Genetic evaluation of Locus of enterocyte effacement pathogenicity island (LEE) in Enteropathogenic Escherichia coli isolates (EPEC).

BACKGROUND AND OBJECTIVES: Enteropathogenic Escherichia coli (EPEC) divided into two groups typical and atypical (aspect). The main virulence genes are located in a pathogenicity island called LEE (Locus of Enterocyte Effacement). LEE frequently inserted in tRNA genes of selC, pheU and pheV in the bacterial chromosome. tEPEC and aEPEC strains have some differences in their pathogenicity. The purpose of this was to investigate the possible differences between tEPEC and aEPEC strains according to the virulence genes encoding by LEE and their relation to insertion sites.
MATERIALS AND METHODS: In this study 130 E. coli isolates confirmed by biochemical analysis from diarrheal patients, were evaluated for EPEC pathotype by PCR. All EPEC strains tested for presence of some LEE encoded virulence genes and sites of LEE insertion by PCR method.
RESULTS: Among 50 strains of EPEC 28 (56%) and 22 (44%) were typical and atypical strains respectively. 19 strains (30%) showed insertion in selC, 7 (14%) in pheU, 4 (8%) in pheV, 8 (16%) in pheU and pheV, 1 (2%) in selC and pheU, 6 (12%) in pheV, pheU and selC and 5 (10%) had no insertion in these sites. Moreover, spa (n = 8, 16%), espB (n = 16, 32%), espD (n = 18, 36%), espF (n = 8, 16%), espG (n = 13, 26%), espH (n = 12, 24%), map (n = 11, 32%) and tir (n = 4, 8%) were present among the strains.
CONCLUSION: Results showed that most of the virulence genes are present in tEPEC isolates. However, aEPEC isolates may acquire other virulence factors. The majority of tEPEC strains showed insertion at selC and aEPEC strains in pheV and pheU.

INTRODUCTION

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries (1, 2). The EPEC strains characterized by intestinal epithelium microvillus destruction which called attaching and effacing (A/E) lesions (3, 4). The A/E lesions is encoded by a 35 Kb pathogenicity island named locus of enterocyte effacement (LEE) (3, 5). LEE contains the genes encoding Intimin, a type III secretion system, a number of secreted (Esp.) proteins, and the translocated intimin receptor named Tir (5). Intimin, a 94-kDa outer membrane protein encoded by the eae gene, is responsible for the intimate adherence between bacteria and enterocyte membranes (6, 7). Type III secretion system effectors interfere with diverse cell signaling processes (8, 9). EspA is the structural protein of a filamentous structure of the bacterial surface which interacts with the epithelial cell in the early stages of the A/E lesion. The EspB and EspD located at the distal end of the EspA filament, and suggested that these proteins involved in pore formation in the host cell membrane (8, 9, 11). Effector proteins such as Tir, EspB, EspG, EspF, EspH and Map translocated through this structure and translocation of these molecules into the host cells results in changes of the cytoskeleton of the underlying epithelial cells (8, 11, 12). Tir also acts as a receptor to intimin (9, 11). LEE PAI inserted in chromosome by insertion in tRNA genes (11, 13). LEE inserted at the selC locus, encoding tRNA for synthesis of selenocysteine and at the pheU and pheV locus, encoding tRNA for synthesis of phenylalanine (11, 13, 14). EPEC is a pathotype with eae virulence gene but lacking shigatoxin (stx) (1). EPEC divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The basic difference between the two groups is the presence of the EPEC adherence factor plasmid (pEAF) in tEPEC and its absence in aEPEC (1, 15). pEAF encodes the bundle-forming pilus (BFP) a type IV pilus which interconnects bacteria within microcolonies, promoting their stabilization (1, 5). Recent epidemiological studies suggest an increasing identification of aEPEC in both developed and developing countries (1).

The aim of this study was to investigate the existence of differences between tEPEC and aEPEC strains according to the virulence genes encoding by LEE and their relation to insertion sites.

MATERIALS AND METHODS

Bacterial isolates

One hundred thirty isolates of E. coli from diarrheal cases were used in this study.

PCR assay

DNA from all isolates was extracted by phenol-chloroform method and then tested for presence of eae gene (Intimin) using primers designed by Zhang et al. (16) (Table 1). E. coli E2348/69 strain was used as a control strain. Presence of stx gene (Shigatoxin) was checked by primers designed by Toma et al. (17) and E. coli O157:H7 used as a control strain. For detection of eaf gene (Bundle forming pilli), primers designed by Kobayashi et al. (18) were used. The insertion site of the LEE PAI was determined by PCR using primers for selC, pheU and pheV sites, and E. coli K12 strain was used as control strain (Negative PCR results indicated insertion at tRNA sites under investigation). Presence of spa, espB, espD, espF, espH, map and tir genes were tested by PCR. The PCR conditions for amplification of the genes and expected size are presented in Table 1.

Table 1

Primers and PCR conditions.

Genes	Primers	PCR conditions	Size	References
eae	5’-CCGAATTCGGCACAAGCATAAGC-3’	94°C, 45s; 45°C, 45s; 72°C, 45s.	863 bp	[17]
	5’- CCCGGATCCGTCTCGCCAGTATTCG-3’
stx	5’-GAGCGAAATAATTTATATGTG-3’	94°C, 45s; 52°C, 45s; 72°C, 45s.	518 bp	[18]
	5’-TGATGATGGCAATTCAGTAT-3’
eaf	5’-CAGGGTAAAAGAAAGATGATAA-3’	94°C, 45s; 55°C, 45s; 72°C, 45s.	399 bp	[19]
	5’-TATGGGGACCATGTATTATCA-3’
selC	5’-GAGCGAATATTCCGATATCTGGTT-3’	94°C, 45s; 60°C, 45s; 72°C, 45s.	527 bp	[15]
	5’-CCTGCAAATAAACACGGCGCAT-3’
pheU	5’-CATCGGCTGGCGGAAGATAT-3’	94°C, 45s; 55°C, 45s; 72°C, 45s.	300 bp	[15]
	5’-CGCTTAAATCGTGGCGTC-3’
pheV	5’-CTGGGTATTGCGGTATCGGTGA-3’	94°C, 45s; 55°C, 45s; 72°C, 45s.	604 bp	[3]
	5’-GCTGGAGTTTGGACGGGGGTAA-3’
spa	5’-GCGGATCCATGGATACATCAACTACAG-3’	94°C, 45s; 60°C, 45s; 72°C, 45s.	639 bp	[8]
	5’-GCAAGCTTTTATTTACCAAGGGATATTCC-3’
espB	5’-GCGGATCCATGAATACTATCGATAATAAC-3’	94°C, 45s; 62°C, 45s; 72°C, 45s.	1111 bp	[8]
	5’-GCGAATTCTTACCCAGCTAAGCGAGC-3’
espD	5’-GCGGATCCATGGTTAATGTAAATAACG-3’	94°C, 45s; 61°C, 45s; 72°C, 45s.	1159 bp	[8]
	5’-GCGAATTCTTAAACTCGACCGCTGAC-3’
espF	5’-GCGGATCCATGCTTAATGGAATTAGTAAC-3’	94°C, 45s; 63°C, 45s; 72°C, 45s.	650 bp	[8]
	5’-GCGAATTCTTACCCTTTCTTCGATTGCTC-3’
espG	5’-GCGGATCCATGATACTTGTTGCCAAATTG-3’	94°C, 45s; 61°C, 45s; 72°C, 45s.	1112 bp	[8]
	5’-GCGAATTCTTAAGTGTTTTGTAAGTACG-3’
espH	5’-GCGGATCCATGCGTTATATAGGGAGG-3’	94°C, 45s; 61°C, 45s; 72°C, 45s.	506 bp	[8]
	5’-GCAAGCTTTTAAACTGTCACACCTG-3’
map	5’-GCTCTAGACATGTTTAGTCCAACGGCAATG-3’	94°C, 45s; 66°C, 45s; 72°C, 45s.	629 bp	[8]
	5’-GCAAGCTTCTACAGCCGAGTATCCTG-3’
tir	5’-GCGGATCCATGCCTATTGGTAACCTTG-3’	94°C, 45s; 61°C, 45s; 72°C, 45s.	1669 bp	[8]
	5’-GCAAGCTTTTAAACGAAACGTACTGG-3’

Statistical analysis

SPSS software version 16.0 was used and P values < 0.05 was considered as significant.

RESULTS

Of 130 isolates of E. coli, 50 were classified as EPEC based on the presence of eae and absence of the stx. Among all 50 EPEC isolates, 28 (56%) were positive for eaf, therefore were considered as tEPEC and other 22 (44%) were aEPEC. Determination of tRNA insertion site of LEE PAI revealed that selC, pheU and pheV (i.e. 24 (48%), 28 (56%) and 28 (56%), respectively)were site of insertion (negative PCR results indicated as insertion).In this study among 50 EPEC isolates spa, espB, espD, espF, espG, espH, map and tir detected in 8 (16%), 16 (32%),18 (36%), 8 (16%), 13 (26%), 12 (24%), 11 (22%) and 4 (8%) isolates respectively. p value calculated for spa (0.05), espB (0.002), espD (0.014), espG (0.016), espH (0.029), map (0.001) which suggest significant correlation between type of EPEC with presence of these genes (p < 0.05). The espF (0.263) and tir (0.089) were not associated with type of EPEC. The presence of the virulence genes in tEPEC and aEPEC strains were listed in Table 2.

Table 2

Distribution of virulence genes in tEPEC & aEPEC isolates.

Gene EPEC	spa	espB	espD	espF	espG	espH	map	tir
tEPEC (28 strains)	7 (25%)	14 (50%)	13 (46.4%)	7 (25%)	11 (39.2%)	10 (35.7%)	11 (39.2%)	4 (14.2%)
aEPEC (22 strains)	1 (4.5%)	2 (9%)	3 (13.6%)	3 (13.6%)	2 (9%)	2 (9%)	-	-

Also among 50 EPEC isolates 19 strains (30%) showed insertion at selC, 7 (14%) in pheU, 4 (8%) in pheV, 8 (16%) in pheU and pheV, 1 (2%) in selC and pheU, 6 (12%) in pheV, pheU and selC and 5 (10%) had no insertion in these sites. In present study comparison of tEPEC and aEPEC isolates showed that the LEE insertion sites were more associated with selC (p = 0.012) at tEPECs, whereas in aEPECs pheU (p = 0.05) and pheV (p = 0.007) were sites of insertion (Table 3).

Table 3

tRNA insertion sites of tEPEC and aEPEC isolates.

Insertion sites EPEC	selC	pheU	pheV
tEPEC (28 isolates)	18 (64%)	8 (29%)	8 (29%)
aEPEC (22 isolates)	9 (40.9%)	13 (59.1%)	13 (59.1%)

DISCUSSION

Enteropathogenic E. coli (EPEC) are organisms that cause diarrhea, especially in children in developing countries (20). EPEC contains a pathogenicity island called LEE which encodes several virulence factors that related with human enterocyte destruction (2, 20). There are many studies about presence of virulence genes in EPEC strains. Among 49 EPEC strains which was studied by Rodrigo et al. (21) spa, espB in 40 (81.6%) isolates and espD in 49 (100%), espF in 22 (42.8%) and tir in 45 (91.8%) were present. In a study by Kim et al. (22) in 10 EPEC isolates spa in 4(40%), espB and espD in 7 (70%) respectively were detected.

Contreras et al. (23) showed that spa (97.8%), espB (93.9%), espD (83.4%) and tir (92%) were present in EPEC isolates. Mairena et al. (24) studied on LEE4 encoded virulence genes in tEPEC and aEPEC and detected spa (94%), espB (50%), espD (40%), espF (78%), sepL (90%) of all strains. They indicated that spa and sepL should be more conserved between EPECs, while espB, espD, and espF should be more diverse. Gartner et al. (25) compared tEPEC 2348/69 with aEPEC 3431 and O181 and indicated that LEE encoded virulence genes showed very variable pattern. Muller et al. (26) studied on LEE encoded virulence genes in aEPEC including escV, bfpB, stx1, stx2, invE, elt, estIa, estIb, astA, aggR, pic, uidA, α-hly,e-hly, lifA(efa1) and ent showed that virulence gene pattern is very different. Also demonstrated that aEPEC mostly inserted in pheU and pheV. Bouzari et al. (3) showed that in 17 EPEC isolates (8 tEPEC and 9 aEPEC) 6 (35%) showed insertion in selC, 7 (41%) in pheU and 2 (11.8%) in pheV. In 2 (11.8%) isolates no insertion at these sites were observed.

In other study, Vieira et al. (27) showed that potentially virulent aEPECs were associated with presence of PAI O122. Our study showed that the presence of the LEE encoded virulence genes that are essential for attaching & effacing are dominant in tEPEC isolates. We have also found that the patterns of these genes are very variable. The majority of virulence genes were present in isolates that their LEE pathogenicity island was inserted at selC site. Also most of the tEPEC isolates had insertion in selC. These results have two conceptions: The aEPEC isolates have reduced virulence in compare to tEPEC isolates, or aEPECs possess other virulence factors than reported so far. Some of our isolates in this study also showed insertion in selC, pheU and pheV sites but no virulence genes could be detected. These results collectively could point at evolutionary differences of these isolates in regards to genome rearrangement due to horizontal gene transfer.