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The presence of the pathogenicity island (PAI) O122 genes, efa1 (lifA), sen, pagC, nleB, and nleE, in typical and atypical enteropathogenic Escherichia coli (EPEC) strains was investigated. The simultaneous occurrence of all genes was statistically associated with diarrhea due to atypical EPEC. Detection of the complete PAI O122 could aid in the identification of potential pathogenic strains of atypical EPEC.
Enteropathogenic Escherichia coli (EPEC) and Shiga toxin (Stx)-producing E. coli (STEC) are important human enteropathogens (9). EPEC is further subgrouped into typical (tEPEC) and atypical (aEPEC) EPEC (8, 21). tEPEC strains are major causative agents of acute diarrhea in infants in developing countries, whereas aEPEC strains affect children and adults worldwide (5, 7, 9, 21). The main difference between tEPEC and aEPEC is the presence of the EPEC adherence factor (EAF) plasmid in tEPEC (8, 21). This plasmid encodes the bundle-forming pilus (BFP), which mediates localized adherence to intestinal cells (9), which is an essential property to differentiate tEPEC from aEPEC strains (1, 7, 21).
Formation of the attaching-and-effacing (AE) lesion is the major virulence mechanism of EPEC and an additional virulence property of enterohemorrhagic E. coli (EHEC) strains, a subset of STEC strains (9). This lesion consists of intimate bacterial adherence to the intestinal epithelial cells, cytoskeleton remodeling, and microvillus effacement (9). The genes encoding the AE lesion are located in a pathogenicity island (PAI) known as the locus of enterocyte effacement (LEE) (9). Despite the recognized importance of AE lesion formation, other putative virulence genes among AE-producing E. coli strains have been described.
Efa1 (EHEC factor for adherence) is an adhesin originally described in some EHEC strains (16). The efa1 gene is almost identical to lifA, an EPEC gene encoding lymphostatin (LifA) (13). LifA inhibits the proliferation of mitogen-activated lymphocytes and the synthesis of proinflammatory cytokines (13). Efa1/LifA contributes to EPEC adherence to epithelial cells and is critical for intestinal colonization by Citrobacter rodentium, which is an AE lesion-producing bacterial pathogen of mice (12). Although, in the prototype EHEC O157:H7 strain EDL933, efa1 (lifA) lacks the 3′ region (efaC), the adherence ability of this strain is preserved (20).
efa1 (lifA) is outside the LEE and inside PAI O122 (16). This island also harbors genes which are very similar to pagC of Salmonella enterica serovar Typhimurium, sen of Shigella flexneri, and two C. rodentium non-LEE genes, nleB and nleE. PagC is required for bacterial survival within macrophages and is immunogenic to humans, while sen encodes an S. flexneri enterotoxin (10). NleB is linked to colonization and disease in mice (11), and NleE induces polymorphonuclear (PMN) transepithelial migration and is involved in the blockage of NF-κB activation (15, 24). Afset et al. (2) found that, of 182 virulence genes searched for among aEPEC strains, 12 were statistically associated with diarrhea, including efa1 (lifA) (having the strongest association with the disease), nleB, and nleE.
In STEC the association of LEE, efa1 (lifA), sen, and pagC was strongly correlated with virulence and disease severity (10). As the presence of PAI O122 genes have been searched for in only a limited number of tEPEC serotypes and in a few aEPEC strains (2, 4, 10, 14, 16), in this study we investigated the presence of efa1 (lifA) and its location in PAI O122 as well as the presence of sen, pagC, nleB, and nleE among well-characterized tEPEC and aEPEC strains isolated from diarrheic and nondiarrheic patients.
A total of 152 strains (45 tEPEC and 107 aEPEC strains) of various serotypes were studied. These strains were isolated from diarrheic (114 strains) and nondiarrheic (38 strains) patients (5, 6, 22). Prototype tEPEC strain E2348/69 and EHEC strain EDL933 were used as positive controls, and E. coli HB101 was used as a negative control.
The presence of efa1 (lifA) was screened by colony blot hybridization assays under stringent conditions (18). The efai and efa5′ DNA probes were fragments amplified from strain E2348/69 with primers described elsewhere (16) corresponding to both the internal and the 5′ regions of the efa1 (lifA) gene, respectively.
For better characterization of PAI O122 and the efa1 (lifA) gene, PCR schemes and primers described previously were used (2, 3, 10, 14) to search for the presence of the efa 3′ region, sen, pagC, nleB, and nleE using Mastermix (Promega, Madison, WI). Localization of efa1 (lifA) in PAI O122 was investigated with Elongase (Invitrogen Life Technologies, Carlsbad, CA).
The efa1 (lifA) gene was found in 60 (39.5%) out of 152 strains studied and was more prevalent among tEPEC strains than aEPEC strains (62.2% versus 30.0%, respectively). Among all the strains analyzed only one (aEPEC) presented a truncated efa1 (lifA) gene, like EHEC strain EDL933, as evidenced by lack of amplification with primers annealing to the 3′ region (efaC) of efa1 (lifA) (3) (Tables (Tables11 and and2).2). This strain was isolated from a diarrheic patient and belonged to serotype O145:nonmotile, thus corroborating a previous finding of a truncated efa1 (lifA) gene in an EHEC strain of the same serogroup (14).
In more than 95% of the efa1 (lifA)-positive strains, this gene was located in PAI O122, as detected by the presence of the 6.5-kb region between efa1 (lifA) and open reading frame (ORF) Z4326 (14) (Tables (Tables11 and and2).2). Failure to detect efa1 (lifA) in PAI O122 in five strains may indicate either that it is located in another spot on these bacterial genomes or that recombination events have occurred in their primers' annealing regions.
Although 92 strains (17 tEPEC and 75 aEPEC strains) were devoid of efa1 (lifA), one of these strains carried only nleB, four carried only sen, three carried only nleB and nleE, six presented sen, nleB, and nleE, and only one carried sen, pagC, nleB, and nleE (Table (Table2).2). In other studies (2, 17) aEPEC strains carrying PAI O122 genes but lacking efa1 (lifA) were also found.
Karmali et al. (10) demonstrated that the severity of disease caused by STEC and EHEC is due to the association of PAI genes. Another study describing PAI O122 genes in outbreak-associated non-O157:H7 STEC strains showed the existence of modules (different combinations of virulence genes) that determined the outcome of infection in vivo (23). In one of these modules, the simultaneous presence of efa1 (lifA), sen (also named ent), pagC, and nleB provided a higher virulence potential (23). Unfortunately, in this study we could not associate different virulence degrees with PAI modules because the strains analyzed were isolated during epidemiological retrospective studies of acute diarrhea. Moreover, EPEC strains usually cause a smaller spectrum of virulence than EHEC.
A complete PAI O122 (carrying efa1 [lifA], pagC, sen, nleB, and nleE) was found in 27.0% of the strains studied and was more prevalent in tEPEC than in aEPEC (48.9% versus 17.8%, respectively). The predominance of four PAI O122 genes in tEPEC was also observed by Scaletsky et al. (19). In the present study, occurrence of a complete PAI was observed in most tEPEC serotypes (Table (Table1)1) whereas incomplete PAIs were found only in tEPEC strains from serotypes O88:H25 and O127:H40. tEPEC O55:H6 strains carried either complete or incomplete PAIs (Tables (Tables11 and and22).
Considering the strains in both EPEC groups altogether, the presence of a complete PAI was significantly associated with diarrhea (P = 0.005 by Fisher's exact test). Significant association was also observed in the aEPEC group (P = 0.04 by Fisher's exact test) (Table (Table3)3) but not in the tEPEC group (P = 0.22 by Fisher's exact test), because of the higher number of tEPEC strains presenting complete PAI in controls.
This study shows that, although tEPEC and aEPEC strains may harbor complete and incomplete versions of PAI O122, a strong association between the presence of a complete PAI O122 and diarrhea was observed only in aEPEC. In fact, Bielaszewska et al. (4) reported that the presence of PAI O122 associated with LEE in aEPEC strains of the O26 serogroup would aid in their virulence potential. One can expect that the association of PAI O122 modules with LEE in aEPEC strains increases the pathogenicity of these strains circulating in our setting. Therefore, detection of complete PAI O122 could aid in the identification of potentially pathogenic strains within this pathotype.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant 08/53812-4) and Programa de Apoio a Nucleos de Excelência (PRONEX MCT/CNPq/FAPERJ) (to T.A.T.G.). A postdoctoral position at Universidade Federal de São Paulo (for M.A.M.V.) was funded by FAPESP.
Published ahead of print on 24 February 2010.