Molecular detection of lactose fermenting enteroinvasive Escherichia coli from patients with diarrhea in Tehran-Iran.

BACKGROUND AND OBJECTIVES: Enteroinvasive Escherichia coli (EIEC) is one the cause of acute diarrhea and bacillary dysentery in developing countries. Routine diagnostic microbiology tests are not capable to distinguish EIEC from other pathogenic or non-pathogenic E. coli. PCR, targeting ipaH, virF, virB and other virulence genes, is a diagnostic method for detecting E. coli pathotypes. Using PCR, we identified EIEC by PCR targeting ipaH and virF genes among E.coli isolates from patients with diarrhea at the selected hospitals in Tehran.
MATERIALS AND METHODS: Isolates of E. coli were cultured from 140 specimens of patients with diarrhea using culture and IMViC test. DNA was extracted using commercial kits and and tested for uidA, ipaH and virF genes by PCR.
RESULTS: Totally, 140 E. coli isolates were confirmed by IMViC tests and PCR targeting uidA gene. Of 140 E. coli isolates, 5 (3.6%) were positive for the ipaH gene, 4 (2.9%) contained virF gene and 4 (2.9%) were positive for both ipaH and virF genes.
CONCLUSION: These results indicated that EIEC is a considerable acute diarrheagenic pathogen in adults and infants. Moreover, virF gene is suggested for evaluation of invasiveness of EIEC.

INTRODUCTION

Enteroinvasive Escherichia coli (EIEC) is one of the intestinal pathotypes of E.coli, that cause diarrhea, very similar to Shigellosis (1, 2). This organism is capable to invade and penetrate to the epithelium of the colon leading to dysentery that is an important cause of morbidity and mortality among children in developing countries (3, 4). Dysentery can be characterized with abdominal pain, fever and diarrhea, sometimes vomiting, and the stool contains blood and leukocyte. In addition EIEC might cause an invasive inflammatory colitis, small bowel syndrome and watery diarrhea that is not distinguishable from infections by other E. coli pathotypes (5).

Pathogenesis and especially the ability to invade the epithelium of the colon is dependant on a 220kb plasmid, called pInv, which carries the genes for a type III secretion system that is used as the virulence factor (6, 7).

There are no biochemical tests for distinguishing of EIEC from other E. coli pathotypes and routine laboratory tests can not discriminate E. coli pathotypes from other non-infectious E. coli and very few biochemical tests can distinguish EIEC from Shigella. Therefore, in many cases EIEC pathotype is missreported as Shigella (8). Regarding to remarkable EIEC acute diarrhea and poor isolation tests, molecular methods like PCR are more applicable for detection of this pathotype. Since detection of EIEC leads to appropriate therapeutic strategies.

The PCR of virulence genes has been used for detection of EIEC and Shigella, which all are located in pInv plasmid. The invasion associated pathogen antigen or ipaH is present both on the chromosome and the invasion plasmid of Shigella and EIEC strains (9, 10). The virF is a member of the AraC family gene, which is a transcription activator and their action is necessary for virulence regulon (11). The uidA gene is a housekeeping gene that encode beta D-glucuronidase in E. coli. This gene can be used for confirmation of E. coli isolates and differentiation of E. coli from non-E. coli isolates by PCR (12).

Considering the high prevalence of gastrointestinal disorders, including diarrhea in populations of developing countries, appropriate detection and treatment of diarrhea has high value of importance. However, there is a very limited study concerning E. coli pathotypes in these regions. The aim of this study was to study the EIEC distribution in patients with diarrhea referring to the selected governmental hospitals in Tehran during 2013 to 2014 using molecular techniques.

MATERIALS AND METHODS

Studied population and sampling.

In this descriptive study a total of 140 patients with diarrhea were investigated by culture. Diarrhea was defined as three or more watery, loose or mucoidal stool in one day in the current study. Prior to sampling, the medical information was collected from each patient. The stool samples were collected from all age groups between April 2013 to September 2013 from selected hospitals of Tehran considering standard protocols for microbial analysis of stool (13). Diarrhea samples were evaluated for PMN, RBC and microscopic situation.

Bacterial culture and identification.

Diarrhea samples initialy were cultured on MaCconkey and Hekton enteric agar (Merck, Germany) and incubated for 24 hours at 37 °C. The lactose fermented colonies were tested by biochemical routine tests including citrate, Methyl Red, Voges Proskauer, indole production, and lysine decarboxylasion for identification of E. coli. All E. coli isolates were saved at −20 in Louria Bertani broth (Merck, Germany) with 20% glycerol for molecular procedures.

Nucleic acid extraction.

DNA was extracted with a commercial genomic DNA extraction kit (Bioneer, South Korea) according to the manufacturer's recommendations. Total DNA was extracted from 0.5 ml of Luria Bertani broth culture grown overnight for all the bacterial isolates. The concentration of DNA samples was measured as micrograms per milliliter based on A260 on A280 values by the nanodrop system (Thermo, USA).

PCR assays.

All E. coli isolates were evaluated for the presence of the uidA, ipaH and virF genes by PCR assay using specific primers (Table 1).

Table 1.

The sequences of primers and sizes of PCR produtcs of the oligonucleotide primers to amplify specific, fragments of the uidA, ipaH and virF genes.

Primer	Sequence (5′–3′)	Product size bp	Reference
uidA	5-GCGTCTGTTGACTGGCAGGTGGTGG-35- GTTGCCCGCTTCGAAACCAATGCCT-3	503 bp	(14)
ipaH	5-CTCGGCACGTTTTAATAGTCTGG-35-GTGGAGAGCTGAAGTTTCTCTGC-3	933 bp	(14)
virF	5-AGCTCAGGCAATGAAACTTTGAC-35-TGGGCTTGATATTCCGATAAGTC-3	618 bp	(14)

PCR amplification was performed in a 25μL reaction mixture containing 200 ng of DNA template (1μL), 12μL ready to use Mastermix (Fermentas, Germany), 10 μL of distilled water and 1μL of 20 pmols forward and reverses primers. DNA amplification was carried out with a thermal cycler (Eppendorf, Germany) with the following thermal cycling program: initial denaturation at 95°C for 5 min followed by 30 cycles of amplification (denaturation at 95°C for 30 sec, annealing for 30 sec at 67°C for uidA, 58°C for virF and 61°C for ipaH and extension at 72°C for 30sec) ending with a final extension at 72°C for 5 min. The E. coli K12 was used as positive control for uidA and Shigella flexnery ATCC 12122 was used as positive PCR control for ipaH and virF. PCR products were visualized after electrophoresis on 1.5% agarose gel stained with ethidium bromide and the product size estimated using a 50bp DNA ladder (Fermentas, Germany).

Data analysis.

Chi squared and Fisher's exact tests were used for analysis of categorical data and ANOVA was used for containing data. Analyses were done using Sigma Stat for Windows V2.03 (SPSS, Chicago, IL, USA). A P value less than 0.05 was accepted as statistically significant.

RESULTS

A total of 140 (100%) E. coli isolates were detected from 140 patients with diarrhea troughout three general hospitals. The patients age range was between 0 to 66 years and mean age of the patients was 32 years. Among 140 patients, 31(22.1%) had abdominal pain and 13 (12.1%) had a fever.

Among diarrhea samples, PMNs and RBCs were seen in 38 (27.1%) and 14 (10%) of samples, respectively (Table 2).

Table 2.

Microscopic situations of diarrheal samples and clinical symptoms in patients infected with EIEC and non-EIEC isolates.

	Sex		Age	WBC	RBC	Abdominal pain	Fever
	Female	Male
EIEC n=5	2(40%)	3(60%)	44±22	23±18	28± 20	4(80%)	4(80%)
Non- EIEC E. coli n=135	51(38%)	84(62%)	32±34	4±1	6±2	27(20%)	9(6.6%)
P value	0.581		0.544	0.021	0.003	0.009	0.001

As we expected, all E.coli isolates were positive for uidA. Of 140 E. coli isolates, 5 (3.6%) were positive for ipaH, while virF was detected in 4 ipaH positive isolates. On the other hand, 4 (2.9%) isolates were positive for both of ipaH and virF genes.

All patients infected with ipaH+/virF+ E. coli isolates, had abdominal pain and fever while one patient infected with ipaH+/virF− showed no symptoms (Table 3).

Table 3.

Microscopic situations of diarrheal samples and clinical symptoms in patients infected with virF+ and virF− EIEC isolates.

EIEC	PMN	RBC	Abdominal pain		Fever
			Yes	No	Yes	No
ipaH+, virF+ n=4	22±16	28±18	4	0	4	0
ipaH+, virF−n=1	4±1	2±1	0	1	0	1
P value	0.007	0.001	0.002		0.001

DISCUSSION

EIEC strains are known as one of clinically important E.coli pathotypes that cause watery diarrhea to dysentery (2, 3). EIEC are closely related to Shigella spp in terms of pathogenicity (1, 3). Given the importance of diarrhea caused by the EIEC, and the inability of conventional cultivation methods in the distinguishing pathogenic E. coli from non-pathogenics, the EIEC is usually ignored and most of the time it is missreported as Shigella spp. Thereby molecular methods seem to be an ideal option for this purpose. Due ipaH wide distribution in EIEC chromosome and plasmid, often was used for detecting of this pathotype (4).

A few studies have been done on EIEC in Iran. In the present study, ipaH gene was used for targeting EIEC in E. coli isolates from diarrheal samples. Our results showed 3.6% of isolated E. coli form diarrheal samples were EIEC.

Recently Alizadeh et al (15) in a study in the south of Iran showed EIEC in 1.4% of total E. coli isolates from <5 years children with diarrhea by PCR on ipaH. Akbari et al (201) in a similar study was evaluated EIEC in under 5 years, infants with acute diarrhea in Tehran (16). They were reported EIEC in 2.7% of samples by amplifying ipaH gene.

Mostafa et al (17) studied diarrheagenic E. coli pathotypes in Libyan children with diarrhea in 2012 using ipaH gene but did not detected EIEC in any sample. Hein et al (2009) were found only 0.8% of isolates as EIEC <5 years Vietnamese children (18).

Hegde et al (19) detected EIEC from diarrhea samples among Indian children <5 years by amplifying iaL gene. They indicate EIEC were present in 3.5% of total E. coli isolates.

ipaH gene was also used for detection of EIEC for the same purpose in Ecuador. EIEC were found in 3.2% of isolates and diarrhea samples among <5 years children in that country which is comparable to the results of our study.

Considering our result and data extracted from literature, the frequency of EIEC varies from 0 to 3.5% (15, 20). This prevalence shows that EIEC in the last years and in different geographic areas are generally considered to be one of the causes of acute diarrhea. Because of differences in time, place and the study conditions it seems to be reasonable for different reported results in reviewing studies.

In spite of majority of studies carried out on children under 5 years, we included patients without any age limitation (age 32±34). However, there were no significant differences between current study results with others. Our study indicates that EIEC is a considerable acute diarrhea producing pathogen in adults as well as infants.

The invasive EIEC illness is characterized by abdominal cramps, diarrhea, vomiting, fever, chills, and a generalized malaise (5). In the current study, we studied clinical symptoms of patients as well as microscopic parameters. In the 80% of EIEC isolates from diarrheal samples PMNs and RBCs were present and patients, which were infected with this isolates had fever and abdominal pain that is evidence of invasive intestinal infection.

The EIEC virulence genes are carried by pInv. Two pInv-encoded activators, the VirF and VirB, are involved in the transcriptional control of the invasion genes. The VirF activates transcription of the secondary regulator virB gene, which activates the operons that are associated with EIEC invasion (21). In addition to ipaH gene, the virF gene was used as a gene involved in the EIEC invasion in our study. Among 5 EIEC isolates 4 (2.8%) isolates were virF+ and one isolate was virF-. The virF negative EIEC was isolated from a sample which has any PMNs and RBCs and associated patient had no fever and abdominal pain. Also the presence of RBCs in samples and abdominal pain and fever in patients was associated with virF+ isolates. This can confirm the role of virF in EIEC invasion.

The present study showed that EIEC can be cause of acute diarrhea in all age groups. Also applying the molecular diagnostic methods for detecting EIEC and other diarrheagenic E.coli pathotypes and subsequent appropriate treatment can be helpful.