Circulation of Shiga Toxin-Producing Escherichia coli Phylogenetic Group B1 Strains Between Calve Stable Manure and Pasture Land With Grazing Heifers

Abstract

Escherichia coli strains carrying Shiga toxins 1 and 2 (stx ₁ and stx ₂), intimin (eae), and hemolysin (ehxA) production genes were found in grass shoot, rhizosphere soil, and stable manure samples from a small-scale cattle farm located at the center of Netherlands, using cultivation-dependent and -independent microbiological detection techniques. Pasture land with grazing heifers in the first year of sampling in 2014 and without grazing cattle in 2015 was physically separated from the stable that housed rose calves during both years. Manure from the stable was applied to pasture via injection into soil once per year in early spring. Among a variety of 35 phylogenetic distinctly related E. coli strains, one large group consisting of 21 closely resembling E. coli O150:H2 (18), O98:H21 (2), and O84:H2 (1) strains, all belonging to phylogenetic group B1 and carrying all screened virulence traits, was found present on grass shoots (10), rhizosphere soil (3), and stable manure (8) in 2014, but not anymore in 2015 when grazing heifers were absent. Presence and absence of these strains, obtained via enrichments, were confirmed via molecular detection using PCR-NALFIA in all ecosystems in both years. We propose that this group of Shiga toxin-producing E. coli phylogenetic group B1 strains was originally introduced via stable manure injection into the pasture. Upon grazing, these potential pathogens proliferated in the intestinal track systems of the heifers resulting in defecation with higher loads of the STEC strain onto the grass cover. The STEC strain was further smeared over the field via the hooves of the heifers resulting in augmentation of the potential pathogen in the pasture in 2014, whereas in 2015, in the absence of heifers, no augmentation occurred and only a more diverse group of potentially mild virulent E. coli phylogenetic group A and B1 strains, indigenous to pasture plants, remained present. Via this model, it was postulated that human pathogens can circulate between plants and farm animals, using the plant as an alternative ecosystem. These data indicate that grazed pasture must be considered as a potential carrier of human pathogenic E. coli strains and possibly also of other pathogens.

Keywords: Escherichia coli; Shiga toxin; Shiga toxin-producing Escherichia coli (STEC); animal production systems; calves; human pathogen; pasture; stable manure.

FIGURE 1

Schematic overview of the surveyed farm near Nijkerk including the stable with 50 calves and the pasture land with 15 heifers (grazing heifers were only present in 2014). The pasture was separated by electric wiring into four sectors (A through D) and a drinking corridor to allow the heifers to drink from a water reservoir near the stream. The heifers were weekly, clockwise, rotated to another sector with freshly grown grass, and at the moment of the first sampling (August 2014), the heifers were located in sector A.

FIGURE 2

Prevalence of Escherichia coli 16S rRNA and virulence genes, as determined by PCR-nucleic acid lateral flow immunoassay (NALFIA), in DNA extracts from grass shoots, rhizosphere soil, and stable manure taken in August 2014 (A) and August 2015 (B) and expressed as fractions of total sample numbers from each ecosystem. Number above bars represents the absolute numbers of positive reactions per target gene for each ecosystem.

FIGURE 3

Number of positive PCR-NALFIA reactions to E. coli 16S rRNA and virulence genes in DNA extracts from grass shoots, rhizosphere soil, and stable manure taken in different sectors of the pasture in August 2014 (A). Numbers on vertical axis represent the number of positive NALFIA reactions for each target gene in grass shoots and rhizosphere soil DNA extracts for each sector (n = 5).

FIGURE 4

Maximum likelihood phylogenetic comparison of 35 STECs from different ecosystems on the Nijkerk cattle farm and 15 E. coli/Escherichia fergusonii strains. The tree was rooted using E. fergusonii (ATCC35469) as outgroup. Phylogenetic relationships between E. coli strains were inferred by making use of 10 concatenated household genes (adk, fumC, glpK, gyrB, icd, lysA, mdh, metG, purA, and recA) with 1,000 bootstrap iterations. Relevant phylogenetic groups (A, B1, B2, D, E, and F) are indicated in the tree. Reference strain abbreviations: HS, O9:H4 strain HS; K12, O16:H48 K12 strain MG1655; O104, O104:H4 strain 2011C-3493; 55989, O104:H4 strain 55989; O103, O103:H2 strain 12009; SE11, O173:H4 strain SE11; O26, O26:H11 strain 11368; O111, O111:H8 strain 11128; IAI1, O8:H9 strain IAI1; SE15, O150:H5 strain SE15; UT189, O18:H7 strain UT189; UMNO26, O17:H18 strain UMN026; O157, O157:H7 Sakai strain; IAI39, O7:H45 strain IAI39; E. fergusonii, E. fergusonii ATCC35469.