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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 (bla TEM , bla SHV , and tetA) were detected, except for bla CTX-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The global emergence of antimicrobial resistance (AMR) needs no emphasis. In this study, the in vitro stability, safety, and antimicrobial efficacy of nanosilver-entrapped cinnamaldehyde (AgC) against multi-drug-resistant (MDR) strains of enteroaggregative Escherichia coli (EAEC) were investigated. Further, the in vivo antibacterial efficacy of AgC against MDR-EAEC was also assessed in Galleria mellonella larval model. In brief, UV-Vis and Fourier transform infrared (FTIR) spectroscopy confirmed effective entrapment of cinnamaldehyde with nanosilver, and the loading efficiency was estimated to be 29.50 ± 0.56%. The AgC was of crystalline form as determined by the X-ray diffractogram with a mono-dispersed spherical morphology of 9.243 ± 1.83 nm in electron microscopy. AgC exhibited a minimum inhibitory concentration (MIC) of 0.008–0.016 mg/mL and a minimum bactericidal concentration (MBC) of 0.008–0.032 mg/mL against MDR- EAEC strains. Furthermore, AgC was stable (high-end temperatures, proteases, cationic salts, pH, and host sera) and tested safe for sheep erythrocytes as well as secondary cell lines (RAW 264.7 and HEp-2) with no negative effects on the commensal gut lactobacilli. in vitro, time-kill assays revealed that MBC levels of AgC could eliminate MDR-EAEC infection in 120 min. In G. mellonella larvae, AgC (MBC values) increased survival, decreased MDR-EAEC counts (p < 0.001), had an enhanced immunomodulatory effect, and was tested safe to the host. These findings infer that entrapment enhanced the efficacy of cinnamaldehyde and AgNPs, overcoming their limitations when used individually, indicating AgC as a promising alternative antimicrobial candidate. However, further investigation in appropriate animal models is required to declare its application against MDR pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]