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Phenylpropionic Acid Metabolism: a Marker for Enteropathogenic Escherichia coli Clonal Group 2 Strains

Enteropathogenic Escherichia coli (EPEC), a leading cause of infantile diarrhea worldwide, includes 12 O serogroups expressing mainly flagellar antigens H2, H6 and H34. Other serotypes are known, but their role in diarrhea has not been firmly established (4). Typically, EPEC strains of these serotypes harbor a pathogenicity island, designated LEE (for locus of enterocyte effacement), that contains all the genes necessary to produce the attaching and effacing (A/E) lesion and a 60- to 70-MDa plasmid (EAF plasmid) that contains fimbrial and regulatory genes (6). Some other EPEC serotypes do not possess the EAF plasmid, and for this reason they have been called atypical EPEC (5). In 1995, Whittam and McGraw (8) showed by multilocus enzyme electrophoresis that strains of serotypes bearing antigens H2, H6, and H34 could be divided into two clusters of related electrophoretic types, which they designated EPEC 1 and EPEC 2. EPEC 1 strains possess H6 and H34 antigens, and EPEC 2 strains possess H2 antigens (8). Other differences between EPEC 1 and EPEC 2 have been reported; for example, distribution of intimin types (1) and the site of insertion of the LEE pathogenicity island (7). In this letter, we report that, in addition to those differences between the two EPEC groups, EPEC 2 strains have the ability to form colored products originating from the metabolism of phenylpropionic acid (Table ​(Table1).1). The fact that enteroaggregative E. coli (EAEC) strains of serotype H2 gave negative results in this test suggests that this ability to metabolize phenylpropionic acid in this way is exclusive to EPEC strains. 
 
 
 
TABLE 1 
 
Formation of colored products from 3-phenylpropionic acid by EPEC serotypes of clonal groups 1 and 2 
 
 
 
The routine test to verify whether an E. coli stain has the ability to metabolize phenylpropionic acid is very simple and easy to perform. One needs to inoculate the E. coli strain on a nutrient agar slant containing 0.2 g of 3-phenylpropionic acid per liter and incubate the slant at 37°C for 24 h. The production of colored compounds is indicated by a change in the color of the medium to reddish-dark brown. This color change is apparently due to the incomplete degradation of 3-phenylpropionic acid, with accumulation of 3-(2,3-dihydroxyphenyl)propionic acid which suffers autooxidation to semiquinone or quinone, giving the red color (2). 
 
The phenylpropionic acid test is not usually included in the series of biochemical tests used to identify E. coli, but it has been used previously by D’Allessandro and Comes (3) and Zuliane and Trabulsi (9) to identify EPEC biotypes. The phenylpropionic acid test now appears to provide an additional tool in epidemiological studies to distinguish between the EPEC clonal groups, EPEC 1 and EPEC 2.