Association of genes encoding P fimbriae, CS31A antigen and EAST 1 toxin among CNF1-producing Escherichia coli strains from cattle with septicemia and diarrhea
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Fifty-six CNF1-producing Escherichia coli strains isolated from cattle with diarrhea or septicemia were screened by PCR for the detection of pap, sfa, afa, clpG, or f17 adherence factor and EAST 1 toxin genes. All the isolates were pap-positive, in accordance with the close association of pap, CNF1 and α-hemolysin genes observed on human and porcine E. coli. Only the gene encoding the P adhesin of class III (PrsG) was detected. Genes encoding CS31A antigen (71%) and S fimbriae (34%) (but not Afa or F17) were detected among the bovine isolates. E. coli producing both CNF1 and plasmid-encoded CS31A is a new example of association between bacterial clones and plasmid-mediated virulence factors. The EAST 1 toxin-encoding gene was detected on 66% of the CNF1-producing isolates but was linked to CS31A rather than to CNF1. These results suggest a close association between EAST 1 toxin and the adherence factor CS31A among pathogenic bovine E. coli.
Virulence factors (cytotoxins, aerobactin, lipopolysaccharide or adhesins) are associated with the capacity of Escherichia coli strains to cause extraintestinal infections such as urinary tract infection (UTI), meningitis or septicemia . The cytotoxic necrotizing factor type I (CNF1) is a chromosome-encoded toxin produced by E. coli strains isolated from diarrhea, septicemia or UTI in humans, piglets or calves . The cytotoxic necrotizing factor type II (CNF2) expressed by E. coli strains isolated from calves and lambs with enteritis or bacteremia is highly homologous to CNF1 . More recently, a low-molecular-mass peptide named enteroaggregative E. coli heat-stable enterotoxin I (EAST 1) produced by enteroaggregative E. coli (EAggEC) has been described . The plasmid-encoded EAST 1, also detected on human or porcine enterotoxigenic E. coli, is genetically and immunologically distinct from other E. coli ST enterotoxins .
Some human CNF1-producing E. coli express P fimbriae . P or P-related (Prs) fimbriae are the most frequent E. coli adhesins associated with human UTI. The G adhesin subunit located at the tip of P fimbriae mediates adhesion to an α-d-galactosyl-(1–4)-β-galactopyranose (Gal-Gal)-containing receptor present on host epithelial cells . Recently, it has been demonstrated that porcine CNF1-producing E. coli also express P or S but not Afa adhesins . The S adhesin expressed by E. coli strains causing newborn meningitis and human UTI mediates adhesion to an α-sialyl-β-2,3-Gal-containing receptor present on bovine and human erythrocytes . The Afa I and Afa III afimbrial adhesins are expressed by uropathogenic E. coli and diarrhea-associated human E. coli, and mediate adhesion to the Dr blood group antigen .
In a previous report, we have demonstrated that bovine CNF2-producing E. coli were genetically associated with fimbriae belonging to the F17 family . In contrast, these CNF2-positive E. coli did not express the afimbrial CS31A adhesin whereas F17 fimbriae were often associated with CS31A in bovine isolates [10, 11]. The CS31A adhesin, first described on E. coli strains isolated from calves with septicemia or diarrhea, is closely related to K88 fibrillae expressed by porcine ETEC and promotes adhesion to an N-acetylneuraminic acid-containing receptor present in human Int-407 cells [12–14]. More recently, CS31A-related antigens have been described on E. coli strains isolated from stools of hospitalized patients with diarrhea and on Klebsiella pneumoniae strains involved in nosocomial infections [14, 15]. Fimbriae belonging to the F17 family were produced by E. coli strains isolated from septicemic or diarrheic calves and lambs and promote inhibitable N-acetyl-d-glucosamine in vitro adhesion on calf intestinal villi [10, 11].
In this report, genetic detection of the EAST I toxin and different adhesive factors was performed on 56 CNF1-producing bovine E. coli isolated from feces or organs. The association of the gene encoding the CNF1 toxin with those encoding the CS31A, F17, P, S and Afa adhesive factors or EAST 1 toxin was analyzed.
2Materials and methods
2.1Bacterial strains and growth conditions
The 31A reference strain producing the CS31A antigen was isolated from the intestinal content of a calf with diarrhea, and causes experimental septicemia in colostrum-deprived calves . The HB101 E. coli strain was used as a negative control. The P4271 E. coli strain was the reference strain producing K88 fibrillae . The 47 CS31A-positive strains and the 28 CS31A-negative strains used to validate the PCR detection of the clpG gene were from our own collection.
The 56 CNF1-producing strains isolated from intestinal contents or organs of cattle with diarrhea have been described elsewhere . The bacterial strains were isolated in 1977–1990 from different herds . Most of these strains showed properties of septicemic strains (resistance to the bactericidal activity of serum, aerobactin and α-hemolysin production). None of the strains reacted with DNA probes specific to enterotoxins STaP, STb, LT-I and LT-IIa or to verotoxins VT-I and VT-II . Some of the isolates adhere in vitro to calf intestinal villi but do not produce K99, F17a or F111 adhesins . The 51 CNF-negative E. coli strains (22 CS31A-positive and 29 CS31A-negative E. coli strains) were isolated from the intestinal content of 51 calves less than 3 weeks old with diarrhea. Fecal samples were received in our laboratory in 1996–1997 for identification of possible enteropathogens.
The bacterial strains tested by PCR were grown for 16 h at 37°C in Luria-Bertani broth medium containing (per liter) 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl (pH 7.0). Phenotypic assays (dot blotting) were performed from strains grown on Minca medium .
2.2Detection of clpG gene and CS31A antigen
The nucleotide sequence of the genes encoding the CS31A, K88ab, K88ac and K88ad structural subunits were aligned for maximum homology using the FASTA program (GenBank accession numbers: M55389, V00292, M35954 and M35637 respectively). The nucleotide primers clpG1 (GGGCGCTCTCTCCTTCAAC) and clpG2 (CGCCCTAATTGCTGGCGAC) specific for the clpG gene encoding the structural subunit of CS31A were designed by direct inspection of multialigned sequences. The primers synthesized and purified by Eurogentec (Seraing, Belgium) were located at 153 bp and 537 bp respectively on the ORF encoding clpG. DNAs to be amplified were released from whole cells by boiling as previously described . The PCR conditions have been described elsewhere . The parameters were denaturation for 2 min at 94°C, annealing of primers at 55°C for 1 min and primers extension for 1 min at 72°C. The 25 cycles of amplification were performed on a GeneAmp 2400 thermal cycler (Perkin Elmer).
Phenotypic detection of the CS31A antigen was performed by dot blotting using antiserum directed against the native form of CS31A as previously described .
2.3Genotypic detection of toxin and adhesins
The PCR methods used to detect the gene encoding the EAST 1 toxin and the F17-family fimbriae have been described elsewhere [4, 10]. The genetic detection of pap, afa and sfa adhesin-encoding operons was performed by a multiplex PCR method previously described . The genes encoding the three allelic variants (classes I, II and III) of the G adhesin (located at the tip of P fimbriae) were also detected by a multiplex PCR method . The strains used as positive controls are summarized in Table 1.
Table 1. Reference E. coli strains used to detect genes encoding adherence factors and toxin by PCR
|PapG (class I)||HB101 (pHRU845)|||
|PapG (class II)||HB101 (pPILL110-35)|||
|PrsG (class III)||HB101 (pJFK102)|||
3Results and discussion
3.1Validation of the PCR method used to detect the clpG gene encoding the CS31A structural subunit
An amplified product with the expected size (402 bp) was obtained from the 31A E. coli reference strain when the clpG1/clpG2 primer pair was used. In contrast, no PCR product was amplified with the HB101 or P4271 bacterial strains (results not shown). To validate the protocol, 47 CS31A-positive and 28 CS31A-negative strains were also tested by PCR. The results were as expected and a strict correlation between genotypic and phenotypic assays was observed (results not shown).
3.2Genetic detection of adhesins and toxin genes
All the CNF1-producing strains were genetically associated with the pap operon (Table 2). These strains isolated from intestinal contents or organs are resistant to the bactericidal effect of serum and produce aerobactin and α-hemolysin (α-Hly) . It is well documented that in human uropathogenic isolates the cluster of genes encoding CNF1 toxin, P fimbriae and α-Hly are closely linked on a pathogenicity island (PAI) . In accordance with our results, similar PAIs have been recently described on the chromosome of bovine CNF1-producing E. coli strains . Although all the isolates were pap-positive, the gene encoding the class III allelic variant of the Pap G adhesin was observed on 64% of the bovine CNF1-producing E. coli, whereas genes encoding class I and class II adhesins were not detected. These results suggest that new variants of the adhesin subunit encoded by the pap operon may be undetectable by the highly specific PCR method or that these pap-positive strains contain partial or modified copies of P fimbrial gene clusters. The papG or prsG genes are not detected among fecal isolates collected from healthy cows  suggesting a role of G adhesin in the infectious process of the CNF1-producing isolates. The presence of class III adhesin (frequently associated with human acute cystitis) may facilitate the spread from the intestine to extraintestinal sites such as the bladder. In addition, genetic detection of S and Afa fimbriae also expressed by E. coli associated with UTI was performed (Table 2). Analysis of the results revealed that S (34%) but not Afa adhesive factors were detected among the CNF1-producing strains. These results were in accordance with the recent study on septicemic CNF1-producing E. coli strains isolated from pigs .
Table 2. Genotypic detection of adherence factors and toxin among bovine E. coli strains
|CNF1-positive||56||56 (100)||19 (34)||0||40 (71)||37 (66)||35 (62.5)||87%|
|CNF-negative||51||18 (35)|| 2 (4)||0||22 (43)||20 (39)||16 (31)||72%|
A high prevalence of the gene encoding the CS31A structural subunit (71%) was observed among the bovine CNF1-producing strains (Table 2). These results were confirmed by a phenotypic assay. The CNF1 toxin is chromosome-encoded , whereas the genes encoding CS31A are located on large plasmids carrying additional virulence factors such as aerobactin production and antibiotic resistance . Therefore, strains producing both the CNF1 toxin and the CS31A antigen represent a new example of association between bacterial clones and plasmid-mediated virulence factors. The gene encoding the F17 structural subunit was not detected among CNF1-producing strains whereas F17 are usually detected on about 50% of CS31A-producing pathogenic E. coli isolated from calves . In accordance with results previously described , we suggest that genes encoding CNF1 and F17 are not present together on the bacterial chromosome. Interestingly, CS31A was prominent among CNF1-producing strains but was not detected among bovine E. coli strains producing the closely related CNF2 toxin . In contrast, previous studies have demonstrated that CNF2 toxin is often associated with F17 fimbriae [3, 10].
The gene encoding the EAST 1 toxin was detected on 66% of CNF1-producing isolates (Table 2). In addition, the EAST 1 gene was detected among 87% of CS31A-positive E. coli strains producing CNF1 (Table 2). To validate the close association between the EAST 1 and CS31A genes, EAST 1 toxin gene detection was performed on a collection of 51 bovine CNF1-negative strains (22 CS31A-positive and 29 CS31A-negative E. coli strains). Analysis of the results confirms the strong association between CS31A and the EAST 1 toxin, since the EAST 1 gene was also detected among 72% of the CS31A-positive but CNF1-negative strains (Table 2). These results suggested that the EAST 1 gene is linked to clpG gene rather than to the CNF1-encoding gene.
In addition to AeggEC, the EAST 1 toxin was found associated with O157:H7 enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), diffusely adhering E. coli (DAEC) and enteropathogenic E. coli (EPEC) [4, 20]. A strong association of EAST 1 was observed with porcine ETEC that possesses the K88 adherence factor and with human ETEC associated with the colonization factor antigen CFA/II [4, 20], suggesting that EAST 1 is distributed among ETEC isolated from human or domestic animals and seems to be associated with the adherence factor type . Interestingly, the CS31A antigen is closely related to the porcine K88 adhesin, also associated with EAST 1 toxin .
In conclusion, we have demonstrated that CS31A is strongly associated with the CNF1 toxin and the P fimbriae among bovine pathogenic E. coli strains. Furthermore, the prevalence of EAST 1 among bovine CS31A-positive strains may be a new example of association of the EAST 1 toxin with an adhesive factor type. These results confirm the wide distribution of EAST 1 among different pathogenic E. coli strains isolated from human and domestic animals. We are investigating the putative genetic linkage between EAST 1 and CS31A.
We thank Majlis Svensson (Lund, Sweden) for providing P-positive E. coli strains and Arlette Darfeuille-Michaud (Clermont-Ferrand, France) for providing the H10407 bacterial strain.