Abstract
Four of six adhesin-encoding genes (lpfA, paa, iha, and toxB) from Shiga toxin-producing Escherichia coli strains were detected in typical and atypical enteropathogenic E. coli (EPEC) strains of various serotypes. Although the most prevalent gene was lpfA in both groups, paa was the only potential diarrhea-associated gene in atypical EPEC.
TEXT
The enteropathogenic Escherichia coli (EPEC) pathotype is subdivided into typical EPEC (tEPEC), which carries the large virulence EPEC adherence factor (EAF) plasmid (pEAF), and atypical EPEC (aEPEC), which lacks this plasmid (14). The pEAF encodes the bundle-forming pilus (BFP), which mediates a localized adherence (LA) pattern on HeLa/HEp-2 cells (13).
Both EPEC subgroups produce attaching and effacing (A/E) lesions on enterocytes, a phenotype associated with the chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin is an outer membrane protein encoded by the eae gene and is the fundamental LEE-encoded adhesin determining establishment of A/E lesions (reviewed in reference 13).
The LEE is also found in enterohemorrhagic E. coli (EHEC), a subgroup of the Shiga toxin-producing E. coli (STEC) pathotype (19). Various additional adhesins have been described in STEC, including Saa (STEC autoagglutinating adhesin) in LEE-negative STEC strains (18); Paa (porcine A/E-associated adhesin), involved in the initial adherence of porcine EPEC strains (4); Lpf (long polar fimbriae), an adhesin found in EHEC and other pathogenic E. coli (26, 27); ToxB (EHEC pO157 plasmid-encoded protein), which is involved in adherence of EHEC O157:H7 (24); Iha (IrgA homologue adhesin), an adhesin similar to Vibrio cholerae IrgA (23); and EspP (extracellular serine protease), which contributes to bovine intestinal colonization (9).
aEPEC strains are a highly heterogeneous group, with many strains presenting an assorted repertoire of virulence genes from various E. coli pathotypes (8, 11, 20, 25, 30). Interestingly, aEPEC strains carrying certain virulence genes or specific combinations are significantly associated with diarrheal disease (2, 11, 20, 21, 30). There is evidence indicating that some aEPEC strains are in fact tEPEC or EHEC strains, which spontaneously lost either pEAF or the Shiga toxin-encoding genes (stx genes) (28). Furthermore, some comparative studies of the aEPEC genome have shown a closer relationship with EHEC than with tEPEC (16, 28), and others have shown that some clonal aEPEC groups are not related to any of these pathotypes (25).
In previous studies, we have searched for a number of E. coli adhesin-encoding genes in our EPEC collection, including the STEC efa1/lifA gene, but none of them were prevalent (11, 29, 30). Therefore, we aimed at investigating the prevalence of six other STEC adhesin-encoding genes and their potential association with diarrhea in aEPEC strains and compared them with those of tEPEC strains.
We examined a total of 146 E. coli strains, which were isolated from 113 diarrheic and 33 nondiarrheic children and adults in Brazil. The strains were characterized as EPEC based on the presence of the eae and absence of the stx genes (14). Further classification as tEPEC or aEPEC was achieved by confirming BFP production by immunoblotting and by checking the bfpA-positive strains for their ability to produce LA on HeLa cells (after 3 h of incubation) (28). Strains lacking bfpA or carrying bfpA but lacking BFP production and showing an adherence pattern distinct from LA were classified as aEPEC (1, 12, 28). The aEPEC adherence patterns were defined after longer incubation periods (6 h) on HeLa cells (Table 1). The ability of all adherent strains to promote actin accumulation was evaluated by the fluorescent actin staining (FAS) test on HeLa cells (15). While all tEPEC strains were FAS positive (FAS+) (data not shown), this property was detected in only 83% of the aEPEC strains isolated from patients and control subjects (Table 1). Interestingly, none of the aEPEC adherence patterns were associated with diarrhea, but FAS+ strains producing the LA-like (LAL) pattern were statistically associated with diarrhea.
Table 1.
Adherence patterns and FAS test results from 100 aEPEC strains isolated from 73 patients and 27 controls
| Phenotypic characteristica | No. (%) of strains from: |
||
|---|---|---|---|
| Patients | Controls | Total | |
| LAL | 60 (82.2) | 19 (70.4) | 79 (79.0) |
| FAS+ | 55 (75.3)b | 14 (51.8)b | 69 (69.0) |
| FAS− | 5 (6.8) | 5 (18.5) | 10 (10.0) |
| LAL/AA | 3 (4.1) | 0 | 3 (3.0) |
| FAS+ | 3 (4.1) | 0 | 3 (3.0) |
| FAS− | 0 | 0 | 0 |
| AA | 3 (4.1) | 1 (3.7) | 4 (4.0) |
| FAS+ | 2 (2.7) | 1 (3.7) | 3 (3.0) |
| FAS− | 1 (1.3) | 0 | 1 (1.0) |
| DA | 2 (2.7) | 4 (14.8) | 6 (6.0) |
| FAS+ | 2 (2.7) | 2 (7.4) | 4 (4.0) |
| FAS− | 0 | 2 (7.4) | 2 (2.0) |
| NC | 3 (4.1) | 2 (7.4) | 5 (5.0) |
| FAS+ | 2 (2.7) | 2 (7.4) | 4 (4.0) |
| FAS− | 1 (1.3) | 0 | 1 (1.0) |
| NA | 1 (1.3) | 1 (3.7) | 2 (2.0) |
| D | 1 (1.3) | 0 | 1 (1.0) |
FAS, fluorescent actin staining; LAL, localized adherence-like; AA, aggregative adherence; DA, diffuse adherence; NC, noncharacteristic adherence; NA, nonadherent; D, cell detachment.
Difference was statistically significant (P = 0.0302).
The presence of five STEC adhesin-encoding genes (espP, toxB, saa, iha, and paa) was examined by colony hybridization under stringent conditions, using as probes fragments of these genes obtained by PCR (3, 4, 6, 17, 22) and labeled with [32P]dCTP. The presence of different lpfA alleles was investigated by PCR using primers and conditions previously described (26). Data were analyzed by two-tailed Fisher's exact test, and P values of ≤0.05 were considered statistically significant.
The lpfA genes were the most prevalent, followed by paa, iha, and toxB in both EPEC groups (Table 2). The paa and iha genes were significantly more frequent in aEPEC than in tEPEC strains, while espP was only found in three aEPEC strains, and saa was not detected (Table 2).
Table 2.
Prevalence of STEC adhesin-encoding genes in tEPEC and aEPEC strains from patients and controls
| Adherence gene | No. (%) of strains: |
|||||
|---|---|---|---|---|---|---|
| aEPECa |
tEPECb |
|||||
| Patients | Controls | Total | Patients | Controls | Total | |
| lpfA | 43 (58.9) | 16 (59.3) | 59 (59.0) | 20 (50.0) | 4 (66.7) | 24 (52.2) |
| paa | 35 (48.0)c | 7 (25.9)c | 42 (42.0)d | 6 (15.0) | 0 | 6 (13.0)d |
| iha | 22 (30.1) | 7 (25.9) | 29 (29.0)e | 3 (7.5) | 1 (16.7) | 4 (8.7)e |
| toxB | 4 (5.5) | 2 (7.4) | 6 (6.0) | 1 (2.5) | 1 (16.7) | 2 (4.35) |
| espP | 3 (4.1) | 0 | 3 (3.0) | 0 | 0 | 0 |
| saa | 0 | 0 | 0 | 0 | 0 | 0 |
| None | 7 (9.6) | 8 (29.6) | 15 (15.0) | 17 (42.5) | 1 (16.6) | 18 (39.1) |
The numbers of aEPEC strains studied were 73 from patients and 27 from controls.
The numbers of tEPEC strains studied were 40 from patients and 6 from controls.
P = 0.067.
P = 0.0005.
P = 0.0058.
None of the adhesin-encoding genes were associated with diarrhea in both EPEC groups, but paa was more frequently found in aEPEC isolates from patients compared with isolates from controls, with this difference reflecting a trend to be statistically significant (Table 2). The association of paa with diarrhea has been previously observed in aEPEC isolates from Norwegian and Brazilian children (2, 21), but those studies did not examine tEPEC isolates.
Different combinations of the genes studied were found among diverse aEPEC and tEPEC serotypes (Tables 3 and 4). The distribution of lpfA in tEPEC agreed with our previous study (26); however, this is the first description of isolates lpfA1–1 allele positive and lpfA2 negative in serotype O142:H34 (Table 4). From the 100 aEPEC strains analyzed, 41 lack both lpfA alleles. Of the remaining 59 lpfA-positive strains, 20 contained the combination of lpfA1–2 and lpfA2–1 alleles. With the exception of one strain, all O55:H7 strains were lpfA1–3 and lpfA2–2, thus confirming previous findings with EHEC O157:H7 and LEE-negative STEC strains (10, 26), that demonstrated that the presence of these two alleles is a reliable way to detect O157:H7 strains and the closely related O55:H− or O55:H7 serotypes. Although different combinations of these alleles were detected in aEPEC serotypes, we found that seven O119:H2 isolates carried the lpfA1–2 allele and were negative for lpfA2 (Table 3). Seven other isolates from different serotypes were also positive for the lpfA1–1 allele and negative for lpfA2, and these alleles were found in rare tEPEC and aEPEC serotypes. Finally, a large number of EPEC strains were negative for both lpfA1 and lpfA2, and no correlation with diarrhea was observed (Table 2).
Table 3.
Distribution of STEC adhesin-encoding genes among aEPEC strains of distinct serotypes
| Serotype | No. of strains | STEC adhesin-encoding gene(s) | lpfA1 type | lpfA2 type |
|---|---|---|---|---|
| O2ab:H45 | 1 | paa | 1 | |
| O11:H2 | 1 | 2 | 1 | |
| O11:H16 | 1 | |||
| O16:H− | 1 | iha | ||
| O19:H− | 1 | iha | 1 | |
| O26:H− | 1 | 2 | 1 | |
| 3 | paa | 2 | 1 | |
| 1 | iha | |||
| 1a | iha, paa, espP | 2 | 1 | |
| O34:H− | 1 | paa | 2 | 1 |
| 1 | iha | 2 | 1 | |
| O39:H− | 1b | toxB, paa | ||
| O49:H− | 1 | iha | 1 | |
| O49:H10 | 1b | toxB, paa | ||
| O51:H− | 1 | iha, paa | 2 | 1 |
| O55:H7 | 6 | paa | 3 | 2 |
| 1 | paa | 3 | ||
| O63:H6 | 2 | paa | ||
| O85:H− | 1 | iha | 1 | |
| O93:H− | 1 | iha, paa | 2 | 1 |
| O98:H8 | 1 | iha, paa | 1 | |
| O101:H33 | 1 | |||
| 1 | iha | |||
| O109:H9 | 1 | iha | 1 | |
| O111:H9 | 1 | |||
| 1 | iha | 1 | ||
| O119:H2 | 7b | paa | 2 | |
| O123:H19 | 1 | 2 | 1 | |
| O124:H40 | 1 | 1 | ||
| O125:H6 | 2 | |||
| O128:H2 | 4 | paa | ||
| O132:H8 | 2b | paa | 2 | 1 |
| O145:H− | 1 | paa | ||
| O145:H34 | 1 | iha, paa | ||
| O154:H9 | 1 | iha | 1 | |
| O157:H− | 2 | iha | ||
| O157:H16 | 2 | |||
| O160:H19 | 1 | 2 | 1 | |
| O162:H− | 1 | toxB, iha | ||
| O177:H− | 1 | |||
| NT:H− | 3 | iha | 1 | |
| 1 | ||||
| 1c | espP | |||
| 1 | 2 | 1 | ||
| 1 | paa | 1 | ||
| 1 | iha | 1 | ||
| NT:H2 | 1 | iha | ||
| NT:H7 | 1b | toxB | 1 | |
| NT:H8 | 1 | |||
| 1b | 1 | |||
| 1b | paa | 2 | 1 | |
| 1 | 2 | 1 | ||
| 1 | 1 | |||
| NT:H9 | 1 | iha | ||
| NT:H11 | 1 | |||
| 1 | iha | 1 | ||
| NT:H19 | 1 | 2 | 1 | |
| NT:H25 | 1 | 2 | 1 | |
| NT:H29,31 | 1 | |||
| NT:H33 | 1b | |||
| NT:H34 | 1 | 1 | ||
| 1 | iha | |||
| 1 | paa | |||
| NT:H38 | 1 | 1 | ||
| NT:H40 | 1 | |||
| NT:H40,43 | 1 | 1 | ||
| 1 | ||||
| NT:H46 | 1 | paa | 1 | |
| NT:NT | 1b | paa, toxB | ||
| 1b | paa | 1 | 1 | |
| R:H− | 1 | 1 | 1 | |
| 1c | iha, paa, espP | 2 | 1 | |
| R:H11 | 1 | |||
| R:H28 | 1 | 1 | ||
| R:H33 | 1 | iha | ||
| R:H40 | 1b |
Strain carrying ehxA and katP genes.
bfpA-positive strains lacking BFP production.
Strain carrying ehxA gene.
Table 4.
Distribution of STEC adhesin-encoding genes among tEPECa strains of distinct serotypes
| Serotype | No. of strains | STEC adhesin-encoding gene | lpfA1 type | lpfA2 type |
|---|---|---|---|---|
| O55:H− | 1 | paa | 3 | 2 |
| 1 | 1 | |||
| O55:H6 | 4 | 1 | ||
| 1 | ||||
| O86:H34 | 1 | |||
| 1 | 1 | |||
| O88:H25 | 5 | 2 | 1 | |
| 1 | iha | 2 | 1 | |
| O111:H− | 6 | |||
| O111:H2 | 6 | |||
| O119:H6 | 3 | paa | ||
| 3 | ||||
| O127:H6 | 1 | toxB | 1 | |
| O127:H40 | 1 | |||
| 1 | toxB | |||
| O142:H6 | 2 | iha | 1 | |
| 2b | paa | 1 | ||
| O142:H34 | 2 | 1 | ||
| 1 | iha | 1 | ||
| O145:H45 | 1 | 1, 2 | 1 | |
| 2 | 1 |
E. coli strains carrying eae and bfpA and lacking stx genes, with all strains producing BFP.
Only these two strains lacked the EAF probe sequence.
We also found that all strains of the traditional aEPEC serotypes O55:H7, O119:H2, and O128:H2 carried paa and all O119:H2 strains carried only lpfA1–2 and lacked lpfA2. In contrast, the O26:H− strains showed different allele combinations which may reflect distinct H types (serotypes); however, with one exception, they possessed the lpfA1–2 and lpfA2–1 alleles. In addition, tEPEC strains of serotypes O111:H− and O111:H2, which were the most prevalent isolates in São Paulo, Brazil, in the past (28), carried none of the genes investigated.
The three strains that harbored espP (O26:H−, NT:H−, and R:H−) carried this gene on large plasmids, which also contained ehxA, although only one possess katP (Table 3). These findings suggest that these strains could comprise EHEC strains that have lost the stx genes (EHEC-LST) (5, 7). In six strains (two tEPEC and four aEPEC strains) that carried both bfpA and toxB, Southern blot analysis indicated that toxB is either located in pEAF or in another plasmid of similar size (data not shown).
In conclusion, we showed that a subgroup of aEPEC strains, producing the LAL pattern and FAS positivity on HeLa cells, are statistically associated with diarrhea. In addition, the prevalence of the STEC adhesin-encoding genes studied is higher in aEPEC than in tEPEC, possibly reflecting an apparently closer relationship of some aEPEC strains to the STEC pathotype. Our case control analysis showed a trend of the paa gene to be associated with aEPEC diarrhea. However, more studies are necessary to confirm that these genes are expressed during human infections and to understand how they contribute to host colonization.
ACKNOWLEDGMENTS
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 Núcleos de Excelência (PRONEX MCT/CNPq/FAPERJ). The work in A.G.T.'s laboratory was supported by NIH/NIAID grant no. 5R01AI079154-03.
The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIAID or NIH.
Footnotes
Published ahead of print on 27 July 2011.
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