TITLE PAGE Title: Prevalence and Virulence Gene Profiles of Escherichia coli O157 from Cattle Slaughtered in Buea, Cameroon Authors and affiliations:

Background: Escherichia coli O157 is an emerging foodborne pathogen of great public health concern. It has been associated with bloody diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome in humans. Most human infections have been traced to cattle and the consumption of contaminated cattle products. In order to understand the risk associated with the consumption of cattle products, this study sought to investigate the prevalence and identify virulence genes in E. coli O157 from cattle in Cameroon. Method: A total of 512 rectal samples were obtained and analysed using conventional bacteriological methods (enrichment on modified Tryptone Soy Broth and selective plating on Cefixime-Tellurite Sorbitol Mac-Conkey Agar) for the isolation of E. coli O157. Presumptive E. coli O157 isolates were confirmed serologically using E. COLIPROTM O157 latex agglutination test and molecularly using PCR targeting the rfb gene in the isolates. Characterisation of the confirmed E. coli O157 strains was done by amplification of stx1, stx2, eaeA and hlyA virulence genes using both singleplex and multiplex PCR. Results: E. coli O157 was detected in 56 (10.9%) of the 512 samples examined. The presence of the virulence genes stx2, eaeA and hylA was demonstrated in 96.4% (54/56) of the isolates and stx1 in 40 (71.4%) of the 54. The isolates exhibited three genetic profiles (I-III) with I (stx1, stx2, eaeA and hlyA) being the most prevalent (40/56; 71.4%) while two isolates had none of the virulence genes tested. Conclusion: A proportion of cattle slaughtered in abattoirs in Buea are infected with pathogenic E. coli O157 and could be a potential source of human infections. We recommend proper animal food processing measures and proper hygiene be prescribed and implemented to reduce the risk of beef contamination.


Introduction
Enterohemorrhagic Escherichia coli serotype O157 is a well recognised human pathogen associated with bloody diarrhoea, haemolytic uremic syndrome, and death in both developing and industrialised nations [1]. Human infections with E. coli O157 are often attributed to the consumption of contaminated food or water with cattle at the centre of the transmission cycle.
Cattle serve as the main reservoir from where the bacteria may be disseminated to humans via contaminated products or through the food chain [2,3]. Common sources of human infections include the consumption of infected or contaminated milk, contaminated beef hamburger, vegetables, and drinking water [4] although person-to-person transmission is also possible [5].
Cattle acquire the pathogen via the faecal-oral route through consumption of contaminated feed, water, or by direct contact with the environment or other animals [6]. Infections generally do not result in disease condition and the bacteria typically colonise the lower gastrointestinal tract [3] from where they are shed intermittently in bovine faeces [7]. Shedding is by both adult cattle and weaned calves [8]. E. coli O157 can survive and replicate in cattle dung for more than 20 months [9]. This presents the possibility of the organism to persist in the environment and ensure maintenance and transmission.
Unlike in cattle, E. coli O157 induces injury in humans due to its ability to produce numerous virulence factors, most notably Shiga toxin (Stx), which is one of the most potent toxins reported so far [10]. The Shiga toxin is a phage encoded exotoxin and has two major forms; Shiga toxin 1 (Stx-1) and Shiga toxin 2 (Stx-2) [11] and acts by inhibiting protein synthesis in endothelial and other cells [12]. The major effect is damage to vascular endothelium, leading to thrombotic lesions and disseminated intravascular coagulation. This does not occur in cattle because they lack the toxin receptors in their blood vessels. In addition to toxin production, E. coli O157 produces numerous other putative virulence factors including a protein called intimin, which is responsible for intimate attachment of the bacteria to the intestinal epithelial cells, causing attaching and effacing lesions in the intestinal mucosa and aiding in the attachment and colonisation of the bacteria in the intestinal wall [13]. Intimin is encoded by the chromosomal gene eaeA, which is part of a pathogenicity island termed the locus of enterocyte effacement.
Another factor that may also affect virulence of E. coli O157 is haemolysin (encoded by hlyA gene) which can lyse red blood cells and liberate iron to help support E. coli metabolism [14].
Despite reports of the wide geographic distribution of E. coli O157 in the African continent since its first reported outbreak in 1982 [15], and the role played by cattle in the transmission cycle of the pathogen, there is a paucity of information on the epidemiology of E. coli O157 in Cameroon. This study sought to investigate the prevalence of E. coli O157 in cattle slaughtered in Buea municipality and to determine the virulence profiles. Data generated in this study are necessary to create public awareness as well as inform preventive and control measures.

Sample collection
A total of 512 rectal samples were collected from freshly slaughtered cattle at the two abattoirs Samples were collected once from each cattle. The cattle slaughtered at the abattoirs are brought from all over Cameroon and the meat sold to the inhabitants of the municipality and beyond. All samples collected were transported at 4 o C, within two hours of collection, to the Laboratory for Emerging Infectious Diseases, University of Buea for analyses.

Isolation and identification of presumptive E. coli O157 strains
Each sample was pre-enriched in modified Tryptone Soy Broth (mTSB) [ [18]. PCR products were separated on 1.5% agarose gel stained with SYBR safe DNA Gel Stain (Invitrogen, Thermo Fisher Scientific, USA) and visualised under UV light using a Gel Documentation-XR (BIORAD, Hercules, CA).

Results
A total of 512 faecal samples (one per cattle) were collected from the two abattoirs between    (Table 1). Forty (71.4%) isolates exhibited all the four virulence genes tested (profile I).

Discussion
Food animals are recognised sources of human E. coli O157 infections [19], with domesticated ruminants, especially cattle, established as major natural reservoirs for the pathogen and playing significant roles in the epidemiology [2,3]. The consumption of undercooked meat products from cattle has been often associated with many human infections. Beef constitutes a staple food in Cameroonian cuisines and the consumption of bovine products in various forms such as beef hamburger, milk and fermented yogurt is common on Cameroonian menus [20]. Vegetables, reported as important vehicles for E. coli O157 transmission to humans [21], are cultivated in Cameroon with bovine faeces as manure and irrigated with water sometimes contaminated with bovine faeces [22]. These vegetables are also integral in Cameroonian diet, eaten raw as in salad, coleslaw and beef hamburger as well as in other traditional dishes [23]. Our study which sought to establish the prevalence of E. coli O157 in cattle slaughtered in abattoirs in Buea, Southwest Region of Cameroon confirmed the presence of the highly infectious E. coli O157 pathogen in cattle with a prevalence rate of 10.9%. These cattle are brought into Buea abattoirs from all over Cameroon and are reared mostly by individual households for subsistence using different farming methods mostly inherited as a culture [24].
Our prevalence rate of 10.9% is of great concern for the entire country. Poor sanitary practices in slaughter houses may lead to contamination of carcasses [25] which are eventually used for food [26]. Similar studies around the world have also reported E. coli O157 prevalence in cattle with consistent findings. Luga et al. [27] reported a prevalence rate of 9% in neighbouring Nigeria and Hiko et al., [28] and Bekele et al. [29] respectively reported 8% and 10.2% in Ethiopia.
Callaway et al. [30] reported a prevalence rate of 11.3%, in the United State, Synge et al. [31] and Omisakin et al. [32] reported prevalence rates of 8.6% and 7.5% respectively in the United Kingdom while Hashemi et al. [33] had a prevalence rate of 8.3% in Iran. It is worth noting that, though the prevalence rates in cattle is somewhat similar in developing and developed countries, Cagney et al. [38] showed all 43 E. coli O157 isolates in minced beef and beef burgers from butcher shops and supermarkets in the Republic of Ireland contained the eaeA and hylA genes while 41 of the isolates contained both the stx1 and stx2 genes. Khalid et al. [39] in a study in Egypt, found a combination of the four virulence genes, stx1, stx2, eaeA and hlyA in 46.7% (7/15) of E. coli O157 strains, while Mohammad et al. [40] identified stx2 in 90.91% (10/11) of E. coli O157 isolates in a study that investigated the presence of multiple virulence genes in the pathogen in Tabriz, Iran. Similar findings were reported by Leotta et al. [41] in both New Zealand (89%) and Argentina (91%) where the prevalence of stx2 was found to be much higher than stx1.
The Shiga toxin is the most important toxin associated with the pathogenicity of E. coli O157.
The high prevalence of stx2 (54/56; 96.6%) is of great concern considering that Stx-2 is 1000 times more potent than Stx-1 [10] and is mostly implicated in cases of haemolytic uremic syndrome in humans [42]. The eaeA gene detected encodes the adherence factor intimin, which is required for intimate attachment of E. coli O157 to the host intestinal mucosa [43] and has been identified as an important accessory virulence factor that correlates with disease [44]. The hlyA gene encodes another major virulence factor in E. coli O157 and its detection alongside the other virulence genes in the recovered strains increases the ability of the organisms to cause infection and severe illnesses [45], highlighting the danger posed by the continuous circulation of this pathogen.

Conclusions
Cattle serving as food animals could be carriers of highly virulent E. coli O157 which have potentials to cause severe infections in humans. To the best of our knowledge, this is the first study to delineate the potential of cattle as reservoirs of E. coli O157 in Cameroon. Considering that the circulation of this pathogen in cattle poses an environmental and human risks, there is therefore need to conduct surveillance and understand the epidemiology of this pathogen in Cameroon.