Molecular Strategies for the Detection, Identification, and Differentiation between Enteroinvasive Escherichia coli and Shigella spp.

Abstract

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc−/ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH−) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella flexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.