Isolation and characterization of enteropathogenic and enterotoxinogenic Escherichia coli from dairy products consumed in Burkina Faso

1 Laboratoire de Biologie Moléculaire, d’Epidémiologie et de Surveillance des Bactéries et Virus Transmissibles par les Aliments (LaBESTA), Centre de Recherches en Sciences Biologiques, Alimentaires et Nutritionnelles (CRSBAN), Ecole Doctorale Sciences et Technologies, 03 BP 7021 Ouagadougou 03, Université Ouaga I Pr Joseph Ki-Zerbo, Burkina Faso. 2 Unité de Bactériologie Expérimentale, Institut Pasteur de Dakar, 36, Avenue Pasteur, BP 220, Dakar, Sénégal. 3 Département Génie Chimique et Biologie Appliquée, Ecole Supérieure Polytechnique, Université Cheikh Anta Diop de Dakar, BP 5085, Dakar, Sénégal.


INTRODUCTION
Food-borne diseases represent a public major health problem, and drink-water, juice, meat, and milk products are usually involved (OMS, 2011).Abdominal cramps, vomiting, diarrhea with/without blood, fever (OMS, 2011) are illnesses caused by foods contaminated.Diarrhea causes mortality to a fifth of all people and a third to children younger than five years old worldwide (OMS, 2014).Diarrheagenic Escherichia coli (DEC) remain the ones mostly associated with endemic and epidemic diarrhea, amongst all the enteropathogenic bacteria worldwide (Nataro and Kaper, 1998).In Burkina Faso, DEC is mainly responsible for diarrhea among infants younger than 5 years often associated with vomiting, fever, and dehydration (Bonkoungou et al., 2013).

Study design and sampling
The study was conducted between October 2011 and June 2015, in ten major cities producing and consuming bovine milk products in Burkina Faso (Figure 1a).Sampling was carried out regularly within three steps of milk production: firstly, 69 farms' milk had been collected in eight cities: "Bobo-Dioulasso" in the Southwest (19), "Dori" in the North (5), "Fada N'Gourma" in the East (12), "Kongoussi" and "Sabcè" in the North Central with respectively 4 and 3 farms, "Koudougou" in the West Central (6), "Léo" in the South (12) and "Ouahigouya" in the North (8).All the farms in an area were connected to dairy transformation units in the same city.Secondly, four yogurts and 13 pasteurized milk products samples were collected from the dairy transformation units associated with the above-cited farms.Thirdly, 436 milk products of consumption from distribution chain were gathered in "Ouagadougou" (Figure 1b) and "Ziniaré".These consisted of 84 sets of milk and 89 curds samples (a traditional production) from open markets, 88 pasteurized milks, 88 yogurts and 87 "dégué" samples from food shop and supermarkets.A total of 125 to 500 ml samples of milk products were gathered and transported at 4°C to the "Laboratoire de Biologie Moléculaire, d'Epidémiologie et de Surveillance des Bactéries et Virus Transmissibles par les Aliments (LaBESTA)/ Université Ouaga I Pr Joseph KI-ZERBO", and examined immediately.

Escherichia coli isolation and identification
The ISO 4832 (ISO, 1991) modified method was used for isolating and identifying E. coli.Twenty-five milliliter samples of milk were homogenized into 225 ml of buffered peptone water (Liofilchem, Italy) and incubated at 37°C.Then, after 24 h of incubation, two loopfuls of enriched broth were streaked into violet red bile lactose (VRBL) agar (Liofilchem, Italy) and ChromoCult coliform agar (Merck, Germany), which were incubated at 44.0 ± 0.1°C for 24 h.Suspicious E. coli colonies appear small, purple with purple cloud and blue at violet respectively on VRBL and ChromoCult coliform agar.Three to five presumptive colonies were carefully chosen and tested for lactose and glucose metabolism, indole, urea, citrate, and fermentative gas production.E. coli colonies were confirmed by API 20E system (BioMerieux, France).

Diarrheagenic Escherichia coli characterization
A boiling process was used to obtain the DNA of each strain.This was carried out by homogenizing two loopfuls of each strain into an Eppendorf Tube comprising 250 µl of sterile water.The mixture was boiled afterwards for 10 min and centrifuged for 10 min.The supernatant was collected and used for the PCR reactions.
A multiplex polymerase chain reaction was used for the detection of five major diarrheagenic E. coli (DEC).This characterization was carried out for the intensifying of 16 virulence genes of DEC with specific primers (Table 1).The virulence genes that follow were categorized according to Antikainen et al. (2009): For Enteropathogenic E. coli (EPEC), the presence of eaeA, escv and/or ent and bfpB.The absence of bfpB indicated atypical EPEC; for Shiga Toxin producing E. coli (STEC), the presence of stx1, and/or stx2 with a possible additional genes as eaeA, escv, ent, and EHEC-hly; *Corresponding author.E-mail: sergebagre@gmail.comTel: (+226) 70 01 37 17.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License for Enteroinvasive E. coli (EIEC), the presence of ipaH, and invE genes; for Enteroaggregative E. coli (EAEC), the presence of virF, and/or aggR, and/or pic, and/or astA genes; for Enterotoxinogenic E. coli (ETEC), the presence of elt, and/or sta, and/or stb genes.

Prevalence of E. coli in dairy products
The results show that 33.33% of dairy products are contaminated by E. coli with 315 strains.High rates were observed in traditional dairy products with 98.55% of farm milk, followed by 34.52% of milk sold in Ouagadougou's open markets, and curds (32.58%).This study also showed that pasteurized milk and "déguè" are contaminated by E. coli respectively at 28.71 and 16.09% (Table 3).

Prevalence of diarrheagenic E. coli
The study revealed that the milk products consumed in study sites are contaminated by some diarrheagenic E. coli with variables rates (Table 4).Atypical enteropathogenic E. coli (presence of eaeA and ent genes) and enterotoxinogenic E. coli (presence of stb gene) was detected with 0.38 and 1.53% of total milk products respectively.Atypical enteropathogenic E. coli (aEPEC) was found in 2.3% of "déguè" while 4.45% of curds, 2.97% of pasteurized milk, and 1.15% of "déguè" were contaminated by enterotoxinogenic E. coli.

Class 1 integrons, and resistance genes
Class 1 integrons were detected in eight out of the 10 pathogenic E. coli isolates (80%).Class 2 or 3 integrons  were not detected.Four class 1 integron-containing EPEC, and ETEC strains contained an identical integron harboring a single cassette, dfrA7, encoding resistance to trimethoprim.No classes 2 and 3 integrons were detected in this study (Table 6).

Prevalence of E. coli in dairy products
Investigation showed that the milk products consumed in Burkina Faso are widely contaminated by E. coli with variable rates.Traditional milk products, such as farm milk (98.55%), milk (34.5%), and curds (32.58%), mostly sold in open markets, are highly contaminated by E. coli.
Comparable outcomes have been reported in Ivory Coast and Burkina Faso with slim differences (Katinan et al., 2012;Bagré et al., 2014).These results could be explained by milking conditions in farms, and handling conditions during selling of milk.In fact, Bagré et al. (2015) showed that a majority of farms in Burkina Faso are mainly traditional with unhygienic practices.In these farms, 43.9% do not clean udders before milking, with a calabash being the main collection utensil.These practices, due to poor hygienic training, could explain the traditional milks' contamination during its production.In addition, in this study, fermented and pasteurized milks are contaminated by E. coli.The pasteurized milks consumed in Burkina Faso are contaminated by E. coli (28.71%).These results are lower than those found in Iran (93.75%) (Nazir et al., 2013).The contamination by E. coli could be also explained by a post-contamination during packaging.In unit, packaging is a manual that could cause contamination by workers.The results have shown that the yogurts, which are consumed in Burkina Faso, are less contaminated.In fact, this low contamination could be explained by the acidity of yogurt.
Investigations have revealed lactic bacteria produce bacteriocins, which inhibit pathogens as E. coli, Listeria innocua and reduce bacterial flora (Khay et al., 2011;Yang et al., 2012); but high contamination of milk products by E. coli before transformation can still contain these bacteria.About "déguè", this contamination could be explained by the supplies used to fermented milk with lumps of millet.Lumps, which are not pasteurized and are often exposed to sun, could bring enteropathogens bacteria particularly for the period of the mixture.

Prevalence of diarrheagenic E. coli
Our study reveals a contamination of milk products by some pathotypes of E. coli.In this study, 522 milk products consumed in Burkina Faso were contaminated characteristically enteropathogenic and enterotoxinogenic E. coli.None of enterohemorrhagic, enteroinvasive and enteroaggregative E. coli was found in this study.Indeed, "déguè" (semi-modern milk product) is contaminated by atypical enteropathogenic E. coli (aEPEC) with the presence of eaeA and ent genes.A number of studies in some countries revealed that milk products are contaminated by atypical EPEC.For example, several authors noted that milk are contaminated at 1.2 to 1.6% in Ivory Coast (Dadie et al., 2010); 1.56% and 8.25% in Iran (Rahimi et al., 2012;Mohammadi and Abiri, 2013), and 7.03% in Saudi Arabia (Al-Zogibi et al., 2015).Correspondingly, pasteurized milks are contaminated at 22.1 and 28.12% in Brazil (Da Silva et al., 2001) and India (Nazir et al., 2013); milk cheeses in Brazil (2, 4, and 6% respectively by EPEC O125, O111, and O55), and in Iran (19.48% with the serotype O127) (Najand and Ghanbarpour, 2006;Paneto et al., 2007).About these pathovars in "déguè", none of the studies was carried out in Africa.The contamination of milk products consumed in Burkina Faso may constitute a public health concern particularly for children younger than five years.Studies on diarrhea etiologies in Burkina Faso revealed that atypical EPEC are one of the most typical bacterial causes (Nitiema et al., 2011;Bonkoungou et al., 2013;Dembélé et al., 2015).In addition, Bonkoungou et al. (2013) reported that aEPEC is more predominant than classical EPEC in diarrhea infections from Burkina Faso.
In recent times, studies displayed lower prevalence of EPEC in children younger than five years in Burkina Faso (4%) (Bonkoungou et al., 2013) and Senegal (1.16%) (Sambe-Ba et al., 2013).Among DEC found, enterotoxinogenic E. coli (ETEC) became predominant in traditional (curd) and semi-modern ("déguè", and pasteurized milk) milk products.Studies in Germany (Franke et al., 1984), India (Nazir et al., 2013) and Iran (Bonyadian et al., 2014) revealed that milk products (3.2%), pasteurized milk (3.13%), and cheeses (1.66%) are contaminated by ETEC.It gives the impression that ETEC is associated with travelers' and infantile diarrhea (Nataro and Kaper, 1998).Enterotoxinogenic E. coli can create heat-stable toxin (ST) and heat-labile toxin (LT), which are responsible to profuse water diarrhea and others symptoms such as fever, vomiting, abdominal cramps.In this study, heat-stable toxin (stb) gene was detected in all strains.In Burkina Faso, ETEC is responsible for infantile watery diarrhea, often associated with dehydration.The consumption of water, foods, unpasteurized milk, raw juice, fruits, vegetables and unheated meals are commonly implicated with ETEC infection (CDC, 2005).

Antimicrobial resistance of diarrheagenic E. coli
Antibiogram patterns revealed multidrug resistance of enteropathogenic and enterotoxinogenic E. coli.A small number of studies have been carried out on pathogenic E. coli isolated from a number of dairy products in the world.Resistance of diarrheagenic E. coli to nalidixic acid is comparable to that observed with enterotoxinogenic E. coli (ETEC) in Brazil (40%) (Paneto et al., 2007), and STEC in Nigeria (20%) isolated from milk products (Ivbade et al., 2014).However, the tetracycline resistance is lower than that found in Nigeria (90%) (Ivbade et al., 2014) and Greece (100%) (Solomakos et al., 2009), and higher than that found in Brazil (31%) (Paneto et al., 2007) concerning STEC and ETEC strains.Resistances to norfloxacin and trimethoprim-sulfamethoxazole were observed.Higher results were observed mainly in Nigeria (20%) (Ivbade et al., 2014) and Greece (100%) (Solomakos et al., 2009) for the trimethoprimsulfamethoxazole on E. coli producing Shiga toxin.Additionally, resistances to penicillins (amoxicillin, ticarcillin, and piperacillin), aminoglycosides (tobramycin), sulfonamides and others fluoroquinolones such as ofloxacin were observed.Diarrheagenic E. coli isolated from curds, pasteurized milks and "déguè" resist five antibiotics families, such as tetracycline, penicillin, aminoglycoside, sulfonamide, and fluoroquinolone.Such resistance could be clarified by the presence of genes encoding resistance to these antibiotics.Integrons characterization revealed mostly the presence of dfrA7 genes encoding resistance to trimethoprim.This classification displays resistances to other antibiotics may be encoded by other mechanisms or unwanted genes in this study.Studies carried out on E. coli producing shiga toxin in Egypt showed 3' conserved regions of integrons contains qnrB, qnrS, and floR genes encoding resistance to quinolones (Ahmed and Shimamoto, 2015).In this study, 80% (8/10) of diarrheagenic E. coli harboured class 1 integrons, with dfrA7 gene encoding trimethoprim resistance.Previous studies carried out in China on E. coli isolated from dairy products revealed the presence of dfrA17 and dfrA1 genes encoding trimethoprim resistance (Zhao et al., 2014).In addition, resistance to antibiotics belonging to other families of tetracycline, penicillin, aminoglycoside and quinolone could be due to a selection of resistant strains in dairy products by antibiotic residues.Our earlier data (Bagré et al., 2015) on the same dairy products revealed antibiotics residues belonging to beta-lactam and/or sulfonamides and/or tetracycline and aminoglycosides and/or quinolones and/or macrolides in several magnitudes.For that reason, antibiotic residues could exert selection pressure on pathogenic strains in these dairy products.This study show that curds, pasteurized milk and "dégué" consumed in Burkina Faso are lowly contaminated by enteropathogenic and enterotoxinogenic E. coli.In addition, the results of the Integrons showed that resistance is carried out by plamsids, with risks of a transmission of inter/intra pathogenic species.These mechanisms could help to understand the genetic materials of the DEC resistance isolated from dairy products.Nevertheless, the risk appears low in prevalence terms; special attention should be giving to the dairy products process.Training and awareness should be done with dairy products transformers and farmers, with the view of protecting the health of consumers and avoid the emergence of resistant pathogens.

Figure 1 .
Figure 1.(a): Map of Burkina Faso with sampling sites in the nine (9) majors' cities producing and/or consuming of milk products.(b): Sampling sites in Ouagadougou, Burkina Faso.

Table 3 .
Prevalence of E. coli in milk products consumed in Burkina.

Table 5 .
Resistance of diarrheagenic Escherichia coli to antibiotics used.