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 fiexneri 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.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

“…All bacterial isolates were ipaH + , as expected because this gene has been found in multiple copies on both the chromosome and the virulence plasmid of Shigella (Kingombe et al 2005, Thong et al 2005. Conversely, the ial locus is exclusively observed on the virulence plasmid and was only detected in some Shigella isolates.…”

“…The ipaH, ial, set1A, sen, sat and iuc virulence markers were amplified as previously reported, with slight modifications (Lüscher & Altwegg 1994, Vargas et al 1999, Ruiz et al 2002, Kingombe et al 2005. Approximately 20 ng of bacterial DNA was used as the template for amplification reactions.…”

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR

“…However, Shigella spp. and entero-invasive E. coli [EIEC] are genetically close and exhibit considerable antigenic cross-reactivity, thus differentiating between them using a single method can be difficult (Cheasty and Rowe, 1983;Lan et al, 2001;Kingombe et al, 2005;Yang et al, 2005).…”

Molecular characterization of Salmonella strains in individuals with acute diarrhea syndrome in the State of Sucre, Venezuela

“…Multiplex PCR has also been used for the identification of Salmonella Typhimurium and Enteritidis in environmental swabs and samples taken from chicken farms, thus demonstrating the scope of the PCR technique in epidemiological studies 36 . Oliveira et al 18 used PCR for the detection of strains of Salmonella sp.…”