Bacteriophages specific to Shiga toxin-producing Escherichia coli exist in goat feces and associated environments on an organic produce farm in Northern California, USA

Authors: Lennon, Marion; Liao, Yen-Te; Salvador, Alexandra; LAUZON, CAROL - California State University; Wu, Vivian

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2020
Publication Date: 6/11/2020
Citation: Lennon, M., Liao, Y., Salvador, A., Lauzon, C.R., Wu, V.C. 2020. Bacteriophages specific to Shiga toxin-producing Escherichia coli exist in goat feces and associated environments on an organic produce farm in Northern California, USA. PLoS One. 15(6):0234488. https://doi.org/10.1371/journal.pone.0234438.
DOI: https://doi.org/10.1371/journal.pone.0234438

Interpretive Summary: Bacteriophages (or phages) are viruses that infect bacteria and can be isolated from the same environments as the their bacterial hosts. Shiga toxin-producing Escherichia coli (STEC) are pathogenic and can cause human illness through consumption of food contaminated with animal sources, such as undercooked meat and ruminant feces. Phages capable of lysing STEC (STEC-specific phages) have been frequently isolated from cattle-associated environment, such as feedlots, animal waste water, and feces. However, the information regarding the presence and diversity of the phages in goat feces is not thoroughly explored. Here, we isolated STEC-specific phages and their STEC hosts from fecal (goat and cattle) and soil samples from an organic farm for 6 months. We found that goat feces and the surrounding soil consistently contained diverse STEC-specific phages which collectively lysed O157 and most of the top 6 non-O157 STEC strains. No STECs were isolated from goat fecal samples but these samples contained STEC-specific phages throughout every sampling period. On the other hand, one cattle fecal sample, without presence of STEC-specific phage, was found to contain one E. coli strain harboring stx2 gene that did not belong to any of the known top 6 non-O157 serogroups. Additionally, one soil sample contained both E. coli O174 strain and a phage lytic against STEC O145 strains. The lack of isolation of STECs and presence of phages lytic against STECs frequently implicated in foodborne outbreaks could indicate that these phages are pontentially exerting control over the STECs in the environment and reducing their presence.

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) contamination on produce is primarily associated with ruminant feces, which can result in severe foodborne illness. Although numerous STEC-specific bacteriophages (phages) have been isolated from cattle feces, the presence of STEC-specific phages and any correlation with O157 and the top six non-O157 STECs (the top seven STECs) in goat feces have not been thoroughly investigated. The objective of this research was to investigate the presence and diversity of the top seven STECs and STEC-specific phages in ruminant feces and surrounding environments on an organic produce-growing farm. One sample each from three sites (goat feces, cattle feces, and soil) was collected monthly for six months (soil was collected for five months; n=17). Individually, the samples were used to isolate STEC by culture and PCR-based methods and to isolate STEC-specific phages using a cocktail of the top seven STEC strains, followed by purification via plaque assays. The isolated phages were subjected to host range tests, PCR screening for Shiga toxin (stx) genes, and morphology observation. Ten samples (6 goat, 3 soil, and 1 cattle) contained various STEC-specific phages belonging to three families (Myoviridae, Siphoviridae, and Podoviridae), none of which contained stx genes. The most frequently-isolated phages showed lytic activity against STEC O103; several of which exhibited a wide host range lytic against other top seven STECs. All the goat fecal samples contained phages without isolation of STECs. Two STEC strains, E. coli O174 and an untypable E. coli strain, were isolated from one soil and one cattle fecal sample, respectively, and both strains contained stx2 gene but lacked gene eae. Additionally, the soil sample containing the STEC O174 isolate was also positive of the phage lytic against STEC O145 strains. The findings of this study indicate that STEC-specific phages were consistently isolated from goat feces. Additionally, the presence of phages with the wide and complimentary host range against the top seven STECs and the lack of top seven STEC isolation could be indicative of potential environmental biocontrol by the isolated phages.