Chicken liver is a potential reservoir of bacteriophages and phage-derived particles containing antibiotic resistance genes

Abstract

Poultry meat production is one of the most important agri-food industries in the world. The selective pressure exerted by widespread prophylactic or therapeutic use of antibiotics in intensive chicken farming favours the development of drug resistance in bacterial populations. Chicken liver, closely connected with the intestinal tract, has been directly involved in food-borne infections and found to be contaminated with pathogenic bacteria, including Campylobacter and Salmonella. In this study, 74 chicken livers, divided into sterile and non-sterile groups, were analysed, not only for microbial indicators but also for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Both bacteria and phages were detected in liver tissues, including those dissected under sterile conditions. The phages were able to infect Escherichia coli and showed a Siphovirus morphology. The chicken livers contained from 10³ to 10⁶ phage particles per g, which carried a range of ARGs (bla_TEM , bla_CTx-M-1 , sul1, qnrA, armA and tetW) detected by qPCR. The presence of phages in chicken liver, mostly infecting E. coli, was confirmed by metagenomic analysis, although this technique was not sufficiently sensitive to identify ARGs. In addition, ARG-carrying phages were detected in chicken faeces by qPCR in a previous study of the group. Comparison of the viromes of faeces and liver showed a strong coincidence of species, which suggests that the phages found in the liver originate in faeces. These findings suggests that phages, like bacteria, can translocate from the gut to the liver, which may therefore constitute a potential reservoir of antibiotic resistance genes.

Fig. 1

Transmission electron micrographs of phages purified from a pool of 10 chicken liver samples from group 2 (sterile inner tissue). Two morphologies were observed: phages with icosahedric capsids and a long non‐contractile tail (A) or a short and curly non‐contractile tail (B). Bar 50 nm.

Fig. 2

Antibiotic resistance genes (ARGs) in the phage DNA fraction of chicken liver samples. Percentage of positive samples for each ARG (bla _TEM, bla _CTX‐M‐1, sul1, armA and tetW) in each matrix. Two types of samples were analysed; group 1: the whole liver (non‐sterile) and group 2: inner tissues of the liver (sterile), recovered by dissection with a sterile scalpel.

Fig. 3

Abundance of each antibiotic resistance gene (ARG) in phage DNA isolated from chicken liver samples of group 1: non‐sterile (47 samples) and group 2: sterile (27 samples). Box plot of the average values (log₁₀ GC g⁻¹ sample) of all ARGs in the positive samples. The cross‐pieces of each box represent (from top to bottom) the maximum, upper‐quartile, median, lower quartile, and minimum values. The diamond (black) shows the mean value. The upper boxes (white) in the box plot include samples showing values within the 75th percentile and lower box samples (grey) show values within the 25th percentile.

Fig. 4

Pie graphs showing the diversity of the viral fraction of chicken liver simples according to Kraken classification of metagenomics data. The graphs compare the distribution and relative abundance of viruses identified in the viromes with regard to the total viral sequences identified. Interactive pie graphs are available upon request.

Fig. 5

Venn diagram showing the number of unique and sharing sequences between the chicken liver virome (PL) and the chicken faecal viromes HP1 or HP2 respectively.