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. Keywords: Phytochemical, Docking, Enteroaggregative E. coli, Non-typhoidal Salmonella
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.
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.
Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result ...in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.
•The study describes development of the ddPCR for the detection of Chlamydia psittaci.•The ddPCR was highly sensitive, and LOD, LOQ were 2.4, 17.8 copies of DNA, respectively•The ddPCR could be a screening tool for clinical samples with low shedding of C. psitaci
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•Synthesised Ag/ZnO NCs using methanolic extract of Curcuma longa.•Verified green synthesis of Ag/ZnO NCs by UV–vis spectroscopy and FTIR.•TGA/DTA revealed thermal stability of Ag/ZnO ...NCs.•PXRD, SEM, and TEM confirmed poly-crystalline morphology of Ag/ZnO NCs.•Exhibited MIC (31.25 μM) and MBC (62.50–125 μM) for MDR- EAEC, NTS and MRSA.
This study attempted green synthesis of silver/zinc oxide nanocomposites (Ag/ZnO NCs) using methanolic extract of stem and leaves of Curcuma longa and its antibacterial efficacy against multi-drug-resistant (MDR) pathogens. UV–Vis spectroscopy suggested green synthesis of Ag/ZnO NCs which was verified by Fourier transform infrared spectroscopy, while thermogravimetric analysis with differential thermogravimetric analysis revealed its thermal stability between 400 °C and 700 °C. Powder X-ray diffraction results revealed irregular poly-crystalline morphology of Ag/ZnO NCs with certain extent of agglomeration, which was further confirmed by field emission-scanning electron microscopy and transmission electron microscopy. Further, minimum inhibitory concentration of 31.25 μg/mL and minimum bactericidal concentration ranging from 62.50 to 125 μg/mL were observed against MDR strains of non-typhoidal Salmonella spp., enteroaggregative Escherichia coli and methicillin-resistant Staphylococcus aureus. This study demonstrated facile synthesis of Ag/ZnO NCs with promising antibacterial activity which could further be explored as potential antibiotic alternative.
In wake of antimicrobial resistance (AMR), the present study evaluated the antimicrobial efficacy of chitosan-encapsulated Cecropin-A (1–7)-melittin-cell penetrating peptide (ENC CAMA-CPP) against ...multi-drug-resistant (MDR) Salmonella Enteritidis. The ENC CAMA-CPP synthesized by ionic gelation technique revealed a particle size of 597.5 ± 13.20 nm and zeta potential of 2.80 ± 0.12 mV. The functional groups including encapsulation confirmation and morphology were evident by Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. On HPLC analysis, an encapsulation efficiency of 75.12 ± 1.52% was observed. Further, a pH-dependent sustained release along with a maximum release of 97% was evident at pH ≥ 8.0 up to 120 h. In vitro, ENC CAMA-CPP was found stable against protease enzymes (trypsin and proteinase K) and biological fluids (simulated gastric fluid and simulated intestinal fluid) and was also tested safe for sheep RBCs, mammalian cells, and beneficial lactobacilli. In the in vitro time-kill assay, ENC CAMA-CPP eliminated intracellular MDR S. Enteritidis at 12 h p.i., while in the in vivo Galleria mellonella larvae model, an improved survival rate, reduced bacterial count, with significant (p < 0.001) immunomodulatory effect and minimal cytotoxicity were evident for ENC CAMA-CPP. Overall, ENC CAMA-CPP appears to be a promising therapeutic candidate against MDR S. Enteritidis and warrants further validation in an appropriate animal model.
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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 (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.
•E. coli isolates from broiler chicken farms exhibited alarming MDR profile.•E. coli isolates harbored tetA, blaTEM, blaSHV genes.•Majority of the isolates clustered together suggesting co-sharing of resistance.•PFGE pattern exhibited extremely diverse genotypic profile.•In-depth risk analysis is warranted to ensure prudent use of antimicrobials in poultry.
Listeria
contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting ...analysis-based differentiation of the genus
Listeria
and
Listeria monocytogenes
. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LOD
abs
and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LOD
rel
of the assay was found to be 1%
Listeria
DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the
Listeria
contamination in the food safety contexts.
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.
This study attempted synthesis and characterization of zinc oxide nanoparticles (ZnO NPs) using the aqueous extract of Piper longum catkin and its in vitro antibacterial efficacy against ...multi-drug-resistant strains (MDR) of non- typhoidal Salmonella (NTS) spp. (S. enterica Typhimurium and S. Enteritidis). The green synthesized ZnO NPs exhibited an absorbance peak at 340 nm by UV-Vis spectroscopy, which was confirmed by FTIR analysis. The TGA/DTA revealed a progressive thermal degradation of the ZnO NPs between 250 °C and 400 °C; however, good thermal stability was exhibited for annealing temperatures between 900 °C and 1300 °C. The ZnO NPs exhibited hexagonal wurtzite crystalline structure by PXRD analysis, which was further confirmed by electron microscopy. Microbroth dilution technique exhibited an MIC and MBC values of 125 and 250 µg/mL, respectively. Overall, the study demonstrated a facile, eco-friendly method for the synthesis of ZnO NPs, which could be employed as a potential antimicrobial alternative candidate.
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