Stool samples from 132 children aged 0 to 5 years old with acute diarrheal or not were collected in the city of Koula-Moutou over a two-year period (May 2016-Fevrier 2018). Samples were screened for Enterobacteria and parasites. Among the various presumptive pathogens cryopreserved in the Cellular and Molecular Biology Laboratory (LABMC), 63 strains of E. coli formed the biological material, of which 53 strains were isolated from children with diarrhea and 10 from healthy children (control). The details of the various characteristics and criteria of the patients recruited were previously described [18].

Phenotypic research of presumptive strains of E. coli O157:H7

Phenotypic research for enterohemorrhagic E. coli (EHEC) serotype O157: H7 in diarrheal and normal stool sample was carried out on the agar medium chromIDTM O157:H7. The young colonies of the E. coli strains were subcultured on the same medium and incubated at 37˚C for 18 - 24 h. E. coli O157:H7 colonies gave the characteristic Green color.

Molecular identification and classification of diarrheagenic E. coli (DEC)

All strains of E. coli were analyzed as described in previous studies with some modifications [19] [20] and the summary description is detailed below.

DNA Extraction

Genomic DNA was extracted bacterial colonies as described below. Colonies were resuspended in 200 µl of 1X DNA/RNA reagent (Biolabs New England). The bacterial suspensions were lyzed in 200 µl of 2X lysis buffer (1% SDS, 20 mM Nacl, 20 mM Tris pH 8, 20 mM EDTA) and 40 µl of 100 µg/ml proteinase K solution. Then, the preparations were incubated in a heat block at 56˚C for 1h. The resulting lysates obtained were centrifuged at 12,000 rpm for 10 min. Then, released DNA was precipitated by adding 350 μl of isopropanol and 50 μl of sodium acetate at 3M and centrifuging at 14,000 rpm for 15 min. The DNA containing pellets were resuspended in 70 μL of ultra-pure water and used later for PCR amplification.

Multiplex Polymerase Chain Reaction

E. coli pathogens (EAEC, EIEC, EHEC, EPEC, ETEC and STEC) were identified using primers listed in the Table 1.

PCR was performed in a final volume of 20 μl containing 1X master mix PCR multiplex (Qiagen^® Multiplex PCR Kit), 1X Q-Solution (Qiagen^® Multiplex PCR Kit) and 2 µl of genomic DNA diluted 1000 fold, and 0.3 µM of each primer set. The amplification was carried out in a thermocycler (Bio-RAD, T100TM, USA) under the program described by Vilchez et al. [20]. The E. coli strains carrying both eaeA and stx genes were considered as STEC O157: H7 (Table 1). On the other hand, those carrying only the eaeA gene were considered as atypical EPEC (aEPEC). While those carrying the bfpA gene or both (eaeA and bfpA) were

identified as typical EPEC (tEPEC). The amplicons were separated on a 1.5% (m/v) agarose gel for 2 h at 300 V. The 100 bp ladder was used as a molecular weight marker (100 bp; Qiagen^®, GelPilot^®). The DNA bands were visualized and photographed under UV light following the gel staining with ethidium bromide.

Diversity of β-lactamases produced by characterized diarrheagenic E. coli

The Antibiogram profile was determined by the agar dilution method according to the reference technique of diffusion in agar medium (Mueller-Hinton) against 12 specific antibiotics of the β-lactam family [23] to determine their sensitivity profiles and categorize phenotypically the different β-lactamases likely to be produced by the DEC. The detection of extended-spectrum β-lactamase isolates (ESBL) was not only done by screening on MacConkey agar supplemented with concentrated Cefotaxime at 2 mg/ml; but also, by the double disc synergy test (phenotypic confirmation test for ESBL) according to the standards of the Antibiogram Committee of the French Society of Microbiology v.2.0. Mai 2019.

Statistical analyzes were performed using R software version 3.2.2. The significance threshold set was 5%. Fisher’s exact test compared the prevalence of DEC isolates between the two population categories.

The characterization of the 63 E. coli strains isolated from children with diarrhea and controls was carried out phenotypically by looking for serovar O157: H7 and genotypically by looking for genes of interest. The results of the phenotypic detection of the E. coli O157: H7 strains are presented in Figure 1.

In total, 63.5% (40/63) of the E. coli isolates were phenotypically positive for the serotype O157:H7, of which 61.9% were detected in diarrheic children and 1.6% in healthy ones. In diarrhea cases, out of 53 E. coli strains screened, 39 strains (74%) were presumptive of the O157:H7 phenotype compared to 1 strain (10%) in control cases (see Figure 1).

Of the 63 strains of E. coli, the detection rate of genes of interest for DEC characterization was 68.3% (43/63). The various pathovars thus detected after PCR screening are presented in Table 2.

The results of Table 2 revealed that 67.9% (36/53) of the isolates from diarrheal cases were positive DEC against 10% (1/10) of the isolates for the control

OR: odds ratio; NA: not applicable.

group for mono-detection. In addition, Fisher’s exact test showed a significant difference between the two groups (p = 0.001). Among the different DEC detected in diarrheic children, ETEC and EHEC pathotypes were the most common with a detection rate of 28.3% each. The least representative pathotype were the EPEC and the EAEC with frequencies of 7.5% and 3.8%, respectively. While for the control group, the only type of DEC identified was the EHEC pathotype with a frequency of 10%. On the other hand, no significant difference was observed between the cases and the control group for the type of DEC (p = 0.4). Furthermore, no strain is STEC or producer of the virulence factor of Shigella ipaH (Table 2).

The co-detection of DEC pathogroups was also recorded in the two groups with an overall prevalence of 9.4% for diarrheal cases and 10% for the control group without any significant difference (p = 1.0) (see Table 2) ETEC/EHEC hybrids were detected in 7.5% of cases of diarrhoea and 10.0% for controls with no significant difference (p = 0.5). The detection of EAEC/ETEC hybrids was found only in diarrheic cases with a rate of 1.9%.

The classification of DEC into standard pathovars was carried out by specific research into their virulence determinants, the profile of which is presented in Table 3.

Table 3 analysis revealed a heterogeneity in the virulence factors of the four

tEPEC: EPEC typical; aEPEC: EPEC atypical.

(4) pathotypes within the different E. coli strains. The EHEC pathogen was mainly carrier of the vit2 verotoxin gene (68.8%), followed by the combination of vit1 and vit2 verotoxin genes (18.8%) and finally those carrying the vit1 gene (12.5%). Eighty percent (80.0%) of ETEC were carriers of the thermostable ST toxin genes versus 20.0% for the thermolabile LT toxin genes. Finally, for the EPEC pathotype, only typical EPEC were recorded, 75.0% of which possesses the Bfp determinant gene while 25% the Bfp and Eae determinant genes (see Table 3). Regarding E. coli hybrid pathovars, the association of EA-LT virulence genes (100%) was the most common for EAEC/ETEC, followed by the association of the vit1-ST determinant genes (75.0%) for ETEC/EHEC.

Among the 41 characterized diarrheagenic E. coli strains, a classification of the β-lactamases they produce was performed, the results are presented in Table 4.

The results of Table 4 revealed a diversity of β-lactamases produced by DEC. Thus, it appeared that the EAEC of this study produced the association of ESBL + Case and Carbases enzymes with frequencies of 50% each. The EHEC produce

PHN: high-level penicillinases; PRI: inhibitor resistant penicillinase; Case: cephalosporinase; ESBL: extended spectrum β-lactamase; Carbase: carbapenemase; EAEC: enteroaggregative E. coli; EHEC: enterohemorragicE. coli; EPEC: enteropathogenicE. coli; ETEC: enterotoxinogicE. coli.

mostly PHN (26.7%) and the association ESBL + Case (26.7%). These strains also produced ESBL + Carbase, and Case with frequencies of 18.8% and 6.3%, respectively. All EPEC were producers of Carbases and the ETEC in this study were mostly producers of ESBL (40.0%). The other isolates in this group produced 26.7% of Carbases, 13.3% of each PRI and Case, and 6.7% of ESBL + Case (see Table 4). ETEC and EHEC hybrids produced the enzymes of the PNH, ESBL, Case and Carbase types with the same frequency (25.0%). Whereas, the only EAEC/ETEC hybrid only produced the association ESBL + Case (100.0%).

The characterization of virulence factors not only makes it possible to classify the different pathotypes of the E. coli strains, but also these factors are of particular interest as potential targets in therapeutic and/or vaccine trials [57]. For the ETEC pathotype, the prevalence of the enterotoxin ST gene was highest and there were no isolates combining both enterotoxin types in this study. This profile is comparable to that obtained by Saka et al., in Nigeria [58], from Cabal et al., in Spain [59], and other studies [25] [57]. Although, in the latter ones isolates combining the two types of enterotoxins were found

The low prevalence of verocytotoxic O157 EHEC isolates in developing countries makes epidemiological characterization of circulating strains difficult [43]. However, the verocytoxin vit2 gene and the combination of both types were the most common genetic markers in the EHEC pathogen in this study. This profile is similar to previous studies [20] [57]. In addition, the virulence markers of the EHEC island-O like shiga toxins (stx/vt) marked by a high diversity of variants of this family [7] [32]. Virulence factor stx2/vt2 is most frequently reported in the literature [44] [60] corroborating the results of this study on the most recurring virulence determinant.

EPEC is one of the most frequently isolated DEC pathotypes in developing countries, particularly in children under 2 years of age [46] [47] [61]. Current advances in their characterization demonstrate the emergence of atypical strains (aEPEC) in addition to the so-called typical strains [50] [62]. The EPEC strains detected in this study were all typical EPEC and were significantly higher in children with diarrhea than in controls. Their high prevalence compared to atypical would be the fact that they are still prevalent in the poor regions of sub-Saharan Africa [63] [64] while atypical are predominant in developed countries [65]. Our results are in agreement with those reported by Saka and collaborators in Nigeria [58]. But, different from those found by Zhou et al., in China, Singh et al., in India and Darbandi et al., in Iran who reported aEPEC prevalences of 77.8%; 20.0%, and 39,0%, respectively, [25] [46] [64].

The E. coli O157: H7 serotype is the main agent implicated in hemorrhagic diarrhea, the molecular characteristic is the presence of the genes coding for a shiga-toxin (stx) by these so-called STEC strains and the capacity for attachment and effacement induced by intimin, an adhesion receptor encoded by the eaeA gene [66] [67]. Although 63.5% of strains had phenotypic characteristics, no PCR detection of associated genetic markers coding for the stx1 and stx2 variants associated with the eae gene was recorded in this study. Our results are different from several studies that found high levels of stx genes [44] [68]. However, 28.3% of the strains were carriers of the vit1 and vit2 genes coding for verocytotoxins whose O157 serotype is also one of the E. coli strains producing verocytotoxins known to be harmful to humans [69]. The strong presence of this serotype of DEC suggests that the origin of diarrhea is food. Indeed, an incidence of 801 cases of foodborne E. coli O157 infection in the province of Alberta in Canada was estimated by the public health database [60] and the Centers for Disease Control and Prevention estimated that in the United States, E. coli O157: H7 causes more than 63,000 cases per year, resulting in more than 2100 hospitalizations and 20 deaths [67]. The rural nature of the study city, consumption of local and imported meats, stream water would contribute to the high rate of E. coli O157 recorded.

The expansion of antibiotic resistance, particularly in enteric isolates in developing countries, is of great concern, and an improvement in our knowledge of the different susceptibility profiles in the latter is essential [70] [71]. Literature reports many cases of multiresistant DEC strains [14] [61] [72] [73]. The phenotypic characterization of the possible resistance mechanisms responsible for the multidrug resistance observed in the DEC of this study reveals significant levels of isolates producing carbapenemase type β-lactamases (31.7%), extended spectrum β-lactamases (ESBL) (24.4%) and the ESBL-cephalosporinases association (17.1%). Indeed, previous studies on the analysis of multidrug resistance phenotypes of DEC certainly favor the production of various ESBLs with variable prevalences; however, other β-lactamases are also involved. In Burkina-Faso, a study revealed 38.7% of DEC-ESBL, 6.4% DEC-ESBL + Case and 9.7% DEC-ESBL + carbapenemases [5]. In India, Mandal et al. reported a DEC-ESBL prevalence of 37.6% of [74], while two different studies in Iran reported prevalences of 43.8% [73] and 69.2% [75], comparable to that found in our study. However, the ETEC pathotype was the most common producer of ESBL, a profile similar to other studies [57] [74]. The frequency of carbapenemases recorded in this study, particularly for EPEC, ETEC and EHEC pathotypes, is likely related to the current pattern of E. coli enzyme release in Enterobacteriaceae. In addition, this diversity of β-lactamases could be the consequence of horizontal transfer of resistance and virulence genes involving mobile genetic elements such as resistance plasmids and class 1 integuments [76] [77] which contribute to the increase of new pathogens and their multiresistance.

The data from this study do not allow for clear profiling of the different pathovars of circulating E. coli. In addition, the low detection of DEC in control children, probably correlated with the sample size, does not allow a strict distinction between strains with high clinical importance and those with minor clinical importance.