Gene profile of virulence, antimicrobial resistance and action of enterocins in Campylobacter species isolated from broiler carcasses.

Abstract

Campylobacteriosis is among the most reported zoonoses in the world, caused by species of Campylobacter, this disease is characterized by gastroenteritis in humans. The main species involved is Campylobacter jejuni, followed by Campylobacter coli. Contaminated chicken meat is often identified as an important source of infection related to human cases and Brazil is the largest exporter of chicken meat in the world, which makes the characterization of brazilian isolates crucial for the establishment of control measures. The objective was to evaluate the contamination of chilled and frozen carcasses sold in the Northeast of Brazil, by Campylobacter species, identify virulence genes, evaluate bacterial resistance to antibiotics and verify the antimicrobial action of the Crude Extract Containing Enterocins (CECE) produced by a probiotic strain of Enterococcus faecium. In this study, 12 chilled carcasses and 12 frozen carcasses were collected, sold retail in supermarkets. The following regions of the carcass were sampled: breast skin, wing skin, belly skin, neck skin, gizzard and liver. Samples of chicken carcasses were analyzed following ISO 10272-2 guidelines for the isolation of Campylobacter spp. The isolates were tested by PCR to identify genus, species C. jejuni, C. coli and C. lari and genes cdtA, cdtB, cdtC, sodB, dnaJ, cmeA, cmeB, cmeC. The assessment of susceptibility to antibiotics was carried out using the standard disk diffusion method and the antimicrobial activity of CECE was determined using the Minimum Inhibitory Concentration (MIC), the methodologies followed the recommendations and cutoff points according to EUCAST and CLSI. A total of 376 isolates of Campylobacter spp. were obtained, among these, 26 (7.0%) were positive for C. jejuni and no isolates were detected for C. coli and C. lari. The highest frequency of C. jejuni was obtained in chilled carcasses with 23 isolates (88.5%, p < 0.0001), in frozen carcasses three isolates were obtained (11.5%). The most frequency site of C. jejuni was the chest skin (7/27.0%), followed by skin of the wing (6/23.0%), skin of the cloaca (5/19.0%), gizzard (4/15.0%), skin of the neck (2/8.0%) and liver (2/8.0%), no significant differences were found between the sites sampled. The gene frequency was determined in: cdtA (3/11.5%), cdtB (3/11.5%), cdtC (5/19.0%), sodB (9/34.5%), dnaJ (3/11.5%), cmeA (4/15.0%), cmeB (4/15.0%) and cmeC (4/15.0%). The three efflux pump genes were amplified in four isolates (15.3%) and all tested genes were amplified in three isolates (11.5%). All C. jejuni isolates (26/100.0%) were found to be multiresistant to three or more classes of antimicrobials. The index of multiple resistance to antimicrobial drugs (IRMA) ranged from 0.4 to 1.0 among isolates of C. jejuni. The antimicrobial activity of CECE was able to inhibit at least 98.5% of the growth of all C. jejuni isolates. Therefore, chilled chicken carcasses present a greater risk of contamination than frozen carcasses, for this reason it is necessary to adopt practices that avoid cross-contamination during the preparation of chicken meat, in order to prevent campylobacteriosis. Furthermore, the presence of multiresistant and potentially virulent isolates highlights the need for further investigations to better understand the use of enterocins as alternative methods in the control of Campylobacter.