Abstract
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.
Highlights
In recent times, enteroaggregative Escherichia coli (EAEC) has been regarded as an emerging foodborne pathogen, and it has been frequently associated with the epidemic as well as endemic diarrheal episodes (Lima et al, 2018)
E. coli ATCC 25922 was used as the quality control strain for antibiotic susceptibility testing
Lfcin (17–30) evaluated in this study was retrieved from biofilm active AMPs (BaAMPs) (Di Luca et al, 2015), commercially synthesized from Shanghai Science Peptide Biological Technology, China, resuspended in phosphate-buffered saline (PBS; pH 7.40) with a final stock concentration of 10 mg/ml and stored at -20◦C until further use (Supplementary Table S1)
Summary
Enteroaggregative Escherichia coli (EAEC) has been regarded as an emerging foodborne pathogen, and it has been frequently associated with the epidemic as well as endemic diarrheal episodes (Lima et al, 2018). The biofilm formation by EAEC is correlated well with the persistence of infection and recalcitrance to empirical antimicrobial treatment (Lin et al, 2017; Petro et al, 2020). This persistent colonization of EAEC leads to carrier status, enabling antibiotic pressure, which results in frightening levels of multi-drug resistance. Multi-drug-resistant (MDR)-EAEC strains were recovered from food handlers (Oundo et al, 2008), diarrheal children, travelers’ diarrhea (Hebbelstrup Jensen et al, 2018), mangrove estuaries (Ghaderpour et al, 2015), and surface water (Canizalez-Roman et al, 2019)
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