Abstract
The presence of subAB was investigated for 3,453 Escherichia coli strains of various pathogenic categories. The occurrence of other virulence genes in subAB-positive strains was investigated. The subAB operon was detected among some Shiga toxin-producing E. coli (STEC) serotypes devoid of eae and carrying ehxA. Most subAB-positive strains also harbored stx2, iha, saa, and lpfAO113.
Subtilase cytotoxin, a new member of the AB5 toxin family, was identified for the first time in 2004 in a virulent O113:H21 Shiga toxin-producing Escherichia coli (STEC) strain that caused an outbreak of hemolytic-uremic syndrome in South Australia (16, 18). The presence of subAB genes was further detected in other STEC strains belonging to different serotypes (19). Subsequently, subAB genes were identified among STEC strains isolated in other countries (3, 8, 9, 14, 25).
To evaluate how widely distributed the subAB operon is, we studied a large collection of STEC serotypes from nonhuman sources and E. coli strains of different pathogenic categories associated with human infections. The subAB-positive strains were further characterized regarding the presence of other virulence genes.
A total of 2,255 E. coli strains isolated from humans and belonging to enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), extraintestinal pathogenic E. coli (ExPEC), and E. coli strains not belonging to the diarrheagenic categories described so far were randomly selected. STEC strains isolated in Brazil from humans have previously been tested for the presence of subAB by our group (3). The 1,198 STEC strains from nonhuman sources were isolated from dairy cattle, beef cattle, buffaloes, and goats. Overall, 109 different STEC serotypes were tested. An STEC strain of serotype O113:H21 (3) was used as a reference strain for subAB, cdt-V, and lpfAO113, and E. coli strain DH5α was used as a negative control.
The strains were screened for the presence of the subAB operon (encoding subtilase cytotoxin) using colony hybridization assays (21). The 1,823-bp subAB-specific DNA probe was derived from the STEC serotype O113:H21 (3) strain by PCR as previously described (19). Hybridization assays were performed under stringent conditions, and the probe was labeled with [α-32P]dCTP (Amersham), using the Ready-To-Go DNA labeling kit (Amersham). All strains which yielded a positive or weak signal in hybridization assays with the subAB probe were retested by PCR (18, 19), and only those confirmed by PCR were considered to be carrying this sequence.
The genetic profiles of the subAB-positive strains were determined using our previously reported data for the same strains (6, 7, 12, 13, 20, 24) regarding the presence of the ehxA, eae, stx1, stx2, and adhesin-encoding genes (1, 10, 11, 15, 17, 22, 23).
A total of 130 STEC strains carrying the subAB operon, representative of each serotype and isolated from different animals, were analyzed by PCR for the presence of genes encoding LpfO113 and cytolethal distending toxin (Cdt-V) (2, 5).
Expression of the SubAB and Cdt-V toxins was investigated using Chinese hamster ovary cells according to the methods of Paton et al. (18) and Bielaszewska et al. (2), respectively. Cells were exposed to filter-sterilized bacterial culture supernatants and observed daily for a period of 7 days. To confirm the loss of viability or morphological changes, trypan blue, violet crystal, and/or 4′,6-diamidino-2-phenylindole (DAPI) staining were performed. Control strains were included in all assays.
As shown in Table 1, the subAB operon was detected exclusively among STEC strains and corresponded to 25.5% (306/1,198) of the STEC collection. The presence of subAB was identified in 44.2% (141/319), 27.1% (29/107), and 23.8% (129/542) of STEC strains isolated from dairy cattle, buffaloes, and beef cattle, respectively. Only 3% (7/230) of STEC strains isolated from goat carried subAB. Among the 109 different STEC serotypes tested, 21 carried the subAB operon. The presence of the subAB operon probably is associated with some STEC serotypes. In the present study, the rate of carriage of this sequence within each serotype ranged from 5.5 to 100% (Table 2). Among caprine STEC strains, only those belonging to O113:H21 carried the subAB operon. A total of 306 (306/1,198) STEC strains carrying subAB were detected. We have previously reported that among 49 human STEC strains isolated in Brazil, none carried the subAB sequence (3).
TABLE 1.
E. coli strains of different pathogenic categories searched for the subAB operon
| E. coli category | Total no. of strains studied | No. (%) of strains positive for subAB |
|---|---|---|
| Enteropathogenic E. coli (EPEC)a | 402 | 0 |
| Enterotoxigenic E. coli (ETEC)b | 264 | 0 |
| Enteroinvasive E. coli (EIEC)c | 266 | 0 |
| Enteroaggregative E. coli (EAEC) | 100 | 0 |
| Extraintestinal E. coli (ExPEC)d | 205 | 0 |
| E. coli strains other than the diarrheagenic categories described so far | 1,018 | 0 |
| Shiga toxin-producing E. coli (STEC) from nonhuman sourcese | 1,198 | 306 (25.5) |
| Total | 3,453 | 306 |
EPEC, strains of serogroups O25, O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158.
ETEC strains ST/LT or both toxin producers.
EIEC strains of serogroups O28, O29, O112, O124, O121, O135, O136, O143, O144, O152, O159, O160, O164, O167, and O173.
ExPEC E. coli strains isolated from blood, cerebrospinal fluid, and/or urine.
STEC strains isolated from humans in Brazil have been previously tested by us (3).
TABLE 2.
Distribution and frequencies of subAB operon-positive STEC serotypes isolated from dairy/beef cattle, buffalo, and goat
| Serotype | No. of strains tested | No. (%) of subAB- positive strains |
|---|---|---|
| O39:H49a | 11 | 9 (81.8) |
| O44:H25b | 5 | 4 (80) |
| O59:H8c | 1 | 1 (100) |
| O74:H28a | 1 | 1 (100) |
| O74:H−a | 3 | 3 (100) |
| O77:H18b,c | 20 | 14 (70) |
| O79:H14a,b | 35 | 34 (87.1) |
| O79:H28c | 5 | 5 (100) |
| O79:H−a | 8 | 8 (100) |
| O96:H19a | 1 | 1 (100) |
| O96:H21a | 5 | 5 (100) |
| O105:H18b | 9 | 1 (11.1) |
| O113:H21a,b,d | 52 | 36 (69.2) |
| O116:H21a,b,c | 32 | 32 (100) |
| O141:H49b,c | 18 | 18 (100) |
| O153:H25b | 5 | 5 (100) |
| O163:H−a | 1 | 1 (100) |
| O163:H19a,b | 3 | 3 (100) |
| O174:H28a | 1 | 1 (100) |
| O176:H18a | 2 | 2 (100) |
| O178:H18b | 4 | 2 (50) |
| O178:H19a,b,c | 46 | 29 (63.0) |
| O179:H8a,b | 9 | 3 (33.3) |
| ONT:H−a | 18 | 1 (5.5) |
| ONT:H2a | 21 | 2 (9.5) |
| ONT:H7a,c | 53 | 6 (11.3) |
| ONT:H8a,b | 38 | 7 (18.4) |
| ONT:H10a | 4 | 3 (75) |
| ONT:H11a | 4 | 2 (50) |
| ONT:H18c | 14 | 4 (28.6) |
| ONT:H19a,b | 59 | 34 (57.6) |
| ONT:H21a,b,c,d | 64 | 8 (12.5) |
| ONT:H25a | 4 | 2 (50) |
| ONT:H46a,b | 16 | 15 (93.7) |
| ONT:H49a | 4 | 3 (75) |
| OR:H19a | 3 | 1 (33.3) |
| Total | 579 | 306 |
Isolated from beef cattle.
Isolated from dairy cattle.
Isolated from buffalo.
Isolated from goat.
All strains carrying subAB were devoid of eae, and 59.8% (183/306) were associated with strains possessing the stx2 gene alone, 39.9% (122/306) were carrying stx1 plus stx2, and only 0.3% (1/306) carried stx1 alone, as previously reported (3, 8, 9, 14, 19). The most frequent adhesin-encoding genes among STEC strains carrying the subAB operon were lpfAO113, iha, and saa, and all strains also carried ehxA.
Among the 130 selected STEC strains carrying the subAB operon, 98.5% (128/130) and 20% (26/130), respectively, harbored the lpfAO113 and cdt-V sequences. In STEC strains carrying the cdt-V gene, 54% and 23% of the isolates, respectively, belonged to serotypes O116:H21 and O113:H21. Expression of the subtilase cytotoxin was detected in 40.7% (53/130) of the studied strains, while the cdt-V gene was expressed in 30.8% (8/26) of the strains. We observed that 24.5% (13/53) of the strains that expressed SubAB also harbored the cdt-V gene; however, none of the isolates coexpressed both cytotoxins. This result is in contrast with previous data in which coexpression of SubAB and Cdt-V in STEC isolates of serotype O113:H21 occurred (4). The expression of subAB genes in a collection of STEC strains belonging to several serotypes is reported here for the first time. The production of this toxin had been seen previously only in O113:H21 STEC (18).
To the best of our knowledge, the search for subAB in other E. coli categories has not been described before, and the present results showed that among E. coli strains, the subAB gene sequence was distributed only among some STEC serotypes.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant 07/53313-05, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília), and Programa de Apoio a Núcleos de Excelência PRONEX MCT/CNPq/FAPERJ.
Footnotes
Published ahead of print on 20 January 2010.
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