Shiga Toxin-Producing Escherichia coli in Faecal Samples from Wild Ruminants

Simple Summary Wildlife is an important source of infectious diseases, including those caused by Shiga toxin-producing Escherichia coli (STEC). STEC are one of the most frequent bacterial agents associated with outbreaks of foodborne disease. This article analyses STEC in faecal samples from red deer and roe deer. The identified O146:H28, O146:HNM, O103:H7, O103:H21, and O45:HNM serotypes and eae/stx2b, stx1a, stx1NS/stx2b, stx2a, stx2b, and stx2g virulence profiles may be potentially pathogenic to humans. The STEC detected in faecal samples from wildlife poses risks to humans, animals, and agricultural production due to the possibility of direct contact with faeces. In conclusion, the pathogenic potential of STEC should be monitored in the context of the ‘One Health’ approach which links human health with animal and environmental health. Abstract Wildlife can harbour Shiga toxin-producing Escherichia coli (STEC). In the present study, STEC in faecal samples from red deer (n = 106) and roe deer (n = 95) were characterised. All isolates were non-O157 strains. In red deer, STEC were detected in 17.9% (n = 19) of the isolates, and the eae/stx2b virulence profile was detected in two isolates (10.5%). One STEC strain harboured stx1a (5.3%) and eighteen STEC strains harboured stx2 (94.7%). The most prevalent stx2 subtypes were stx2b (n = 12; 66.7%), stx2a (n = 3; 16.7%), and stx2g (n = 2; 11.1%). One isolate could not be subtyped (NS) with the applied primers (5.6%). The most widely identified serotypes were O146:H28 (n = 4; 21%), O146:HNM (n = 2; 10.5%), O103:H7 (n = 1; 5.3%), O103:H21 (n = 1; 5.3%), and O45:HNM (n = 1; 5.3%). In roe deer, STEC were detected in 16.8% (n = 16) of the isolates, and the eae/stx2b virulence profile was detected in one isolate (6.3%). Two STEC strains harboured stx1a (12.5%), one strain harboured stx1NS/stx2b (6.3%), and thirteen strains harboured stx2 (81.3%). The most common subtypes were stx2b (n = 8; 61.5%), stx2g (n = 2; 15.4%), non-typeable subtypes (NS) (n = 2; 15.4%), and stx2a (n = 1; 7.7%). Serotype O146:H28 (n = 5; 31.3%) was identified. The study demonstrated that the zoonotic potential of STEC strains isolated from wildlife faeces should be monitored in the context of the ‘One Health’ approach which links human health with animal and environmental health.


Introduction
Wildlife is an important source of infectious diseases transmitted to humans. It has the potential to harbour foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC), Campylobacter spp., Yersinia enetrocolitica, or Salmonella spp. [1][2][3][4][5][6][7]. Human activities have led to the loss of wildlife habitats and have increased the interactions between wildlife, humans, domestic animals, and livestock. The above has increased the prevalence of zoonotic diseases, and the 'One Health' holistic approach was introduced to sustainably regulate and optimise the health of humans, animals, and the ecosystem by meeting the need for clean and nutritious food, water, and air. 'One Health' is an A total of 201 faecal samples were collected from wild animals including 106 red deer and 95 roe deer between May 2020 and May 2021 in north-eastern Poland (Warmian-Masurian and Podlaskie Voivodeships). Fresh faecal samples were collected in designated feeding locations in the forest. 'Point 0 was the observation of faeces upon arrival at the feeding site. From then on, freshly deposited faeces were collected every 3-4 h, always within 24 h of being passed by the target species at each location. Both hunters and foresters have specialised knowledge and are able to correctly identify both faeces and animal species. The samples were kept in a field cooler and stored at 4 • C until processing (within 24 h). In the laboratory, faecal samples of one g each were ground, combined with 10 mL of buffered peptone water (BPW) (BTL, Łódź, Poland) in aseptic conditions, and incubated overnight at 37 • C, with an aeration rate of 180 rpm. The resulting culture was used to extract DNA and isolate STEC. An amount of 500 µL of each enrichment culture was stored at −80 • C in 30% sterile glycerol.

Extraction of DNA and STEC screening by PCR
DNA was extracted from 1 mL of each BPW (BTL, Łódź, Poland) enrichment culture using a Genomic Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions. All samples were tested for stx 1 , stx 2 , and eae using the procedure recommended by the European Union Reference Laboratory for E. coli (EU-RL VTEC_Method 01 for E. coli) and aggR genes described previously [13,[25][26][27][28][29][30][31]. An amount of 50 µL of BPW (BTL, Łódź, Poland) enriched cultures of the samples with amplified stx 1 , stx 2 , eae or aggR genes were plated on CHROMagar STEC (1381)-a selective medium for the isolation of STEC (GrasoBiotech, Starogard Gdański, Poland), and incubated at 37 • C for approximately 24 h. Mauve colonies were isolated, and the presence of stx 1 , stx 2 , eae, or aggR was confirmed by conventional PCR. After confirmation, each STEC isolate was stored at −80 • C in 30% sterile glycerol.

Statistical Analysis
The Clopper-Pearson 'exact' method based on beta distribution at a significance level of α = 0.05 was used to evaluate the prevalence of STEC strains in red deer and roe deer populations. Statistical comparisons were conducted using EpiTools-free epidemiological calculators [38].

Discussion
In the present study, the prevalence and pathogenic potential of STEC strains isolated from the faeces of 106 red deer and 95 roe deer were examined. Wildlife faeces were analysed in this study because they directly enter the environment, fields, and water. Agricultural products such as vegetables may become contaminated with STEC pathogens through direct or indirect contact with wildlife. STEC strains were isolated from 17.92% of faecal samples collected from red deer and 16.84% of faecal samples collected from roe deer. All STEC strains were serotyped as non-O157 and were aggR-negative. Serotypes O146:H28, O146:HNM, O103:H7, O103:H21, and O45:HNM, and the eae/stx 2b , stx 1a , stx 1NS /stx 2b , stx 2a , stx 2b , and stx 2g virulence profiles were identified. According to the EFSA, ECDC, and the European Union One Health 2020 Zoonoses Report, STEC serogroups (based on the O antigen) O26, O157, O103, O145, O146, O91, O80, and O128 were most commonly identified in humans, whereas stx 2a and stx 2b virulence profiles were reported in patients with HUS, bloody diarrhoea, and hospitalised patients. Virulence profile stx 1a was also identified in patients with bloody diarrhoea and patients requiring hospitalisation [15].
The study demonstrated that red deer and roe deer are potential carriers of non-O-157 STEC isolates that may be pathogenic to humans. This is an important consideration because the red deer population has been increasing steadily in Europe, and wildlife have direct and indirect contact with humans, domestic animals, livestock, water bodies, and the environment [39,40]. The results also confirmed that wildlife, including red deer and roe deer, could play a role as reservoirs and shedders of STEC in the environment [3,[40][41][42][43]. The prevalence and pathogenic potential of STEC strains isolated from rectal swabs from red deer and roe deer were examined in our previous study. Four O157:H7 (4.1%) strains were isolated from red deer and one O157:H7 (0.75%) strain was isolated from roe deer. STEC strains were identified in 21.65% and 24.63% of rectal swabs from red deer and roe deer, respectively [13]. The most dangerous human STEC virulence profile, stx 2a was identified in one STEC strain from roe deer and one strain from red deer [13]. The STEC strains isolated in this study appear to have a lower pathogenic potential than those isolated from rectal swabs in our previous experiment. However, pathogenic potential exists. The environmental spread of STEC should be monitored and controlled, especially since rare stx human subtypes, such as stx 2g , have been detected in human clinical samples in Denmark and Germany [44,45], and the stx 2g virulence profile was associated with the outbreak of HUS in France [46].
The results of studies conducted in different European countries indicate that wildlife such as red deer and roe deer could carry mainly STEC belonging to serogroups other than the top five serogroups (O26, O103, O111, O145, and O157). In Italy, the prevalence of STEC in free-ranging red deer was 19.9%, O146:H28 was the most frequently detected serotype (32.3%), and all isolated strains were non-O157:H7 [42]. In Spain, the prevalence of STEC was 24.7% in red deer and 5% in roe deer, O146 was the most frequently detected serogroup, and all strains were non-O157 [1]. However, in another Spanish study, the prevalence of STEC in faecal samples collected from red deer was 35%, four isolates (4.3%) belonged to O157:H7, and the remaining ones were non-O157 (95.7%) [47]. In Portugal, the prevalence of STEC was 9.5% in red deer and 25% in roe deer; all STEC strains were non-O157, and serogroup O146 was the second (after O27) most frequently identified O-group [2]. In Germany, none of the STEC isolated from faecal samples collected from roe deer belonged to the top-five serogroups [48]. In the current study, two strains isolated from faecal samples collected from red deer belonged to group O103 (one of the top five serogroups) with the virulence profiles stx 1a and stx 2g . These results corroborate the findings of Spanish and Italian authors who found that STEC strains isolated from red deer were mainly eaenegative (89.47%), and stx 2 was the most common stx type (84.21%) [2,42,47]. Nevertheless, there is a need for more accurate and complete data about the pathogenic potential of STEC isolated from wildlife, in particular red deer and roe deer populations. In this study, STEC strains isolated from roe deer were mainly eae-negative (93.75%), and stx 2 was the most common stx type (75%). Further research is needed to determine why STEC strains isolated from faecal samples collected from red deer and roe deer seem to have lower pathogenic potential than STEC strains isolated from rectal swabs taken from red deer and roe deer in our previous study [13]. More information is needed to accurately characterise STEC strains present in the environment.
There is no doubt that wildlife can carry STEC that are dangerous to human life and health. Due to the loss of natural wildlife habitats and rapid population growth, wild animals are increasingly observed in fields, near farms, and in other areas that are used by people and other animals. Humans, livestock, and wildlife belong to the same ecosystem, and the health of each should be equally important. Emergent zoonotic bacterial diseases should be identified and controlled under the 'One Health' approach, which integrates the efforts of physicians, veterinarians, epidemiologists, microbiologists, public health workers, and hunters. To prevent infections, the food chain should be controlled at all levels, from agricultural production, processing, and food preparation to production facilities and domestic kitchens. Hygiene education is indispensable because environmental strains should be monitored to minimise the risk of infection and outbreaks of foodborne diseases.

Conclusions
The majority of STEC strains isolated from faecal samples collected from red deer and roe deer did not belong to the top five serogroups. O146 was the most prevalent O-group. All STEC isolates were aggR-negative, and most of them were eae-negative. stx 2 was the most common stx gene type, and stx 2b was the most common subtype of the stx 2 gene. stx 2a , stx 2b , and stx 2g virulence profiles were identified, and these profiles have been reported in patients with HUS and bloody diarrhoea. Wild ruminants such as red deer and roe deer are reservoirs of potentially pathogenic STEC. These animals can shed STEC in faeces and contaminate agricultural production systems, water, and the environment. According to the 'One Health' concept, naturally occurring STEC strains should be monitored in the environment, agriculture, and water to evaluate the risks to public health. New information about serogroups and virulence profiles is needed to expand our knowledge about the epidemiology and circulation of STEC in the environment. Knowledge and communication are necessary for prevention and control strategies.