Characterization of endolysin gene of bacteriophages infecting Listeria spp isolated from dairy industry wastewater.

BLANCO FERNANDEZ, MARIA DOLORES; BARRIOS, MELINA ELIZABETH; CAMMARATA, ROBERTINA VIVIANA; TORRES, CAROLINA; MBAYED, VIVIANA ANDREA

Berlin

Workshop; 23rd International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology; 2018

EVBC, KU LEUVEN

Bacteriophages and their endolysins, enzymes that degrade cell wall of bacteria, are emerging as alternative tools to detect and inhibit growth of pathogen bacteria. Listeria monocytogenes is a foodborne pathogen that causes listeriosis: a serious invasive disease that affects both humans and a wide range of animals. Listeria spp is ubiquitous in the dairy farm environment, and could be present in dairy processing plants and wastewater. All Listeria-specific bacteriophages found to date are members of the Caudovirales, of the Siphoviridae or Myoviridae families. Myophages infecting Listeria have been recently classified by the ICTV in the Spounavirinae subfamily, and in the P100virus genus.The aim of this work was to isolate Listeria spp. bacteriophages and their endolysin codifying genes from wastewater of a dairy industry. Wastewater with and without treatment, was sampled during a year and isolation of bacteriophages was performed after an enrichment step using as hosts L. innocua, L. ivanovii and L. monocytogenes serotypes 1/2a, 1/2b and 4b. Bacteriophages infecting L. innocua and L. ivanovii were isolated (n=24) from 3 out of 12 samples. Bacteriophages were purified and host range was performed using spot test and EOP against 5 collection strains and several field isolates of Listeria spp. Two bacteriophages of narrow and broad host range, vB_Lino_VEfB7, and vB_Liva_VAfA18, were selected for further characterization. High titer stocks of bacteriophages were purified by centrifugation with ammonium acetate and morphological information of the purified bacteriophages was obtained by negative staining and transmission electronic microscopy. Their morphology, size and contractile tails indicate that these bacteriophages belong to the Myoviridae family. Bacteriophage genomes were extracted using phenol-chloroform followed by ethanol precipitation and tested by digestion with RNAsa A and DNAse I. RFLP was performed digesting genomes with restriction enzymes HindIII and NcoI. Consistent with the morphological findings, bacteriophages contain dsDNA genomes, but showed different RFLP pattern. A PCR designed to amplify conserved domains of endolysins: PGRP and CwlA, was applied to characterize this gene. Another PCR was designed to amplify the complete endolysin gene, and the complete sequence of this gene was obtained and analyzed. Substitution model selection and a phylogenetic ML tree of the endolysin gene was carried out using IQ-Tree software. The sequence of endolysin gene indicated that the codified enzyme it?s an N-acetyl-muramoyl-L-alanine amidase, related to A511 and P100 species of the recently described P100virus genus. Further evolutionary analysis are needed to evaluate their belonging to this species or their taxonomy within this genus.