Abstract
Composting is a complex biodegradable process that converts organic materials into nutrients to facilitate crop yields, and, if well managed, can render bactericidal effects. Majority of research focused on detection of enteric pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in fecal composts. Recently, attention has been emphasized on bacteriophages, such as STEC-specific bacteriophages, associated with STEC from the fecal-contaminated environment because they are able to sustain adverse environmental condition during composting process. However, little is known regarding the isolation of STEC-specific bacteriophages in non-fecal composts. Thus, the objectives were to isolate and genomically characterize STEC-specific bacteriophages, and to evaluate its association with STEC in non-fecal composts. For bacteriophage isolation, the samples were enriched with non-pathogenic E. coli (3 strains) and STEC (14 strains), respectively. After purification, host range, plaque size, and phage morphology were examined. Furthermore, bacteriophage genomes were subjected to whole-genome sequencing using Illumina MiSeq and genomic analyses. Isolation of top six non-O157 and O157 STEC utilizing culture methods combined with PCR-based confirmation was also conducted. The results showed that various STEC-specific bacteriophages, including vB_EcoM-Ro111lw, vB_EcoM-Ro121lw, vB_EcoS-Ro145lw, and vB_EcoM-Ro157lw, with different but complementary host ranges were isolated. Genomic analysis showed the genome sizes varied from 42kb to 149kb, and most bacteriophages were unclassified at the genus level, except vB_EcoM-Ro111lw as FelixO1-like viruses. Prokka predicted less than 25% of the ORFs coded for known functions, including those essential for DNA replication, bacteriophage structure, and host cell lysis. Moreover, none of the bacteriophages harbored lysogenic genes or virulence genes, such as stx or eae. Additionally, the presence of these lytic bacteriophages was likely attributed to zero isolation of STEC and could also contribute to additional antimicrobial effects in composts, if the composting process was insufficient. Current findings indicate that various STEC-specific bacteriophages were found in the non-fecal composts. In addition, the genomic characterization provides in-depth information to complement the deficiency of biological features regarding lytic cycle of the new bacteriophages. Most importantly, these bacteriophages have great potential to control various serogroups of STEC.
Highlights
In recent years, produce outbreaks associated with contamination of enteric pathogens, such as Shiga toxin-producing Escherichia coli (STEC), have drawn significant attention of health administrations and the public
With regard to phage isolation, the spot assay results showed that both C1 and C2 contained phages lytic against the same serogroups of STEC strains, including O111, O121, O145, and O157, regardless of lysis levels on the plates
The phages against non-pathogenic E. coli strains, including DH5α, ATCC 13706, and ATCC 43888 (E. coli O157:H7 without stx genes), were detected by spot assay in the C2 (Table 1)
Summary
Produce outbreaks associated with contamination of enteric pathogens, such as Shiga toxin-producing Escherichia coli (STEC), have drawn significant attention of health administrations and the public. During 2011, a severe outbreak in Europe was associated with contamination of fenugreek sprouts with E. coli O104:H4. A massive recall occurred in the United States regarding contamination of romaine lettuce with E. coli O157:H7 in 22 states, causing illness in 98 people, 46 hospitalizations, and 10 cases of hemolytic uremic syndrome (CDC, 2018). Produce is often served raw and, is susceptible to any contamination from farm to fork. Contamination of pathogenic STEC strains on produce firstly occurs on the farm mostly derived from the contact with fecal-contaminated sources, such as wild animal, irrigation water and composts (Walters et al, 2011; Cooley et al, 2013)
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