Knowledge of the epidemiology of infections associated with E. coli pathotypes is very crucial for health policymakers [16]. Consequently, the results of the present work may assist to epidemiological and therapeutic perspectives. The prevalence of diarrheagenic E. coli strains in this study is similar to some countries such as Colombia (14.4%) and is comparable with the other reports from India (26%), Iran (34%), USA (5.5% and 19%), etc. [17–21].
In our study, the most prevalent pathotype was STEC, the positive strains for stx1 and/or stx2 genes. There was no significant difference between the prevalence of stx1 and stx2 genes, while some previous studies have been reported the predominance of stx1 genes [22, 23]. STEC strains to harbor the stx1 gene are frequently responsible for non-complicated diarrhea to asymptomatic infections, while the stx2 gene is mainly associated with severe cases of diarrhea and HUS [23, 24].
In this research, some STEC strains (less than 10%) carried eae gene, which were identified as EHEC pathotypes. Reports from EHECs in Iran is rare [17, 25], while annually, more than 70,000 cases of EHEC infections transmitted to human by the routes of foodborne, person-to-person, waterborne, animal contact and laboratory-related are recorded in the USA by Centers for Disease Control and Prevention (CDC) [26]. From 2006 to 2010, there were 254 cases of EHEC infections with an average prevalence rate of 0.11 per 100,000 populations in Korea and 20,883 cases with an average prevalence of 3.26 per 100,000 populations [27].
The strains which are positive for the eae gene and negative for stx genes belong to EPEC. Virulence genes on the LEE (locus of enterocyte effacement) pathogenicity island in EPECs cause attaching and effacing lesions on enterocytes. This pathotype had the second highest frequency in this work; our results are comparable with the results reported by Usein et al. [28]. In a systematic review, the presence of EPECs has been estimated from 0.00% to approximately 60% among E. coli isolated from urinary tract infection, diarrheic and healthy cases in Iran [29]. EPEC pathotype is an important cause of mild-to-severe loose/watery stools, especially in children with fecal-oral, Human-to-human, and animal-to-human transmission.
The third prevalent pathotype of this study was ETEC causing moderate-to-severe diarrhea in human. The profiles st/lt and st were the most common toxigenic genotype among ETECs, which agrees with Bhakat et al. (2019) [30]. None of the strains carried the lt gene alone, which is in contrast with the global prevalence of the gene where the presence of lt is higher than st or st/lt profile [31]. It has been reported that positive ETECs for st/lt profile are more often associated with a higher risk of death in young children [22, 32, 33]. The prevalence of ETECs in this study is similar to some countries such as India (13.6%). It is comparable with the other reports from Argentina (18.3%) and Vietnam (2.2%) [34, 35]. During 2016 to 2017, 244 cases of ETEC has been detected from Minnesota [36]; the incidence rate of ETEC in the USA has been estimated as 250 per 100,000 population in the USA within all age groups and in both males and females [37]. In a systematic review that has been performed in Iran, the frequency of ETEC was 16% according to 5669 isolates in 28 publications [29]. Differences in the prevalence rates of E. coli pathotypes found in the present and previous studies may be associated with geographical locations, study methods, public health, and host factors.
Specific virulence genes of pathotypes are mostly on mobile genetic elements such as phages and plasmids as identified previously in STECs and ETECs [38]. So horizontal gene transmissions between various pathotypes lead to combine the virulence factors and emerge hybrid pathotypes, which in some studies named hetero-pathogenic E. coli strains with more potential to cause more severe infections [5]. There is low information about the incidence of hybrid pathotypes in Iran; one EPEC/ETEC strain (positive for intimin and heat-stable ) and one EHEC/ETEC strain (positive for Shiga toxin, intimin and heat-stable toxins) were detected from healthy cattle in 2016 [39]. In our study, we detected some hybrid pathotypes of E. coli for the first time in human in Iran; we found three virulence profiles, including eae/st (EPEC/ETEC), stx1/st (STEC/ETEC), and stx1/eae/st (EHEC/ETEC). Best of our knowledge, there are still no reports of EHEC/ETEC strains in human from other countries.
There is a report about the identification of EPEC/ETEC strains in the USA which is isolated from diarrheic, asymptomatic, and death cases in children [40]. In Finland and Sweden, STEC/ETEC strains were isolated from 2.05% of clinical strains initially characterized as STEC during 15 years [38, 41]. Many researches have revealed a high frequency of STEC/ETEC isolates from ruminants and piglets, showing the importance of animals as the main reservoirs for the hybrid pathotypes [5]. The emergence of multiple virulent pathogens is a new and very critical public health concern. So, characterization of genotypic and phenotypic virulence and antimicrobial resistance of single and hybrid pathotypes is an important and necessary for surveillance, treatment, and control of related infections.
DEC strains can be sources for antibiotic resistance genes [42]. Interestingly, ESBL production was determined phenotypically in approximately thirty-seven percent of our isolates. In a similar study, 60.6% frequency of ESBL positive strains have been stated by Khairy et al. (2020) in Egypt [16]. In view of the genes encoding ESBLs, Khoshvaght et al. (2014) reported a high prevalence of blaTEM and blaCTX−M genes in EAEC isolates (78.9% and 63.1%, respectively) [43]. Ali et al. (2014) found more than 78% of EAEC and none of the EPEC isolates carried ESBL genes [44]. Findings of a study performed by Zhou et al. (2018) showed a very high percentage (93.3%) of ESBL genes among E. coli pathotypes [45] which confirms our findings, especially about blaTEM. The occurrence of resistant hybrid pathotypes of E. coli could be related to horizontal gene transmission among various clones [11]. For example, García et al. (2018) detected a STEC/ETEC isolate from diarrheic pig harboring a plasmid containing multiple resistance genes and multiple virulence genes [46]. Generally, the emergence of antimicrobial resistance might result from excessive consumption of antimicrobials without a doctor's supervision [47]. The unplanned use of antibiotics over the past decades has exposed the population of bacteria in the human’s normal flora to selective pressure in favor of resistant bacterial strains. Today, this phenomenon has led to antibiotic resistance and even multi-drug resistance among bacterial pathogens [48].