Variability of Escherichia coli O157 strain survival in manure-amended soil in relation to strain origin, virulence profile, and carbon nutrition profile

Abstract

The variation in manure-amended soil survival capability among 18 Escherichia coli O157 strains (8 animal, 1 food, and 9 human isolates) was studied using a single sandy soil sample and a single sample of cattle manure as the inoculum carrier. The virulence profiles of E. coli O157 strains were characterized by detection of virulence determinants (73 genes, 122 probes in duplicate) by using the Identibac E. coli genotyping DNA miniaturized microarray. Metabolic profiling was done by subjecting all strains to the Biolog phenotypic carbon microarray. Survival times (calculated as days needed to reach the detection limit using the Weibull model) ranged from 47 to 266 days (median, 120 days). Survival time was significantly higher for the group of human isolates (median, 211 days; minimum [min.], 71; maximum [max.], 266) compared to the group of animal isolates (median, 70 days; min., 47; max., 249) (P = 0.025). Although clustering of human versus animal strains was observed based on pulsed-field gel electrophoresis (PFGE) patterns, no relation between survival time and the presence of virulence genes was observed. Principal component analysis on the metabolic profiling data revealed distinct clustering of short- and long-surviving strains. The oxidization rate of propionic acid, α-ketobutyric acid, and α-hydroxybutyric acid was significantly higher for the long-surviving strains than for the short-surviving strains. The oxidative capacity of E. coli O157 strains may be regarded as a phenotypic marker for enhanced survival in manure-amended soil. The large variation observed in survival is of importance for risk assessment models.

Fig. 1.

Phylogenetic relation between E. coli O157 PFGE pattern, isolation origin, genetic characteristics, and Weibull parameters b (scale parameter), n (shape parameter), and the TTDL (number of days to reach detection limit as calculated by the Weibull model using the estimated parameter values) for survival in manure-amended soil.

Fig. 2.

Dendrogram based on the Identibac virulence gene microarray results. Only genes that were present in at least one strain have been included. Black boxes indicate genes present. A, animal origin; F, food origin; H, human origin. The survival time is indicated in days. Gene specifics: astA, heat-stable enterotoxin 1; cba, colicin B pore forming; cdtB, cytolethal distending toxin B; celB, endonuclease colicin E2; cma, colicin M (resembles beta-lactam antibiotics); eae, intimin; espF, type III secretion system; espJ, prophage-encoded type III secretion system effector; espP, putative exoprotein precursor; etpD, type II secretion protein; gad, glutamate decarboxylase; hlyA, hemolysin A; iha, adherence protein; iss, increased serum survival; katP, plasmid-encoded catalase peroxidase; nleA, non-LEE-encoded effector A; nleB, non-LEE-encoded effector B; nleC, non-LEE-encoded effector C; stx1A, Shiga toxin 1 A subunit; stx2A, Shiga toxin 2 A subunit; stxA₂, Shiga toxin 2 subunit A; stxB₂, Shiga toxin 2 subunit B; tccP, Tir-cytoskeleton-coupling protein; tir, translocated intimin receptor; toxB, toxin B potential adhesion.

Fig. 3.

Principal component analysis (PCA) ordination diagram of Biolog data for the different E. coli O157 strains used in this study. The length and direction of the lines represent the contribution of the oxidative activity of specific substrates to the first two dimensions (i.e., the explained variance). Only substrates showing a significant different oxidative activity between both groups of short and long survivors are shown. Black squares represent strains surviving less than 200 days, and gray squares more than 200 days. The numbers represent the days of survival; the letters indicate whether the strain is from animal (A), human (H), or food (F) origin.

Fig. 4.

Best-fit relation of survival times of E. coli O157 strains in manure-amended soil (closed circles, animal isolates; open circles, human isolates; open square, food isolate) and normalized oxidative activity on α-ketobutyric acid (A), α-hydroxybutyric acid (B), and propionic acid (C).