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

Jess Vergis; Satyaveer Singh Malik; Richa Pathak; Manesh Kumar; Sunitha Ramanjaneya; Nitin Vasantrao Kurkure; Sukhadeo Baliram Barbuddhe; Deepak Bhiwa Rawool

doi:10.3389/fmicb.2020.575917

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

Front Microbiol. 2020 Sep 18:11:575917. doi: 10.3389/fmicb.2020.575917. eCollection 2020.

Authors

Jess Vergis¹, Satyaveer Singh Malik¹, Richa Pathak¹, Manesh Kumar¹, Sunitha Ramanjaneya¹, Nitin Vasantrao Kurkure², Sukhadeo Baliram Barbuddhe³, Deepak Bhiwa Rawool^{1

3}

Affiliations

¹ Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, India.
² Department of Veterinary Pathology, Nagpur Veterinary College, Nagpur, India.
³ ICAR-National Research Centre on Meat, Hyderabad, India.

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.

Keywords: Galleria mellonella; antimicrobial peptide; biofilm; confocal microscopy; enteroaggregative E. coli; lactoferricin (17–30).