Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

Abstract

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

Salmonella Prophages, Their Propagation, Host Specificity and Antimicrobial Resistance Gene Transduction

“…Antimicrobial resistance genes can be transduced by a variety of lysogenic phages. The potential of phages for horizontal gene transfer, including antibiotic resistance genes, has only recently been discussed [ 23 ]. Therefore, we isolated DNA from the total lysates of activated prophages and analyzed it by PCR for 14 common antibiotic resistance genes.…”

“…serovars such as Typhimurium are known to harbor functional and truncated prophages in their genome. Some of these prophages can confer phage resistance, while others can interact with phages infecting the cell and fuse into so-called hybrid phages with a different host specificity or lifestyle (virulent or lysogenic), and the transfer of antibiotic resistance genes is also possible [ 23 ].…”

The human microbiome project at ten years - some critical comments and reflections on “our third genome”, the human virome

“…In contrast, viral fraction and viral sequence reads in clinical and environmental samples contain many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes [59] . However, in another report, the viral fractions in three types of food (chicken, fish, and mussels) were identified as sources of ARG-carrying phage particles.…”

Spatial propagation of temperate phages within and among biofilms

“…Temperate phages, by contrast, have two different modes of propagation after infecting a host bacterium: 1) they may proceed with a lytic infection similar to that of virulent phages, killing the host cell in the process of producing a new burst of phage virions for continued horizontal transmission, or 2) they may propagate vertically along with the host by integrating into the bacterial genome to generate a host lysogen that carries the phage's genome along with it as it undergoes normal growth and division (16). Once integrated into a host's genome, temperate phages can introduce new genes that substantially alter cell physiology, including antibiotic resistance, metabolic capacity, and virulence, among many others (17)(18)(19)(20)(21). Additionally, the movement of temperate phages into and out of host genomes frequently mediates horizontal gene transfer; has powerful, ancient, and widespread effects on microbial evolution (22,23); and can drive lysogenic conversion of pathogens that present major challenges to human health.…”