The Emergence of Extended-Spectrum β-Lactamase Producing and Hybrid Pathotypes of Escherichia Coli Isolates from Diarrheic Human Cases: A New Public Health Concern in Iran


 Background: Intra-intestinal pathogenic strains of Escherichia coli are responsible for mild to severe gastrointestinal lesions, which are mediated by various virulence factors. In this study, we have focused on a comprehensive set of E. coli pathotypes, including STEC, EPEC, EHEC, ETEC, and some hybrid pathotypes including, STEC/ ETEC, EHEC/ ETEC, and EPEC/ ETEC.Methods: Totally, 467 stool samples were obtained from gastrointestinal patients during four years (2016 to 2020). Four pathotypes of E. coli (EPEC, ETEC, EHEC, STEC) were screened due to six virulence genes, including eae, stx1, stx2, st, and lt using the conventional PCR method. Finally, detected E. coli pathotypes were subjected to determine phenotypic and genotypic β-lactam resistance properties.Result: In this study 59/467 (12.63%) strains belonged four pathotypes including STEC (20/59; 50.8%), EPEC (11/59; 18.6%), ETEC (8/59 13.5%), EHEC (5/59; 8.4%) and three novel hybrid pathotypes including STEC/ETEC (3/59; 5%), EHEC/ETEC (1/59; 1.6%), and EPEC/ETEC (1/59; 1.6%). Totally 23/59 (38.9%) isolates were identified as ESBL-producing. Conclusion: Transmission of virulence and antibiotic resistance genes among E. coli strains lead to the emergence of antimicrobial-resistant hybrid pathogenic strains, which is an important health concern. According to food-born and fecal-oral transmission of these E. coli strains, standard methods should be performed to eliminate the possible contamination of food, equipment, and living environment to manure and feces.


Background
Diarrhea is one of the most common syndromes due to gastrointestinal infections, which could lead to hospitalization and death among children and the elderly worldwide [1,2]. Infectious diarrhea caused by more than one bacterial agent can be responsible for severe clinical symptoms in children [1,2]. Among the bacterial species, Escherichia coli can be considered as one of the most prevalent causes (Sjöling, Sadeghipoorjahromi, Novak, & Tobias, 2015). Diarrheagenic E. coli (DEC) could be classi ed into seven main pathotypes, which include Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enteroaggregative E. coli (EAEC), Diffusely adherent E. coli (DAEC), Shigatoxin producing E. coli (STEC) which include a sub-pathotype called Enterohemorrhagic E. coli (EHEC) and adherent-invasive E. coli (AIEC) [5].
In this work, four pathotypes of DEC, including ETEC, EPEC, STEC, and EHEC, were considered for molecular characterization. ETECs cause watery diarrhea in travelers, especially in children in low and middle-income countries. Genetic diagnostic keys are based on the presence of heat-labile and/or heatstable enterotoxin encoding genes, including lt and st, respectively [6]. EPEC infection in human leads to watery diarrhea in children via virulence factors which encoded by the genes on the chromosomal locus of enterocyte effacement (LEE) pathogenicity island such as eae, which encodes intimin [7]. STEC pathotype is usually responsible for self-limiting mild to severe bloody diarrhea. STECs are genetically detected based on the presence of one or both stx1 and stx2 genes, encoding Shiga toxins. STEC has a sub-pathotype named EHEC, which produces Shiga toxin and intimin simultaneously. The strains of EHEC could cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) [1,7,8].
E. coli strains harbor some virulence genes associated with two or more DEC pathotypes called hybrid pathotypes of E. coli. The combination of virulence factors has been mostly led to more severe intestinal infections. Some examples of hybrid pathotypes that were associated with severe diseases are STEC/ETEC, EAEC/STEC, EPEC/ETEC, ExPEC/STEC, ExPEC/EHEC, ExPEC/EPEC, ExPEC/EAEC, and ExPEC/ETEC [9][10][11]. In 2011, an outbreak of EAEC/STEC occurred in Germany and spread out to Europe and North America; in EAEC/STEC hybrid pathotype, stx and aggR (transcriptional regulator of enteroaggregative E. coli) were the main virulence genetic traits that have a role in diarrhea, bloody diarrhea and HUS [12].
Antibiotic therapy in E. coli infections is commonly used for severe cases. Antimicrobial treatment of these infections faces many problems due to the emergence of antibiotics resistant strains [13]. One of the main mechanisms of antimicrobial resistance in E. coli is assessed against β-lactam antibiotics; this mechanism is performed through the hydrolysis of the antibiotics via β-lactamases encoded by large plasmids harboring many different resistance genes [14]. The most prominent extended-spectrum betalactamase enzymes in E. coli are encoded by the genes belonged to bla TEM , bla SHV , and bla CTX−M types [15]. bla TEM and bla SHV genes commonly encode the beta-lactamase enzymes, which hydrolyze penicillins and rst-generation cephalosporins, while the genes from the bla CTX−M group usually encode the βlactamase enzymes, which hydrolyze third-generation cephalosporin antibiotics [15].
In this study, we have focused on a comprehensive set of positive E. coli strains for virulence genes of STEC, EPEC, EHEC, and ETEC, which isolated from diarrheic human. We subjected these strains to investigate molecular virulence properties and antimicrobial resistance of the pathotypes and their hybrid.

Methods
Fecal sampling, E. coli isolation, and DNA extraction A total of 467 stool samples were obtained from patients with diarrhea since from 2016 to 2020. All samples were cultured on McConkey medium and incubated at 37°C. After 24 hours, one suspected E. coli (smooth, round, and pink) colony was picked up from each plate and studied by biochemical tests (IMViC including indole, Methyl red, Voges-Proskauer, and Citrate tests) for diagnostic con rmation. DNA of con rmed E. coli colonies were extracted by boiling method. Each colony was suspended in 350 µl distilled sterile water and boiled for 15 min at 98-100°C by heating block (Eppendorf, Germany). Then, the boiled bacterial suspensions were centrifuged at 13000 rpm for 1-2 min (Eppendorf, Germany) and the supernatant was stored at -20°C for the next steps.

Discussion
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][18][19][20][21].
In our study, the most prevalent pathotype was STEC, the positive strains for stx1 and/or stx2 genes. There was no signi cant 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 identi ed as EHEC pathotypes. Reports from EHECs in Iran is rare [17,25], while annually, more than 70,000 cases of EHEC 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 pro les 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 pro le [31]. It has been reported that positive ETECs for st/lt pro le 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.
Speci c virulence genes of pathotypes are mostly on mobile genetic elements such as phages and plasmids as identi ed 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 rst time in human in Iran; we found three virulence pro les, 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 identi cation 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)  . 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 ora 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].

Conclusion
Genetic factors associated with virulence and antibiotic resistance are exchanged among E. coli strains. Therefore, the emergence of hybrid pathogenic strains and resistance to antibiotics is an important health concern. This study identi ed several ESBL-producing hybrid pathogenic E. coli strains that could be hazardous for public health. Since the transmission of these strains could be food-born and fecal-oral, so standard methods should be considered to eliminate possible contamination of food, equipment, and living environment to manure and feces.

Declarations Ethics declarations
All experimental protocols were approved by the committee for ethics in biomedical research in Veterinary Faculty of Shahid Bahonar University of Kerman, Iran. Also, all methods were carried out in accordance with relevant guidelines and regulations presented by Iran National Committee for Ethics in Biomedical Research.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are available from the corresponding authors on reasonable request.

Competing of interests
The authors declare that they have no competing interests.

Funding
This research received no speci c grant from any funding agency.
Authors' contributions RG, HA, and DK participated in the study design. ZA, RG, and HA performed sampling and the laboratory tests. ZA, RG, and HA wrote the manuscript. All authors read and approved the nal manuscript.