Molecular study to Detect Escherichia coli in Diarrheic Children and its Antibiotic Resistance

Diarrheal diseases can lead to infections and cause morbidity and mortality in children. Diarrheagenic Escherichia coli (DEC) is an etiological agent, which is considered the major causative agent of diarrhea in children in some developing countries. The aims of this work were to estimate Escherichia coli (E. coli ) causing diarrhea in children less than 5 years old, and to detect some biofilm virulence factors and the effect of some antibiotics. For the methodology, a total of 112 specimens were collected from children from two health centers, Al-Zahraa Teaching Hospital and Public Health Laboratory (located in Al-Kut city/ and the Wasit province in Iraq). All specimens were grown on simple and rich media. A total of 43 (38.4%) E. coli isolates were identified using different traditional methods, such as biochemical tests and 16S rRNA sequencing. Polymerase chain reaction (PCR) testing was used to detect some virulence factor genes that play an important role in the pathogenesis of diarrheic E. coli e.g., 16S rRNA, bfpA, and eaeA. In this study, several antibiotics were used to estimate the sensitivity and resistivity of E. coli isolates. A total of 43 isolates were fully identified as E. coli . these samples were used to detect the virulence factor genes, and 31 (72.1%) and 29 (29.4%) isolates carried bfpA and eaeA, respectively. The preponderance of E. coli isolates were completely resistant to penicillin 43 (100%). Additionally, 33 (76.7%) and 27 (62.8%) isolates were resistant to cephalothin and amoxycillin-clavulanic acid, respectively. Furthermore, the isolates of E. coli isolates showed different levels of sensitivity to antibiotics, including polymyxin B 40 (93%), norfloxacin 38 (88.4%), gentamycin 26 (60.4%), and meropenem 22 (51.2%). In conclusion, diarrheagenic E. coli isolates were the prevalent among diarrheic children. Most isolates showed varying results for the presence of virulence factors. In addition, all isolates were resistant to penicillin and sensitive to polymyxin B.

Diarrheal disease is a major global problem, causing more than 2 million deaths annually and primarily affecting children under five years of age.In addition, diarrhea is considered as one of the major disease-contributing factors for infection and death among children and the 2 nd major cause of death globally among different groups of children who are under five years of age, following mortality resulting from respiratory tract infections. 1,2Hebbelstrup et al. 3 mentioned that one of the major bacteria that causes diarrhea is diarrheagenic Escherichia coli (DEC), which causes gastrointestinal infections.In addition, there are six DEC pathotypes, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic (Shiga toxin-producing) E. coli (EHEC STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adhering E. coli (DAEC).ETEC, EPEC, and EAEC primarily targets the gut while DAEC, EHEC-STEC, and EIEC affect the colon.Furthermore, ETEC is a common bacterial agent that causes diarrhea and death in most developing countries.The signs of ETEC are similar to those of other bacterial infections, such as Vibrio cholera, but appear milder.The specific virulence factors resemble the enterotoxins of ETEC from other DEC. 4,5Kaper et al. 5; and Alikhani et al. 6 reported that EPEC is an important pathogenic group of DEC that is linked to diarrhea in children in developing countries.Strains of typical EPEC have an extra-chromosomal DNA (plasmid) named EPEC adherence factor (EAF).It encodes a type 4 pilus called the bundleforming pilus (bfp).Several types of EPEC possess a chromosomal gene called eae gene, which encodes the 'outer membrane protein intimin' and affects the gastrointestinal tract mucosa.In addition, isolates of EHEC-STEC caused bloody and/or non-bloody diarrhea and hemolytic uremic syndrome.Furthermore, the virulence factor key EHEC is Shiga toxin (stx gene), which is recognized as Vero-toxin (Vtx) and consists of two subgroups: stx1 and stx 2 .The most important serotype among the EHEC-STEC strains were shown to be O157:H7. 7AEC causes diarrhea in adults and travelers.This pathway is defined as a novel gut pathogen that causes various disorders worldwide.EAEC adheres to the cells of HEP2, and the mucosa of the gut by fimbria named aggregative adherence fimbria (AAFs), which is encoded by the gene (aggR), which is placed in the essential plasmids of EAEC named pAA. 8,1Jafari et al. 9 clarified that EIEC strains are a patho-type that causes inflammatory invasivE.colitis, and sometimes bacterial dysentery.In most cases, EIEC causes watery diarrhea.
Bonkoungou et al. 10,11 mentioned that these diseases are common in developing countries, particularly in areas with poor sanitation, hygiene, and a limited amount of safe drinking water.In addition, poor health conditions, such as malnutrition, increase the risk of infection with diarrhea.The causative agents of diarrhea, especially in acute cases, involve a wide range of pathogens, including bacteria, viruses, parasites, and fungi.In previous studies, viruses such as Rotavirus and E. coli were the two major causative agents of diarrhea, in addition to other pathogens, such as Campylobacter spp. 12 Zaidi et al. 13 and Vu Nguyen et al. 14 demonstrated that the major causative agents of diarrhea represented by diarrheagenic pathogens include DEC, Rotavirus, bacterial dysentery (Shigella spp.), Salmonella spp., entameba dysentery (Entamoeba histolytica), Bacteroides fragilis (enterotoxigenic), Campylobacter jejuni, parasitic Cryptosporidium spp.Furthermore, DEC is a major causative agent of severe diarrhea and is a major public health. 15EC coli is an important bacterial pathogen that causes diarrhea and death, especially during childhood age. 16In addition, DEC is a remarkable cause of childhood diarrhea and is responsible for 30-40% of acute diarrhea cases in developing countries. 17DEC is a remarkable etiological cause of both sporadic and diarrheal outbreaks worldwide.The most common DEC pathotypes cause increased morbidity and mortality globally. 18esistance to antibiotics has recently emerged as the most common problem worldwide.This has been attributed to random sale of different antibiotics, incentives for healthcare supply to prescribe antibiotics, human expectations, and rising costs due to emergence of antibiotic resistance. 19,20Moreover, Liu et al. 21and Jones et al. 22 stated that the noteworthy benefits of antibiotics in decreasing mortality and morbidity rates were challenged by the emergence of antibiotic-resistant strains in recent years.Apart from the acquisition of virulence genes by E. coli, there are a large number of cases of antibiotic resistance gene possession by the microorganism present in the clinical samples for animal and environment. 23,24Genetic changes associated with phenotypic resistance to several antibiotics, such as tetracycline, gentamycin, quinolones, sulfa-trimethoprim, and β-lactams, have been investigated in DECs. 25 Jafari et al. 26 demonstrated that studies in the Tehran capital (Iran) demonstrated a high frequency of resistance to STEC in populations with EAEC-, STEC-, EPEC-, and ETEC-infected children with diarrhea.In contrast, another study conducted in western Iran reported an increased phenotypic rate of resistance to EHEC in a population of STEC-, EHEC-, and EPEC-infected children. 27Bai et al. 28 and Montealegre et al. 29 stated that phenotype E. coli resistance is highly polymorphic, and this case is attributed to genome flexibility in E. coli, accelerating the emergence of pathogenic types showing individual resistance to the antibiotic phenotype.
The goals of this study were to estimate the incidence E. coli causing diarrhea in children less than five years old, detect some virulence factors, and determine the effect of some antibiotics.

Collect and culture for different specimens
A total of 112 clinical samples were collected from different sites, which included swabs from children's' stools who were hospitalized in two health centers: Al-Zahraa Teaching Hospital and the Public Health Laboratory (located in Al-Kut City, Wasit Province, Iraq).All these specimens were grown on traditional and rich media.Initially, bacteria were cultured on blood agar and nutrient agar, then on selective media, such as MacConkey, eosin methylene blue (EMB), and brain heart infusion broth (BHI B) agar; all samples were incubated 37°C for 18-20 hours.Furthermore, conventional and molecular methods have been used to extract several isolates of bacteria.Microbiological techniques, such as biochemical tests and PCR, were used to detect isolates.Moreover, Mueller-Hinton agar was used to assess antibiotic sensitivity against different E. coli isolates.Strain E-2348 was used as a control for the PCR assay (Center for Vaccine Development, USA).

Extraction protocol for E. coli DNA and PCR technique
The DNA in several isolates of E. coli were diagnosed using the Geneaid Genomic DNA Extraction Kit (U.S.A.).DNA was extracted with commensurate company guidance.Briefly, E. coli specimens were centrifuged, and the pellets were suspended in 0.2 ml buffer for ten min.A total of 0.2 ml of GD buffer was tested for ten min.Subsequently, 0.2 mL absolute of ethanol added to the lysate.A 2 ml tube was used, and then collected and centrifuged using GD columns.A buffer of W1 was added to the GD column and centrifuged.In addition, the wash buffer was tested and eluted from the column.The mixture was added and left for 3 min in order to ensure that pure DNA was obtained.Several virulence genes are required for the detection of DEC, all of which are recognized by PCR testing.

Preparation of reaction master mix for PCR
The PCR master mix was prepared using the GoTag Green Master Mix Kit (Promega, USA), and the master mix was prepared according to the manufacturer's instructions, as summarized in Table 1.

Polymers chain reaction (PCR) thermo cycler program
Additionally, PCR thermocycler conditions for E. coli were achieved using a PCR thermocycler system, which is similar for each gene except for the annealing temperature, as outlined in Table 2.
The specific primers, for example, 16S rRNA, to detect of E. coli isolates eaeA and bfpA were designed by Eurofins MWG Operon (MWG, Germany) (Table 3).The concentration and quality of the DNA specimens were estimated using a NanoDrop.The amplified DNA product was stained with ethidium bromide.

Antibiotics sensitivity test against E. coli
Sensitivity tests were conducted using the disc method, and antibiotics were selected according to the Clinical and Laboratory Standards Institute (CLSI).Mueller-Hinton agar was used for this purpose.Nutrient agar cultured with E. coli (107 CFU/ml) was incubated at 37°C for 24 hours.Discs of different antibiotics were placed on the surface of the agar.The antibiotics used in the current study were as follows: penicillin (PEN) 10U, amoxicillin-clavulanic acid (AMC) 10µg, gentamicin (GEN) 10µg, meropenem (MPM) 10µg norfloxacin (NOR) 10µg, trimethoprim-sulfamethoxazole (SXT) 250µg, cephalothin (INN) 30µg, polymyxin B (PMB) 200U.The results of this method (resistant, intermediate or susceptible) were conducted according to the CLSI system.All E. coli isolates were tested for multidrug resistant (MDR).

Statistical analysis method
All data were subjected to a one-way analysis of variance (ANOVA).We considered a P-value of 0.05 to be statistically significant.

Isolation and identification of E. coli
A total of 43 (38.4%)E. coli, was obtained from stool samples from children.These isolates were identified using conventional and molecular methods, such as culture and microscopic examination, biochemical tests, and PCR, and all results were confirmed using molecular techniques, such as 16S rRNA.All bacterial isolates demonstrated similar results across several  The DNA of all E. coli isolates was extracted, and PCR was performed.All isolates were identified as DEC.

Identification of E. coli by PCR technique
Different E. coli isolates were identified as DEC using 16S rRNA as PCR positive (Fig. 1).
As for virulence factors, in DEC, bfpA was detected in 31 (72.1%)isolates from E. coli as PCR positive, whereas the eaeA gene was detected in 29(67.4%)isolates and considered a PCR positive (Fig. 2 and 3).These genes play a critical role in the pathogenesis of diarrhea caused by E. coli.

DisCUssiON
Most types E. coli are harmless and cause diarrhea.Some strains E. coli (i.e.E. coli O157:H7) can cause dangerous symptoms, such as stomach cramps, vomiting, bloody, and diarrhea.Successful management of any infectious disease requires recognition of the causative agents and treatment of signs manifested by the disease.This study was conducted to detect E. coli isolation rate and virulence factors of pathogenic E. coli that isolated from DEC in the Wasit province (Iraq).Diarrhea is a multifactorial disorder related to a wide range of pathogens, including bacteria, viruses, and parasites. 21,30Most commonly isolated bacteria among DEC is E. coli when applying traditional and molecular methods.The results of the current study are in agreement with those of Begum et al., 31 conducted in Mizoram.In the current study,   the prevalence of DEC was higher than that of the other microorganisms (Table 2).The current study agreed with other studies conducted in Iran/ Tahran and Tanzania by Jafari et al. 26 , and Moyo et al., 32 respectively, who demonstrated that the most common microorganism was DEC (7.9%), which was lower than that reported in other developing countries.In addition, a study by Dias et al. 33 (Brazil and Mexico) observed that EAEC was the primary pathotype DEC, with respective rates of 50%.
Regarding non-DEC and the capacity to cause diarrhea, contagious diseases, which do not first affect the GIT, can cause acute diarrhea.The pathogenesis of this type of diarrhea involves intestinal inflammation, cytokine action, red blood cell (RBC) sequestration, programmed cell death, increased endothelial cell permeability in the GIT microvasculature, and invasion of epithelial cells in the GIT by several agents.Several symptoms, such as fever and diarrhea, occur in patients with respiratory syndrome (SARS), Plasmodium parasites (malaria), and dengue fever.Diarrhea also occurs in patients with acquired lung inflammation when it is suggestive of legionellosis, and those with systemic bacterial infections.Although diarrhea is rare in patients with early borreliosis, the incidence is high in those with other tick-borne infections, such as tick-borne, ehrlichiosis, and others.Unfortunately, it is often not established whether diarrhea is an initial clinical sign and/or whether it progresses during the course of the disorder. 34cently, molecular diagnostic techniques have become common in clinical laboratories.PCR is capable of detecting several pathogens via the amplification of specific genes encoding important virulence factors.If it is difficult to diagnose DEC using traditional laboratory techniques, PCR becomes beneficial in clinical laboratories because of its specificity and sensitivity.In the current study, all isolates produced 43 (100%) as E. coli by PCR method.In contrast, there were 31 (72.1%) and 29 (67.4%)isolates from E. coli contained the bfpA, and eaeA genes, respectively.Furthermore, typical EPEC is the most common cause of watery diarrhea in children, especially in developing countries.The current study is compatible with and nearest to a study carried out on children in Peru (South America) by Contreras et al. 35 , who observed that the most common pathotype in diarrhea was the predominant genes of bfpA (74%) and eaeA (54%).In addition, a study conducted in Yogyakarta/ Indonesia by Harti et al., 36 who observed that the percentage of predominant genes in DEC was espA (85%), bfpA (80%), and eaeA (51%).Another study conducted among young children in South Africa with diarrheal disease, by Galane and Roux, 37 clarified that the PCR results observed 59 (32.6%) isolates of E. coli carried genes (eaeA), and 6(3.3%) possessed bfpA genes, 4 (2.2%)CNF1, and 2 (1.1%) carried Stx 2 genes.These results were different from those of the current study, and this difference between the two studies may be ascribed to the conditions and various geographical areas and/or other related genes or carried on plasmids, among

Table 1 .
Reaction of PCR mixture master mix

Table 3 .
Primers were tested for E. coli in the present work, and selected primer to gene of 16S rRNA * Designed by my self-using the national centre for biotechnology information (NCBI).

Table 4 .
Antibiotic resistivity and susceptibility in different isolates of DEC Antibiotics dosage (µg) Resistant Intermediate Sensitive No*: Number, **: E. coli is MDR.