Prevalence of Genes Encoding Outer Membrane Virulence Factors Among Fecal Escherichia coli Isolates

Objective: Escherichia coli is commensal bacterium of human intestine. The gut is a common pool of E. coli isolates causing urinary tract infections (UTIs). Some of fecal E. coli (FeEC) by the possession of certain virulence factors is able to cause diseases in human and other mammalian models. To evaluate the health threats coordinated with a given fecal source of E. coli strains, we determined the frequency of genes expressing virulence determinants in fecal E. coli isolates collected from human feces in Zabol, southeast of Iran. Methods: Escherichia coli isolates (n = 94) were separated from the feces of patients attending teaching hospitals, and screened for various virulence genes: fimH, his, hlyA, ompT, irp2, iucD, iroN, and cnf1 by using the multiplex polymerase chain reaction (PCR) method. Results: The prevalence of virulence genes was as follows: adhesins (fimH, 98% and iha, 26%), alpha-hemolysins (hlyA, 10%), outer membrane protease (ompT, 67%), aerobactin (iucD, 67%), iron-repressible protein (irp2, 91%) and salmochelin (iroN, 33%) and cytotoxic necrotizing factor 1 (cnf1). According to the diversity of different virulence genes, the examined isolates exhibited 29 different patterns. Conclusion: Our results demonstrated that most of the assessed isolates harbored several virulence factors. Our findings propose possibility of human feces serving as a source for pathogenic organisms, supporting the notion that fecal materials of humans play a role in the epidemiological chain of extra-intestinal pathogenic E. coli. This is the first report of the frequency of virulence factors among E. coli isolates collected from human feces in Iran.

ty-associated islands (PAIs). 8The fecal flora from the hosts provides the most common source for infecting E. coli strain. 9The UTI-causing strains that are commonly called UPEC are obtained by faces, the place of their entrance to the urinary tract via colonization of the vaginal introitus and the periurethral location. 10E. coli strains residing within intestinal tract and promoting UTI typically contain virulence genes necessitated for colonization of the urinary tract.On the other hand, it seems that some of FeEC strains carrying many genes that encode virulence factors and can cause serious disease at diverse extraintestinal sites.2][13] However, there is no information about the frequency of virulence mediators in FeEC isolated from human in Iran.Therefore, this study was conducted to examine the distribution of 8 virulence factors, including type 1 fimbriae (fimH), iron-regulated gene homologue adhesion (iha), alpha-hemolysin (hlyA), outer membrane protease (ompT), aerobactin (iucD), yersiniabactin (irp2), salmochelin receptor (iroN) and cytotoxic necrotizing factor 1 (cnf1) in E. coli strains isolated from human feces in Zabol, southeast of Iran, by using the multiplex polymerase chain reaction (PCR) method.

Bacterial Isolates
The sample size was calculated as described by Charan and Biswas. 14Swabs were collected directly from stool samples of the patients with diarrhea admitted to teaching hospitals in Zabol, Iran during July 2014 through October 2014.Samples were suspended into Cary-Blair transport media (Laboratorios Conda, S.A., Spain) and transported to laboratory on ice where one loop from each sample was streaked directly on MacConkey agar (HiMedia Laboratories) within 4 hours after collection.Plates were incu-bated at 37°C for 18-24 hours, and up to 3 colonies with typical appearance of E. coli were selected and subjected to biochemical tests including oxidase, indole, methyl red, Voges-Proskauer, nitrate reduction, urease production, Simmons' citrate agar (HiMedia Laboratories), and various sugar fermentation. 15,16traction of DNA The bacteria were isolated from 1 mL of the E. coli culture grown for 18 hours at 37°C.The bacterial DNA was extracted by boiling methods. 17Briefly, all E. coli isolates were cultivated overnight (16 hours) in 5 mL Luria-Bertani (LB) broth (HiMedia Laboratories) in a shaking incubator (200 rpm) at 37°C.Two milliliters of bacterial iso lates were then pelleted, suspended in 200 μL of sterile double-distilled water and boiled at 95°C for 10 minutes.The mixture was cooled on ice (5 minutes), and the supernatant was collected following centrifugation (13 000 rpm 5 minutes).After centrifugation, the supernatants were kept as DNA at -20°C until applied for PCR.
Detection of Putative Virulence Genes Using the Multiplex PCR Method Prevalence of putative virulence genes in FeEC isolates was determined by multiplex PCR. 3 Details of primer sequences, target genes and products size are shown in Table 1.Amplification of selected genes was performed by setting a net volume of 25 μL.The reaction contained 2 μL of DNA, 12.5 μL of Taq DNA Polymerase Master Mix Red (amplicon, A/S, Denmark), 1 μL of primers (30 pmol concentration for each) (Pishgam, Iran) and 8.5 30 pmol ddH 2 O.The multiplex PCR was performed considering an initial phase of denaturation (94°C, 5 minutes), followed by 35 cycles consisting of denaturation (94°C for 30 seconds), annealing (59°C for 50 seconds) and extension (72°C for 70 seconds), and followed by a final extension step at 72°C for 5 minutes.Amplification was performed fimH-R ATCAGCAGTACAGCAAACAGGG using a gradient Eppendorf 's Mastercycler ® Pro (Eppendorf, Germany).The multiplex PCR products were separated by agarose gel 2% electrophoresis and visualized under UV-induced fluorescence.A 100 bp DNA ladder (Fermentase) was used as size standard (Figure 1).Amplification identities were confirmed by restriction analysis.
Restriction Analysis Amplified fragments of selected genes were confirmed by restriction analysis.Restriction patterns of 8 sequences were obtained with the Webcutter 2.0, online software (http://rna.lundberg.gu.se/cutter2/).The hlyA, iha, irp2 and iroN gene sequences were restricted with TfiI endonuclease followed ompT, iucD and fimH sequences restricted with AluI endonuclease.MspI endonuclease was chosen for restriction of cnf1 sequence.Restriction conditions were identical in all cases.Each 30 μL reaction mixture contained 1 μL of restriction endonuclease, 8 μL of PCR product, 3 μL of specific endonuclease enzyme buffer and 18 μL of sterilized dH 2 O.After overnight incubation, the restriction products were determined by electrophoresis of the digested DNA in 2% agarose gel.

Discussion
The lower gastrointestinal tract is considered as the richest pool of UTI generating organisms.Some strains can colonize the vagina and urinary tract. 18The variety and heterogeneity of virulence factors, including adhesins, toxins and siderophores emerge to be momentous for E. coli strains and make the development of multiplex PCR method especially significant.In this work, to our knowledge for the first time in Iran, we assessed the prevalence of virulence gene profile in 94 FeEC isolates Understanding the distribution of virulence determinants in FeEC isolates is important to evaluate their relative contribution to the extraintestinal infection.We also assessed multiplex PCR assays to detect virulence factors utilizing a combination of primers previously reported. 3ur findings showed that FimH adhesion was the most prevalent virulence factor detected.This factor occurred in 92 (98%) FeEC isolates as seen in Table 2. Similar results were obtained by previous studies, 18,19 whereas the frequency of the iha gene, as other adhesion, was 26% (24/94).Our laboratory previously showed that the prevalence of iha gene was 29% among UPEC isolates. 20owever, no significant differences were observed in relation to iha gene.According to our knowledge, to date, no reports have been published about the prevalence of iha gene among FeEC isolates.The findings of our study indicated that the presence of the hlyA and cnf1 genes was 10% and 3% respectively.
In an other study, Usein et al 21 demonstrated that the hlyA and cnf1 genes were found in 35% of E. coli bacteria recovered from the fecal flora of healthy adult humans.The results showed the variation geographical distribution of these genes.The prevalence of the cnf1 gene was different in fecal isolates studied by other investigators. 22,23In accordance with our study, Obiet et al 24 also described that the cnf1 gene was expressed in 3.5% of E. coli isolates collected from diarrheic stool samples in South Africa.In an other study, our group demonstrated that cnf1 gene was more frequently detected in UPEC (28%) in comparison with FeEC isolates (3%). 20Particularly, ExPEC expresses a richness of apparently excessive iron obtaining systems, including the salmochelin, yersiniabactin, and aerobactin siderophores.In accordance, we observed in our study a very high prevalence of the iron acquisition genes; irp2 (91%), iucD (67%) and iroN (33%).The irp gene cluster is mapped within the high pathogenicity island (HPI) described primarily in Yersinia spp.and horizontal gene transfer has caused it to be present in intestinal and extra intestinal clinical E. coli strains. 25The iroN gene, that is situated on the iroA gene cluster encodes a receptor which is responsible for iron uptake mediated by the siderophores salmochelins, contributes to the virulence of UPEC.This interaction facilitates transport of the complex into the bacterial cytosol. 26The virulence genes of UPEC bacteria such as iroN, iucD and irp2 have been previously recorded from Iran. 11,12However, according to published data, there is no information on the occurrence of these genes in FeEC isolates.
The distribution of the ompT gene among the studied isolates was also similar to that in the previously reported data. 27OmpT rather seems as a conserved protease executing in metabolism of E. coli derived from secretory proteins.This outer membrane protease contributes to the destruction of several proteins interacting with the outer membrane. 28However, there is no report about the prevalence of ompT gene among FeEC isolates in Iran.
The analysis of the association between the presences of different combinations of virulence genes among FeEC isolates, allowed us to divide the tested isolates into 29 virulence patterns noted FeEC1 to 29.Our results revealed the complexity of the properties of virulence markers in fecal E. coli isolates.The pattern FeEC6 included strains simultaneously posi tive for irp2 + , ompT + , iucD + , iroN + and fimH + (12 isolates).The prevalence of genes coding for the 2 adhesion pathways (type 1 fimbriae and iha) which confers the ability to colonization among 24 isolates were fit with those published by other investigators. 3,29Among FeEC isolates, 89 isolates contain genes encoding an iron acquisition protein (yersiniabactin, aerobactin or salmochelin receptor).The maximum number of detected amplicons in 1 isolate was 6.The main novel finding is the high occurrence of genes of outer membrane virulence proteins among FeEC isolates in Zabol, southeast of Iran.Apart from the high occurrence of virulence genes, various virulence determinants were also revealed in fecal E. coli strains.In our analysis, 74% (70/94) of the bacteria were represented with various virulence genes (4 or more genes).It is comprehensible that the recurrence and prevalence of virulence features of fecal E. coli strains are different in other regions of Iran.Probably, geographical differences, cultural habitants, dietary features, public and hospital health policies, weather climate of each region and even sampling methods may exert high impacts on detection rate of virulence factors of FeEC isolates.Nonetheless, further research on the expression of virulence genes and molecular typing methods covering wider geographical areas in Iran is needed to determine the distribution pattern of virulence determinants and develop effective strategies to treat FeEC -induced diseases.This may constitute a limitation of our study.

Conclusion
The FeEC isolates expressing virulence genes may be regarded as an important organism for extra-intestine infections in Iran.The current work is the first report which identified virulence genes among FeEC isolates in Iran.
The multiplex PCR designed in the present study successfully screened FeEC isolates for various E. coli-related virulence genes.Further studies in other parts of Iran are needed to identify virulent factors and to ascertain the pathophysiology of such infectious agents to consider possible prevention interventions.

Ethical Approval
We obtained informed consent form our participants.

Competing Interests
Authors declare that they have no conflict of interest.

Table 1 .
Primers for the Multiplex PCR Assays

Virulence Factor Target gene Primer Primer Sequences (5'-3') Size of Product(bp)
Electrophoresis of Virulence Selected Genes Among FeEC Isolate Obtained by Multiplex PCR.Abbreviations: FeEC, fecal Escherichia coli; PCR, polymerase chain reaction.Each band is indicated by the names of the virulence genes.Lines 1 and "M" denote 100 bp DNA marker.