Comparative evaluation of in-house developed latex agglutination test (LAT) with World Organisation for Animal Health (WOAH) -recommended methods for the detection of Bacillus anthracis spores from the soil.

In-house developed Bacillus anthracis-specific synthetic peptide-based latex agglutination test (LAT) assay was comparatively evaluated with World Organisation for Animal Health (WOAH)-recommended polymerase chain reaction (PCR)/real-time PCR (qPCR) methods for the screening of B. anthracis spores from the soil to provide a simple, rapid, and economical immunodiagnostic test for field application.

Genetic diversity and risk factor analysis of drug-resistant Escherichia coli recovered from broiler chicken farms.

A total of 38 Escherichia coli isolates were recovered from 120 samples collected from various sources of broiler chicken farms (n = 10 each) in Andhra Pradesh and Telangana states. Though the recovered E. coli isolates were found variably resistant to the tested antibiotics, all the tested isolates were susceptible to meropenem. Alarming multi-drug resistance (MDR) was observed (34/38) among the recovered isolates, wherein antibiotic-resistant genes (blaTEM, blaSHV, and tetA) were detected, except for blaCTX-M-9. The heatmap with cluster analysis exhibited that majority of the E. coli isolates recovered from different sources and regions clustered together based on their phenotypic resistance suggesting co-sharing of resistance. However, the pulsed-field gel electrophoresis (PFGE) typing revealed an extremely diverse genotypic profile. Further, a significant statistical association was not observed between hypothesized risk factors and recovered MDR- E. coli isolates from various sources, although a significant statistical association between antibiotic resistance with large flock size, poor biosecurity practices, poor workers' hygiene, and poor disinfection practices was noticed. Since the study highlighted an alarming level of drug resistance among the recovered E. coli isolates, further in-depth research in similar veins is required to ensure the prudent use of antimicrobials in the poultry sector and the implementation of an antimicrobial surveillance system.

Green Synthesized Silver Nanoparticles Using Lactobacillus Acidophilus as an Antioxidant, Antimicrobial, and Antibiofilm Agent Against Multi-drug Resistant Enteroaggregative Escherichia Coli.

The present study was envisaged to employ the green synthesis and characterization of silver nanoparticles (AgNPs) using the potential probiotic strain Lactobacillus acidophilus, to assess its antibacterial as well as antibiofilm activity against multi-drug-resistant enteroaggregative Escherichia coli (MDR-EAEC) strains and to investigate their antioxidant activity. In this study, AgNPs were successfully synthesized through an eco-friendly protocol, which was then confirmed by its X-ray diffraction (XRD) pattern. A weight loss of 15% up to 182 °C with a narrow exothermic peak between 170 °C and 205 °C was observed in thermogravimetric analysis-differential thermal analysis (TGA-DTA), while aggregated nanoclusters were observed in scanning electron microscopy (SEM). Moreover, the transmission electron microscopy (TEM) imaging of AgNPs revealed a spherical morphology and crystalline nature with an optimum size ranging from 10 to 20 nm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of green synthesized AgNPs against the MDR-EAEC strains were found to be 7.80 mg/L and 15.60 mg/L, respectively. In vitro time-kill kinetic assay revealed a complete elimination of the MDR-EAEC strains after 180 min on co-incubation with the AgNPs. Moreover, the green synthesized AgNPs were found safe by in vitro haemolytic assay. Besides, the green synthesized AgNPs exhibited significant biofilm inhibition (P < 0.001) formed by MDR-EAEC strains. Additionally, a concentration-dependent antioxidant activity was observed in 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. Hence, this study demonstrated potential antibacterial as well as antibiofilm activity of green synthesized AgNPs against MDR-EAEC strains with antioxidant properties and warrants further in-depth studies to explore it as an effective antimicrobial agent against MDR infections.

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence, resistance to biocides, genomic landscape and biocontrol.

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.

Antibacterial efficacy of in-house designed cell-penetrating peptide against multi-drug resistant strains of Salmonella Enteritidis and Salmonella Typhimurium.

The in vitro antibacterial efficacy of an in-house designed cell-penetrating peptide (CPP) variant of Cecropin A (1-7)-Melittin (CAMA) (CAMA-CPP) against the characterized multi-drug resistant (MDR) field strains of Salmonella Enteritidis and Salmonella Typhimurium were evaluated and compared with two identified CPPs namely, P7 and APP, keeping CAMA as control. Initially, the minimum inhibitory concentration (MIC) (µg ml-1 ) of in-house designed CAMA-CPP, APP and CAMA was determined to be 3.91, whereas that of P7 was 7.81; however, the minimum bactericidal concentration (MBC) of all the peptides were twice the MIC. CAMA-CPP and CAMA were found to be stable under different conditions (high-end temperatures, proteinase-K, cationic salts, pH and serum) when compared to the other CPPs. Moreover, CAMA-CPP exhibited negligible cytotoxicity in HEp-2 and RAW 264.7 cell lines as well as haemolysis in the sheep and human erythrocytes with no adverse effects against the commensal gut lactobacilli. In vitro time-kill assay revealed that the MBC levels of CAMA-CPP and APP could eliminate the intracellular MDR-Salmonella infections from mammalian cell lines; however, CAMA and P7 peptides were ineffective. CAMA-CPP appears to be a promising antimicrobial candidate and opens up further avenues for its in vivo clinical translation.

In silico molecular docking and in vitro antimicrobial efficacy of phytochemicals against multi-drug-resistant enteroaggregative Escherichia coli and non-typhoidal Salmonella spp.

BACKGROUND: In the wake of emergence of antimicrobial resistance, bioactive phytochemical compounds are proving to be important therapeutic agents. The present study envisaged in silico molecular docking as well as in vitro antimicrobial efficacy screening of identified phytochemical ligands to the dispersin (aap) and outer membrane osmoporin (OmpC) domains of enteroaggregative Escherichia coli (EAEC) and non-typhoidal Salmonella spp. (NTS), respectively. MATERIALS AND METHODS: The evaluation of drug-likeness, molecular properties, and bioactivity of the identified phytocompounds (thymol, carvacrol, and cinnamaldehyde) was carried out using Swiss ADME, while Protox-II and StopTox servers were used to identify its toxicity. The in silico molecular docking of the phytochemical ligands with the protein motifs of dispersin (PDB ID: 2jvu) and outer membrane osmoporin (PDB ID: 3uu2) were carried out using AutoDock v.4.20. Further, the antimicrobial efficacy of these compounds against multi-drug resistant EAEC and NTS strains was determined by estimating the minimum inhibitory concentrations and minimum bactericidal concentrations. Subsequently, these phytochemicals were subjected to their safety (sheep and human erythrocytic haemolysis) as well as stability (cationic salts, and pH) assays. RESULTS: All the three identified phytochemicals ligands were found to be zero violators of Lipinski's rule of five and exhibited drug-likeness. The compounds tested were categorized as toxicity class-4 by Protox-II and were found to be non- cardiotoxic by StopTox. The docking studies employing 3D model of dispersin and ompC motifs with the identified phytochemical ligands exhibited good binding affinity. The identified phytochemical compounds were observed to be comparatively stable at different conditions (cationic salts, and pH); however, a concentration-dependent increase in the haemolytic assay was observed against sheep as well as human erythrocytes. CONCLUSIONS: In silico molecular docking studies provided useful insights to understand the interaction of phytochemical ligands with protein motifs of pathogen and should be used routinely before the wet screening of any phytochemicals for their antibacterial, stability, and safety aspects.

Comparison of recombinant and synthetic listeriolysin- O peptide- based indirect ELISA vis-à-vis cultural isolation for detection of listeriosis in caprine and ovine species.

The present study evaluated the comparative serodiagnostic efficacy of recombinant listeriolysin-O (rLLO) and synthetic LLO- 2 peptide-based indirect ELISA vis-à-vis cultural isolation using samples (n = 1326; blood, sera, vaginal swabs, and rectal swabs) collected from caprines (n = 350) and ovines (n = 50) having reproductive and/or nervous system disorders and/or healthy animals. On screening the test sera by rLLO- based ELISA, the antibodies against LLO (ALLO) were observed in 17.71% of the caprines and 2% of the ovines, respectively, while synthetic LLO-2- based ELISA revealed ALLO in 6.86% of caprines and not in ovines. Moreover, the adsorption of positive test sera with streptolysin-O (SLO) resulted in a significant reduction (7.43%; p < 0.05) in the seropositivity with rLLO- based ELISA, whereas LLO-2- based ELISA revealed marginal reduction (4.29%; p > 0.05) in the seropositivity. Overall, the seropositivity with LLO-2 synthetic peptide revealed comparatively less cross-reactivity in comparison to rLLO. The cultural isolation yielded five pathogenic L. monocytogenes isolates and three non-pathogenic Listeria spp. from caprine samples; however, Listeria spp. could not be recovered from any of the ovine samples. Further, on comparing seropositivity with the isolation study results, it was found that two out of the five animals from which pathogenic L. monocytogenes isolated were also found seropositive in both the ELISAs even after adsorption with SLO. Interestingly, rLLO- based ELISA detected antibodies against unadsorbed caprine sera even in those samples from which non-pathogenic Listeria spp. were isolated, whereas antibodies were not detected in LLO-2 peptide-based ELISA. In conclusion, it could be inferred that the synthetic LLO-2 peptide serves as a non- cross-reactive, ideal diagnostic antigen in serodiagnosis of capro-ovine listeriosis.

Current perspectives on the occurrence of Q fever: highlighting the need for systematic surveillance for a neglected zoonotic disease in Indian subcontinent.

Coxiellosis or Q fever is an important global occupational zoonotic disease caused by one of the most contagious bacterial pathogens - Coxiella burnetii, which ranks one among the 13 global priority zoonoses. The detection of C. burnetii infection is exhibiting an increasing trend in high-risk personnel around the globe. It has increasingly been detected from foods of animal origin (including bulk milk, eggs, and meat) as well as tick vectors in many parts of the world. Coxiellosis is reported to be an important public health threat causing spontaneous abortions in humans and potential reproductive failure, which would result in production losses among livestock. Further, comprehensive coverage of the reports and trends of Q fever in developing countries, where this infection is supposed to be widely prevalent appears scarce. Also, the pathogen remains grossly neglected and underreported. Moreover, policymakers and funding agencies do not view it as a priority problem, especially in the Indian subcontinent, including Sri Lanka, Bhutan, Pakistan, Nepal, Bangladesh and Maldives. Here, we review the occurrence and epidemiology of the disease in a global context with special emphasis on its status in the Indian subcontinent.

Exploiting Lactoferricin (17-30) as a Potential Antimicrobial and Antibiofilm Candidate Against Multi-Drug-Resistant Enteroaggregative Escherichia coli.

The study evaluated the in vitro antimicrobial and antibiofilm efficacy of an antimicrobial peptide (AMP), lactoferricin (17-30) [Lfcin (17-30)], against biofilm-forming multi-drug-resistant (MDR) strains of enteroaggregative Escherichia coli (EAEC), and subsequently, the in vivo antimicrobial efficacy was assessed in a Galleria mellonella larval model. Initially, minimum inhibitory concentration (MIC; 32 µM), minimum bactericidal concentration (MBC; 32 µM), and minimum biofilm eradication concentration (MBEC; 32 µM) of Lfcin (17-30) were determined against MDR-EAEC field isolates (n = 3). Lfcin (17-30) was tested stable against high-end temperatures (70 and 90°C), physiological concentration of cationic salts (150 mM NaCl and 2 mM MgCl2), and proteases (proteinase-K and lysozyme). Further, at lower MIC, Lfcin (17-30) proved to be safe for sheep RBCs, secondary cell lines (HEp-2 and RAW 264.7), and beneficial gut lactobacilli. In the in vitro time-kill assay, Lfcin (17-30) inhibited the MDR-EAEC strains 3 h post-incubation, and the antibacterial effect was due to membrane permeation of Lfcin (17-30) in the inner and outer membranes of MDR-EAEC. Furthermore, in the in vivo experiments, G. mellonella larvae treated with Lfcin (17-30) exhibited an increased survival rate, lower MDR-EAEC counts (P < 0.001), mild to moderate histopathological changes, and enhanced immunomodulatory effect and were safe to larval cells when compared with infection control. Besides, Lfcin (17-30) proved to be an effective antibiofilm agent, as it inhibited and eradicated the preformed biofilm formed by MDR-EAEC strains in a significant (P < 0.05) manner both by microtiter plate assay and live/dead bacterial quantification-based confocal microscopy. We recommend further investigation of Lfcin (17-30) in an appropriate animal model before its application in target host against MDR-EAEC strains.

Global scenario, public health concerns and mitigation strategies to counter current ongoing SARS-CoV-2 / COVID-19 pandemic.

Severe acute respiratory syndrome Coronavirus- 2 (SARS-CoV-2), the etiological agent of the novel coronavirus disease (COVID-19), has posed a great public health threat to the global community as a pandemic. The origin of the virus has been linked to animals, through a yet-to-be-identified intermediate host. The disease is transmitted to humans mainly through inhalation or contact with infected droplets. The variable clinical presentation of COVID-19 includes fever, cough, sore throat, breathlessness, fatigue and malaise; however, cutaneous, ocular, neurological, and gastrointestinal manifestations have also been reported. There is an urgent need to strengthen One Health surveillance, intervention, and management strategies to understand the ecology of coronaviruses and to prevent epidemics in the future. Global attention toward the development of treatments, immunotherapies, vaccines, and control options to combat the COVID-19 pandemic has been on an increasing trend. Here, we review the current epidemiological status, public health concerns, and mitigation strategies for COVID-19.

Antimicrobial Efficacy of Indolicidin Against Multi-Drug Resistant Enteroaggregative Escherichia coli in a Galleria mellonella Model.

Antimicrobial resistance against enteroaggregative Escherichia coli (EAEC), an emerging food-borne pathogen, has been observed in an increasing trend recently. In the recent wake of antimicrobial resistance, alternate strategies especially, cationic antimicrobial peptides (AMPs) have attracted considerable attention to source antimicrobial technology solutions. This study evaluated the in vitro antimicrobial efficacy of Indolicidin against multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) strains and further to assess its in vivo antimicrobial efficacy in Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 32 µM) and minimum bactericidal concentration (MBC; 64 µM) of Indolicidin against MDR-EAEC was determined by micro broth dilution method. Indolicidin was also tested for its stability (high-end temperatures, physiological concentration of salts and proteases); safety (sheep RBCs; HEp-2 and RAW 264.7 cell lines); effect on beneficial microflora (Lactobacillus rhamnosus and Lactobacillus acidophilus) and its mode of action (flow cytometry; nitrocefin and ONPG uptake). In vitro time-kill kinetic assay of MDR-EAEC treated with Indolicidin was performed. Further, survival rate, MDR-EAEC count, melanization rate, hemocyte enumeration, cytotoxicity assay and histopathological examination were carried out in G. mellonella model to assess in vivo antimicrobial efficacy of Indolicidin against MDR-EAEC strains. Indolicidin was tested stable at high temperatures (70°C; 90°C), physiological concentration of cationic salts (NaCl; MgCl2) and proteases, except for trypsin and tested safe with sheep RBCs and cell lines (RAW 264.7; HEp-2) at MIC (1X and 2X); the beneficial flora was not inhibited. Indolicidin exhibited outer membrane permeabilization in a concentration- and time-dependent manner. In vitro time-kill assay revealed concentration-cum-time dependent clearance of MDR-EAEC in Indolicidin-treated groups at 120 min, while, in G. mellonella, the infected group treated with Indolicidin revealed an increased survival rate, immunomodulatory effect, reduced MDR-EAEC counts and were tested safe to the larval cells which was concurred histopathologically. To conclude, the results suggests Indolicidin as an effective antimicrobial candidate against MDR-EAEC and we recommend its further investigation in appropriate animal models (mice/piglets) before its application in the target host.

Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources.

INTRODUCTION: Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. MATERIALS AND METHODS: A total of 336 samples comprising diarrhoeic stool samples from infants (n=103), young animal (n=106), foods (n=68) and associated environmental sources (n=59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction-based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. RESULTS AND DISCUSSION: Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of 'atypical' EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs.

Characterization and biofilm forming ability of diarrhoeagenic enteroaggregative Escherichia coli isolates recovered from human infants and young animals.

Enteroaggregative Escherichia coli (EAEC) is an important pathotype that causes infection in humans and animals. EAEC isolates (n=86) recovered from diarrhoeal cases in human infants (37) and young animals (49) were characterized as 'typical' and/or 'atypical' EAEC strains employing PCR for virulence associated genes (cvd432, aaiA, astA, pilS, irp2, ecp, pic, aggR, aafA, aggA, and agg3A). Besides, biofilm formation ability of human and animal EAEC isolates was assessed using microtiter plate assay. In addition, the transcriptional profile of biofilm associated genes (fis and ecp) was also evaluated and correlated with biofilm formation assay for few selected EAEC isolates of human and animal origins. Overall, a diverse virulence gene profile was observed for the EAEC isolates of human and animal origins as none of the EAEC isolates revealed the presence of all the genes that were targeted. Nine 'typical' EAEC isolates were identified (6 from humans and 3 from animals) while, the majority of the isolates were 'atypical' EAEC strains. Isolation and identification of three 'typical' EAEC isolates from animals (canines) appears to be the first report globally. Further, based on the observations of the biofilm formation assay, the study suggested that human EAEC isolates in particular were comparatively more biofilm producers than that of the animal EAEC isolates. The fis gene was highly expressed in majority of 'typical' EAEC isolates and the ecp gene in 'atypical' EAEC isolates.

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases.

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.