). Out of these 21 isolates, 13 showed growth (>0.5 OD at 600 nm) 0.3% bile salts. Moreover, these isolates also had the ability of auto-aggregation (20.05 ± 0.62%-50.70 ± 1.40%), and co-aggregation with Salmonella Gallinarum (5.22 ± 0.21%-42.07 ± 0.70%). Results revealed that lactobacilli had a higher level of resistance to vancomycin (100%), streptomycin (100%), ciprofloxacin (95%), gentamicin (90%), doxycycline (90%), oxytetracycline (85%), and bacitracin (80%), and a lower level of resistance to penicillin (33%), erythromycin (28%), chloramphenicol (23%), fusidic acid (23%) and amoxicillin (4%). The Limosilactobacillus fermentum PC-10 and PC-76 were sensitive to most of the antibiotics. The overall results revealed that two Limosilactobacillus fermentum strains (PC-10 and PC-76) fulfill the in vitro selection criteria of probiotics, i.e, tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with Salmonella Gallinarum, and absence of acquired antibiotic resistance. The Limosilactobacillus fermentum PC-10 and PC-76 also inhibited the (>5 log10) growth of Salmonella Gallinarum in co-culture assay. It is concluded that Limosilactobacillus fermentum PC-10 and PC-76 may be further investigated and developed as anti-Salmonella Gallinarum probiotics for poultry.

Aflatoxins posing threat to food safety and security in Pakistan: Call for a one health approach.

Aflatoxins are among the most important mycotoxins due to their widespread occurrence and adverse impacts on humans and animals. These toxins and/or their metabolites cannot be destroyed with cooking or boiling methods. Therefore, consumption of aflatoxin-contaminated food may lead to impaired growth, compromised immunity, stomach and liver cancer, and acute toxicity. These adverse effects along with food wastage might have detrimental consequences on a country's economy. Several studies from Pakistan reported a high prevalence of aflatoxins in food and feed commodities (Range; milk = 0.6-99.4%, cereals, and grains = 0.38-41%, animal feed = 31-100%). Notably, Pakistan reported very high figures of impaired child growth-stunted 40.2%, wasted 17.7% and underweight 28.9%-that could be associated with the higher aflatoxin prevalence in food items. Importantly, high aflatoxins prevalence, i.e. 100%, 69% and 60.5%, in children has been reported in Pakistan. Food and feed are more prone to aflatoxin contamination due to Pakistan's hot and humid climate; however, limited awareness, inadequate policy framework, and weak implementation mechanisms are the major obstacles to effective control. This review will discuss aflatoxins prevalence, associated risk factors, adverse health effects, required regulatory regime, and effective control strategies adopting the One Health approach to ensure food safety and security.

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.

Two phase feature-ranking for new soil dataset for Coxiella burnetii persistence and classification using machine learning models.

Coxiella burnetii (Cb) is a hardy, stealth bacterial pathogen lethal for humans and animals. Its tremendous resistance to the environment, ease of propagation, and incredibly low infectious dosage make it an attractive organism for biowarfare. Current research on the classification of Coxiella and features influencing its presence in the soil is generally confined to statistical techniques. Machine learning other than traditional approaches can help us better predict epidemiological modeling for this soil-based pathogen of public significance. We developed a two-phase feature-ranking technique for the pathogen on a new soil feature dataset. The feature ranking applies methods such as ReliefF (RLF), OneR (ONR), and correlation (CR) for the first phase and a combination of techniques utilizing weighted scores to determine the final soil attribute ranks in the second phase. Different classification methods such as Support Vector Machine (SVM), Linear Discriminant Analysis (LDA), Logistic Regression (LR), and Multi-Layer Perceptron (MLP) have been utilized for the classification of soil attribute dataset for Coxiella positive and negative soils. The feature-ranking methods established that potassium, chromium, cadmium, nitrogen, organic matter, and soluble salts are the most significant attributes. At the same time, manganese, clay, phosphorous, copper, and lead are the least contributing soil features for the prevalence of the bacteria. However, potassium is the most influential feature, and manganese is the least significant soil feature. The attribute ranking using RLF generates the most promising results among the ranking methods by generating an accuracy of 80.85% for MLP, 79.79% for LR, and 79.8% for LDA. Overall, SVM and MLP are the best-performing classifiers, where SVM yields an accuracy of 82.98% and 81.91% for attribute ranking by CR and RLF; and MLP generates an accuracy of 76.60% for ONR. Thus, machine models can help us better understand the environment, assisting in the prevalence of bacteria and decreasing the chances of false classification. Subsequently, this can assist in controlling epidemics and alleviating the devastating effect on the socio-economics of society.

The immunogenicity and efficacy of acommercially available Infectious Bovine Rhinotracheitis (IBR) virus vaccine against a Pakistani field IBR strain.

Infectious bovine rhinotracheitis (IBR) is a highly communicable disease of cattle and wild ruminants that is caused by Bovine alphaherpesvirus 1 (BoHV­1). For IBR control, several developed countries have adopted the immunization and eradication programs focusing on IBR­positive animals. In Pakistan, livestock producers are importing commercially available vaccine of BoHV­1, but no studies on the efficacy of these commercial vaccines against local isolates are available. Therefore, the present study was aimed to evaluate the efficacy of a commercially available vaccine of BoHV­1 against local field isolates of virus. The rabbit model was used and the vaccine was evaluated for immunogenicity and protection after challenge with a highly virulent strain of a field virus. The immune response was measured by virus neutralization titers (VNT). This vaccine induced a humoral response in rabbits but that was not sufficient to completely protect the vaccinated animals against the wild­type BoHV­1 strain challenge. While a low virus titer compared to control rabbits was observed in the vaccinated rabbits (p<0.05), there was no sterilizing immunity or freedom from infection. However, complete freedom from disease, for example, the absence of pyrexia was noticed in the vaccinated group. In conclusion, the present study demonstrated that imported vaccine stock provoked only a partial protection against indigenous isolated of BoHV­1. However, tests performed on rabbits are preliminary, as only those performed on the source species can determine more reliable results.

Assessment of aflatoxin M1 exposure and associated determinants in children from Lahore, Pakistan.

Aflatoxins are potent carcinogenic and immunomodulatory mycotoxins, and exposure may lead to deleterious effects on human health. This study aimed to detect aflatoxin M1 (AFM1) as biomarker of exposure and determine associated risk factors in children attending a specialized-childcare hospital in Lahore. Urine samples collected from 238 children (1-11 years) during winter (January-mid-March 2020) and hot-humid summer (August-September 2020) were tested for AFM1 presence using ELISA. Data on potential risk factors were also collected. Of 238 samples, 156 (65.5%) were positive for urinary AFM1. Season was significantly associated (OR = 2.64; 95% CI = 1.49-4.79; p = 0.001) with AFM1 positivity; prevalence was higher in hot-humid months (74.6%) than winter (57.3%). The place of living was also significantly associated (OR = 2.21; 95% CI = 1.25-3.97; p = 0.007), and urinary AFM1 positivity was higher in urban children (71.1%) compared to rural (58.3%). Median value for creatinine-adjusted AFM1 was 1.9 ng/mg creatinine (Q1-Q3 = 0.82-6.0 ng/mg creatinine), while non-creatinine-adjusted AFM1 was 0.57 ng/mL (Q1-Q3 = 0.23-1.4 ng/mL). Significantly higher urinary AFM1 levels were detected in children; age ≤2 years (p = 0.037), who consumed more milk (p = 0.048), and who presented to the nutrition clinic (p = 0.003). These findings highlight the need for an effective control program to reduce the AFM1 burden in children.

Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice.

Lactobacilli (n = 24), isolated from human infants and yogurt, showed variable in vitro activity against Salmonella enteritidis (8.0 ± 1.0 to 16.6 ± 0.5 mm) and other gut pathogens (9.0 ± 1.0 to 15.3 ± 0.5 mm), as determined by a well diffusion assay. The isolates were identified as Limosilactobacillus fermentum (FY1, FY3, FY4, IL2, and IL5), Lactobacillus delbrueckii (FY6 and FY7), Lactobacillus sp. (IL7), and Lactobacillus gasseri (IL12). All isolates showed variable in vitro tolerance to acidic pH for 3 h and visible growth at pH 4 and in the presence of 0.3% ox-bile. The antibiotic susceptibility profile of Lactobacillus isolates indicated resistance against vancomycin, ciprofloxacin, streptomycin, and lincomycin. Isolates had variable auto-aggregation and showed variable capabilities to co-aggregate with S. enteritidis. Based on all tested parameters, L. fermentum IL2, L. fermentum IL5, and L. gasseri IL12 were selected for co-culture experiments, followed by in vivo evaluation in Balb/c mice. All the selected isolates resulted in a 100% reduction in S. enteritidis in broth. Lactobacillus isolates efficiently colonized mouse guts and inhibited S. enteritidis colonization. Overall, there was ≥99.06% and ≤4.32 Mean log10 reduction in Salmonella counts in mice feces within 7 days. The study, thus, provided characterized lactobacilli that could be considered as potential ingredients for probiotic formulations intended to prevent S. enteritidis infection in humans.

Assessment of aflatoxin B1-lysine adduct in children and its effect on child growth in Lahore, Pakistan.

Aflatoxin B1 is an important toxic food contaminant and there is very little information available about its exposure and effects on the health of the Pakistani population. Therefore, children (n = 238) aged 1-11 years were recruited in this study to estimate the levels of aflatoxin B1-lysine adduct and to measure its adverse effects on growth. Blood samples were analyzed to detect AFB1-lysine adducts through high-performance liquid chromatography. Socio-demographic information and anthropometry measurements were also obtained. All participants had detectable levels of AFB1-lysine adduct with a median concentration of 10.66 pg/mg albumin (95% CI: 8.6-12.4). Differences in area of residence (p < 0.05) and the father's employment (p < 0.05) were significant predictors for aflatoxin concentration levels in ordinary least square and quantile regression models (residence in 75th quantile and father employment in 90th quantile). Children aged from 5 to 11 years in the 5th and 90th quantiles of the regression model had a significant association with aflatoxin levels. A very high (50.4%, 120/238) prevalence of growth impairment (stunting, wasting, and underweight) was also observed in this study. Although we couldn't establish the effect of aflatoxin on growth impairment, children with low serum albumin levels (OR = 0.18; 95% CI: 0.05-0.56; p = 0.004) were likely to be at risk of wasting. Also, low birth weight was strongly associated with wasting (OR = 3.11; 95% CI: 1.36-7.03; p = 0.006) and underweight (OR = 4.60; 95% CI: 2.21-10.05; p= <0.001), while the mother's school level education had a correlation with child stunting (OR = 1.84; 95% CI: 1.07-3.22; p = 0.029). The high prevalence of growth impairment and high concentration of serum AFB1-lysine adduct levels in study participants demand immediate efforts to mitigate the adverse health outcomes in children in Pakistan.

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.