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Journal of Clinical Microbiology, September 2003, p. 4480-4482, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4480-4482.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Multiplex PCRs for Identification of Necrotoxigenic Escherichia coli

Sigrid Van Bost,1* Etienne Jacquemin,1 Eric Oswald,2 and Jacques Mainil1

University of Liège, Faculty of Veterinary Medicine, Laboratory of Bacteriology, Sart-Tilman B43a, B-4000 Liège, Belgium,1 Institut National de la Recherche Agronomique, Unité de Microbiologie Moléculaire, 31076 Toulouse Cedex, France2

Received 5 May 2003/ Returned for modification 13 June 2003/ Accepted 3 July 2003


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ABSTRACT
 
Two multiplex PCRs were developed for the detection of necrotoxigenic Escherichia coli virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference E. coli strains and by multiplex PCRs with necrotoxigenic E. coli strains isolated from different animal species.


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TEXT
 
Most Escherichia coli strains are commensal inhabitants of the gastrointestinal tract, but some strains can be pathogenic. Those are characterized by the production of different factors that contribute to virulence. In the case of necrotoxigenic E. coli (NTEC), these may include fimbrial or afimbrial adhesins, the siderophore aerobactin, and two toxins: the cytotoxic necrotizing factor (CNF) and the cytolethal distending toxin (CDT).

Two different types of NTEC have actually been reported: NTEC1 and NTEC2 (3, 4). NTEC1 strains have been isolated from cases of enteritis in ruminants, pigs, dogs, rabbits, and horses and from extraintestinal infections in pigs, dogs, cats, and humans (5). NTEC2 strains have been mainly isolated from ruminants with intestinal infections or with septicemia (21). They produce two different toxins: CNF1 and CNF2 (3). CNF1 is chromosomally encoded (6), whereas CNF2 is coded for by genes located on the Vir plasmid (19).

In NTEC1 strains, the cnf1 gene can be associated, on a pathogenicity island, with a pap gene cluster (2). The pap/prs gene cluster is composed of genes coding for structural subunits (papA, papE, papF, and papG) and a gene (papC) coding for a transport protein (11). Another adhesin family frequently associated with P fimbriae in NTEC1 strains is the S family (15). The sfa operon is composed of genes coding for a structural subunit (sfaA) and accessory proteins (sfaG, sfaH, sfaS, sfaC, sfaB, sfaD, and sfaE) (9). Several NTEC1 bovine strains can also be associated with the Afa-VIII variant of the Afa family (15).

In NTEC2 strains, the cnf2 gene can be associated, on the Vir plasmid, with genes coding for the fimbrial adhesin F17 (18), for the afimbrial adhesin Afa-VIII (15), and/or for a CDT (20).

The afa operon can have a chromosomal location (in NTEC1 and NTEC2) or be located in the Vir plasmid (in NTEC2) and contains at least four genes: afaB coding for a chaperon, afaC coding for an anchor protein, afaD coding for an invasin, and afaE coding for the structural adhesin (7).

The F17 family includes four antigenic variants based on differences in the structural subunit encoded by the f17A gene of the operon. In NTEC2, the f17 genes can be located on the chromosome or on the Vir plasmid (14).

The CDTs are a family of toxins defined by their ability to block the cell cycle at the G2/M stage, causing the formation of giant elongated cells (20). Two new variants were described: CDT-III in NTEC2 (20) and CDT-IV in NTEC1 strains (I. Tóth, B. Nagy, L. Beutin, B. Szabó, I. Barcs, L. Emody, and E. Oswald, submitted for publication). CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, and cdtC) (20). The cdt-III gene cluster is located on the Vir plasmid (20), whereas the location of the cdt-IV gene cluster appears to be the chromosome (Tóth et al., submitted).

In addition, many NTEC strains possess properties that allow them to be invasive, causing septicemia, bacteremia, and internal organ infections (18). The aerobactin system is encoded by a five-gene operon, with four genes encoding the enzymes needed for aerobactin synthesis (iucA, iucB, iucC, and iucD) and a fifth gene encoding the outer membrane receptor protein (iutA) (8). This operon is frequently located in a plasmid (23).

The goal of this study was to develop a multiplex PCR allowing rapid and specific identification of NTEC and characterization of their specific virulence factors. The approach used some primers already designed in single PCRs (Table 1).


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  		TABLE 1. Sequences of PCR primers and product sizes

Because of the similarity in the sizes of the fragments amplified from f17A and cdt-III genes (Table 1), the primers could not all be used in a single reaction. Two PCR primer sets were then used: M1 and M2. M1 contains the primers for the genes coding for the CNF, CDTs, and aerobactin, and M2 contains the primers for the genes coding for the P, S, F17, and Afa adhesins.

DNA was extracted from bacteria by boiling. Bacteria were harvested from an overnight Luria-Bertani broth culture. Three hundred microliters was centrifuged for 30 s and resuspended in 50 µl of sterile water, boiled for 10 min, and recentrifuged for 30 s.

Two microliters of the supernatant was added to the reaction mixture (50 µl) containing 0.5 µl of each primer (Gibco BRL, Paisley, United Kingdom), 5 µl of the four deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Roosendaal, The Netherlands), 5 µl of the PCR buffer (Roche, Indianapolis, Ind.), and 2.5 U of the Taq DNA polymerase (Roche).

The samples were subjected to 30 cycles of amplification on a thermal cycler (PCR Sprint; Hybaïd). The parameters used in M1 were denaturation for 3 min at 94°C, followed by denaturation for 1 min at 94°C, annealing of primers for 1 min at 60°C, and primer extension for 1 min at 72°C. The final extension was performed at 72°C for 7 min. The parameters used in M2 were denaturation for 5 min at 95°C, followed by denaturation for 1 min at 95°C, annealing of primers for 1 min at 57°C, and primer extension for 1 min at 72°C. The final extension was performed at 72°C for 7 min. Reaction products were then analyzed by electrophoresis on a 1.5% LSI agarose (Life Sciences International, Zellik, Belgium) gel (M1) or a 2% Metaphor agarose (BioWhittaker, Walkersville, Md.) gel (M2), stained with ethidium bromide, and photographed under UV light.

Six E. coli strains were used as controls: 1404, J96, 28c, 239KH89, C600(pABN1) and HS. Strain 1404 is the positive control for the detection of the cnf2, f17A, and cdt-III genes. Strain J96 is the positive control for the detection of the cnf1, pap/prs, and sfa genes. Strain 28c is the positive control for the detection of cdt-IV gene. Strain 239KH89 is the positive control for the detection of the afa-8 gene. Finally, strain C600(pABN1) is the positive control for the detection of iucD gene. HS was used as the negative control (Table 2). Each primer pair was first used in a simplex PCR with the corresponding control strain as a template. Both multiplex reactions were subsequently tested on a pool of appropriate control strains. The fragments amplified from papC and afa8E genes were too close, and use of a 2% Metaphor agarose gel was necessary.


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  		TABLE 2. Colony hybridization and multiplex PCR results for the E. coli strains used in this study

Both multiplex reactions were further tested on E. coli strains isolated from bovines, humans, piglets, and dogs (Table 2). The genotypes of these strains were previously characterized by colony hybridization (16, 17). The results of the multiplex PCRs were in total agreement (Table 2).

The two multiplex PCRs correctly determined the presence of the corresponding genes in all of the strains tested. They were also specific, detecting no other genes than those of the colony hybridization assay. Of course, more strains must be tested before concluding 100% specificity and sensitivity.

Nevertheless, the multiplex PCRs described in this study can be useful methods for rapid identification of the virulence-associated genes of NTEC strains in bovine, porcine, and canine species and humans.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from "Ministère des Classes Moyennes et de l'Agriculture—Recherche et Développement—convention 5936 (2000-2001)."


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FOOTNOTES
 
* Corresponding author. Mailing address: Université de Liège, Faculté de Médecine Vétérinaire, Microbiologie des Denrées Alimentaires B43bis, 20 Bd. de Colonster, B-4000 Liège, Belgium. Phone: 32 4 366 40 14. Fax: 32 4 366 40 16. E-mail: svanbost{at}ulg.ac.be. Back


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Journal of Clinical Microbiology, September 2003, p. 4480-4482, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4480-4482.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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