Istituto Zooprofilattico Sperimentale della Sardegna

Mura Elia, Noli Alessia Caterina, Rossi Maria Lucia, Terrosu Giovanni, Fadda Antonio

In May 2011 an Early Warning Response System (EWRS) message from Germany reported a large outbreak of including a significant number of (276 cases) patients with Haemolytic-Uremic Syndrome (HUS) and bloody diarrhoea, which resulted in the death of two people. Similar outbreaks have been reported in France and Turkey . Entero-aggregative and Shiga -producing (STEC) serotype O104:H4 strains (stx2-positive, eae-negative) identified in patients’ samples were considered to be the causal agent. The isolation of STEC O104:H4 strain is rare with only one previous case previously reported (Korea, 2005 ref). The source of the 2011 outbreak was identified as contaminated Egyptian sprouts (ref) caused by an infrequent hybrid strain of Enteroaggregative Escherichia coli (EaggEC) group generated by the acquisition of a phage encoding the stx2 gene. The of this strain lacks the eae gene, encoding the adherence factor intimin, but includes typical virulence cassettes of the Enteroaggregative Escherichia coli (EaggEC) group (attA, aggR, aap, aggA, and aggC) and a gene encoding for the stx of STEC. It is accepted that the reservoir for STEC strains is ruminants, whilst for EaggEC strains transmission is suggested to be via human reservoirs. However, a few studies on the pathogenic role and epidemiological characteristics of hybrid EaggEC STEC/VTEC strains have suggested a possible mechanism of spread via animals. This has underlined the importance to screen foodstuffs for EaggEC, which may represent an emerging public health concern. This research was focused on collecting epidemiological data for the presence of Enteroaggregative Escherichia coli, stx-producing Escherichia coli and stx2a-converting , in ruminants faeces.

During July 2014 faecal samples from 48 ruminants were collected including 24 dairy and 24 dairy sheep, located in the province of Sassari (Sardinia). The samples were examined for the presence of Enteroaggregative Escherichia coli, stx-producing Escherichia coli and stx2a-converting bacteriophage by conventional microbiological methods and by a Real Time PCR. The identification of serotypes was performed following the ISO/TS 13136:2012 for the top five serogroup (O157, O26, O111, O103, O145), while the O104:H4 was screened following the procedure "detection and identification of Verocytotoxin- producing Escherichia coli (VTEC) O104: H4 in by Real Time PCR “ EU-RL-Rev 1 VTEC_Method_04 (EU reference laboratories for E.coli- Department of Veterinary Public Health and Food Safety, Unit of Foodborne Zoonoses, Istituto Superiore di Sanità). About 200 g of sample (taken from individual animals) were transported at refrigeration temperature to the laboratory. 10 g of faeces was suspended in 90 ml of BPW, incubated at 37 °C for 18-24h. After incubation 1 ml of the culture was performed for DNA extraction and real time PCR (stx and eae), than positive samples were tested for serogroup-associated genes. If the samples were positives for one of the serogroup-associated genes a serogroup-enrichment (SSE) was performed in order to facilitate the isolation. For searching the presence of 15 g of faeces in 40 ml of saline solution were placed and the mixture was incubated at 37 °C for 24h and then centrifuged once at 2000 rpm for 10 min followed by a second centrifugation at 9000 rpm for 10 min. Then DNA was extracted and real time PCR was performed for the detection of bacteriophages (in order to ISO/TS 13136:2012).

SHEEP FARMS CATTLE FARMS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 stx1 - + - - + + - - + + - - + + - - + + + + + - + + stx1 - + - + + ------+ - + - - + - + - - - + stx2 - - - + + + - - + + - + + + + + + - + + + - - - stx2 + + - + - - + + + + - - + - - + + + + + + + - + eae - + - - - + + - + - - + + + + + + - + + + + + - eae + - - + + - + + + + - - + - + + + + - + + + + + aggR ------aggR ------aaiC ------aaiC ------stx1 stx1 - - - - - + - + + ------+ ------phage phage stx2 stx2 ------+ + - + ------+ ------phage phage Table 1. Positives genes in sheep farms Table 2. Positives genes in cattle farms

Real time PCR results showed all samples were negative for enteroaggregative Escherichia coli typical genes (aggR and aaiC), whilst 39/48 (81.25%) were positive for verocytotoxin gene (stx1, stx2, eae) (Figure 1). This included 20 separate sheep and 19 separate cattle farms. 7 sheep samples and one cattle, had acquired the stx2a- converting bacteriophage (tables 1 and 2). Serogroups O157, O145, O103 was most represented with O104: H4 found most frequently in sheep farms (tables 3). These preliminary data agree with previous suggestions that animal reservoirs are unfavourable for the development of enteroaggregative and Shiga toxin-producing Escherichia coli. The presence of both VTEC genes and bacteriophages in some samples was significant and suggests further increased sample sizes are required to assess seasonal variations for the presence of these pathogens in the faeces of ruminants.

Figure 1. Positives genes in sheep and cattle farms • Frank C, Milde-Busch A, Werber D. Results of surveillance for infections with Shiga toxin-producing Escherichia coli (STEC) of serotype O104:H4 after thelarge outbreak in Germany, July to December 2011 . Euro Surveill.2014;19(14):pii=20760. Available Cattle Sheep Total online: http://www.eurosurveillance.org/ViewArticle. aspx?ArticleId=207 Serogroups +/n (%) +/n (%) +/n (%) • ISO/TS 13136:2012. Microbiology of food and animal feed- Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens- Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the O157 16/24 (66,6%) 6/24 (25%) 22/48 (45,8%) determination of O157, O111, O26, O103 and O145 serogroups O26 9/24 (37,5%) 6/24 (25%) 15/48 (31,2%) • O104: H4 in food by Real Time PCR “ EU-RL-Rev 1 VTEC_Method_04 (EU reference laboratories for E.coli- Department of O111 1/24 (4,1%) 2/24 (8,3%) 3/48 (6,2%) Veterinary Public Health and Food Safety, Unit of Foodborne Zoonoses, Istituto Superiore di Sanità) O103 14/24 (58,3%) 11/24 (45,8%) 25/48 (52%)

18/24 (75%) 15/24 (62,5%) 33/48 (68,7%) O145 O104 H4 5/24 (20,8%) 9 (37,5%) 14/48 (29%) Table 3. Positives serogroups in sheep and cattle farms