Loss of Virulence Genes in Escherichia coli Populations during Manure Storage on a Commercial Swine Farm

Abstract

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in Escherichia coli during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (n ‫؍‬ 392) carried estB, fedA, stx 2e , astA, paa, aida-I, and sepA at lower frequencies than isolates obtained from fresh feces (n ‫؍‬ 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal E. coli population was initially dominated by a single genotype, all isolates of which carried fedA and aida-I. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of E. coli that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by E. coli. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.

Comparison of Selected Nutrients and Bacteria from Common Contiguous Soils Inside and Outside Swine Lagoon Effluent Spray Fields after Long‐Term Use

“…Low but detectable culturable levels of bacterial pathogens found earlier in lagoon effluents (McLaughlin et al, 2009) and levels below cultural detection limits found in the present study are similar to results reported by Peu et al (2006), who concluded that fecal bacteria in swine manure effluents applied to field soils were diluted in soil to levels indistinguishable from those of nonmanured soil. Based on studies of E. coli by others (Dubreuil, 2008; Duriez et al, 2008; Kaper et al, 2004; Khac et al, 2006), it is considered possible, but unlikely, because of differences in pathotypes, serotypes, virulence factors, and attenuated virulence potential during manure storage, that E. coli isolated from soils inside and outside the spray fields include virulent human pathogenic strains. All of these enteric pathogens, however, can cause public health problems linked to environmental contamination from field sources that might involve manure or manure‐contaminated soil (Gerba and Smith, 2005).…”

Evaluating the Pathogenic Potential of Environmental Escherichia coli by Using the Caenorhabditis elegans Infection Model

“…The temperature-sensitive C. elegans strain DH26 [fer-15(b26)II] was routinely grown in the presence of E. coli OP50, a uracil auxotroph, using standard practices (52 2 , stx 1 , estIb, estIa, and ast, was determined through multiplex PCR using primers and conditions previously described (8,54,55 The Bingen phylogenetic grouping of each isolate was determined by multiplex PCR screening for the presence of chuA, yjaA, tspE4.C2, and svg as previously described (57,58), using cell lysates prepared by proteinase K digestion. The chuA positive control, ATCC 35381, was purchased from ATCC.…”

Class 1 Integrons, Selected Virulence Genes, and Antibiotic Resistance in Escherichia coli Isolates from the Minjiang River, Fujian Province, China

“…E. coli strains RO8, B44, F107, P16M, AMR-472, and JG280 were used as positive controls for the detection of the virulence genes (1). Selected PCR products were sequenced as described below , and 2 l of DNA extract (7,40). The programs used for multiplex PCR are described by Duriez et al (7).…”

“…Selected PCR products were sequenced as described below , and 2 l of DNA extract (7,40). The programs used for multiplex PCR are described by Duriez et al (7). Class 1 integrons were detected using specific primers (TGATGGCGACGC ACGAC and TTGGGCAGCAGCGAAGT) developed with the Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA) software program (48).…”