TEXT

Top Abstract Text References

Enterohemorrhagic Escherichia coli (EHEC), particularly of serotype O157:H7, have emerged in recent years as the predominant cause of hemorrhagic colitis, which can progress into a hemolytic uremic syndrome in humans (11, 14, 16). Molecular and biochemical studies showed that serotype O157:H7 is a discrete clone (17, 18). Unlike other E. coli, isolates of serotype O157:H7 do not ferment D-sorbitol within 24 h, lack -glucuronidase activity, and do not grow at 45.5°C (2, 5, 6). These traits, especially the absence of sorbitol fermentation, are used extensively to distinguish isolates of serotype O157:H7 from other E. coli serotypes (4, 12). However, the emergence of phenotypic variants has sporadically been observed. It has been reported that serotype O157:H7 can mutate to a sorbitol-positive phenotype either in food products or occasionally during culture in D-sorbitol-containing media (7).

In E. coli, D-sorbitol and some other hexitols are trapped by vectorial phosphorylation (8, 9). Utilization of D-sorbitol is initiated by a specific enzyme II-enzyme III complex of the phosphotransferase system (PTS), which converts the substrate to D-sorbitol-6-phosphate. The phosphate donor in this reaction is phosphoenolpyruvate (PEP). D-Sorbitol-6-phosphate is then converted to D-fructose-6-phosphate by an NAD-linked dehydrogenase. The transport system acts on D-sorbitol with a high affinity and on D-mannitol with a low affinity.

In the present study, we tested the influence of growth temperature on the ability to ferment D-sorbitol in E. coli O157:H7. All strains were from the Centre National de Référence de Typage Moléculaire Entérique at The Institut Pasteur. Non-sorbitol-fermenting O157:H7 strains will be referred to here as Sor. In a first experiment, 13 Sor and one sorbitol-fermenting (Sor⁺; strain 96336) O157:H7 strain were inoculated with 30 µl of culture in D-sorbitol-peptone water containing 10 mM D-sorbitol and bromothymol blue as an indicator (3) and then incubated at either 30, 37, or 40°C. The culture conditions rendered the incubation medium anaerobic. A Sor⁺ non-O157:H7 strain (strain 75-88) was included as a control. Although all isolates were able to grow in this medium at 30, 37, and 40°C, they could not produce acid from sorbitol at 37 and 40°C. We observed that for 13 Sor E. coli O157:H7 strains, acid production from D-sorbitol could be detected at 30°C but not at 37 and 40°C after incubation for 3 to 4 days. For the two Sor⁺ strains (96336 and 75-88), acid production from D-sorbitol was observed within 24 h at all of the temperatures tested. This temperature sensitivity was restricted to strains grown on D-sorbitol. The thirteen Sor strains were able to produce acid from D-mannitol, D-dulcitol, and L-sorbose within 24 h at 30, 37, and 40°C (data not shown).

To verify the temperature effect on sorbitol fermentation, the anaerobic growth behavior of Sor and Sor⁺ strains at 30 and 40°C was studied (Fig. 1). The time course of cell growth and D-sorbitol utilization by one Sor O157:H7 strain (Ec40) and two Sor⁺ strains (96336 and 75-88) grown on peptone-water containing 10 mM D-sorbitol at 30 and 40°C were studied. Enzymatic assay of sorbitol was determined by using a sorbitol test kit (Boehringer GmbH, Mannheim, Germany). At 40°C, strain Ec40 (wild type) was unable to utilize D-sorbitol as a carbon source and grew at the expense of peptones in the growth medium. At 30°C, a biphasic growth curve was obtained. At this temperature, strain Ec40 utilized peptones first and then D-sorbitol with a lag period of 72 h (Fig. 1A). Growth inhibition at 40°C was not observed with Ec40 grown on D-mannitol. Furthermore, anaerobic growth on D-mannitol was faster than on D-sorbitol (data not shown). Whatever growth temperature used, the two Sor⁺ strains (96336 and 75-88) showed high anaerobic growth yields (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Anaerobic growth of Sor and Sor+ strains of E. coli O157:H7 on D-sorbitol at 30 and 40°C. The means of at least three determinations are presented, and the standard deviation was 0.01 to 0.05. (A) Cells of Ec40 (circles) and its sorbitol Sor+ variant (triangles) were grown anaerobically at 30°C (open symbols) and at 40°C (filled symbols) on peptone broth medium containing 10 mM D-sorbitol. (B) Cells of 96336 (circles) and 75-88 strains (triangles) growing anaerobically at 30°C (open symbols) and at 40°C (filled symbols) on the same medium.

A Sor⁺ variant could be isolated from strain Ec40, after four or six overnight transfers at 30°C, in peptone broth medium containing 10 mM D-sorbitol. Anaerobic growth of this variant reached the stationary phase after 24 h at 30°C and after 48 h at 40°C (Fig. 1A). D-Sorbitol was utilized simultaneously. Similarly, Sor⁺ variants have been obtained for all Sor strains studied. These latter variants showed the same growth behavior as the Sor⁺ variant of strain Ec40.

The inability to grow at higher temperatures on hexitols has already been reported (10, 15). Several works suggested that this growth inhibition is apparently due to a temperature-sensitive enzyme. To investigate this possibility, transport and metabolism of D-sorbitol were studied in Sor and Sor⁺ strains of E. coli O157:H7 at 30 and 40°C (Table 1). Since D-sorbitol transport system accumulates both D-sorbitol and D-mannitol, the PTS activities towards both hexitols were investigated as described previously (1). We measured the PEP-dependent phosphorylation of [¹⁴C]sorbitol and [¹⁴C]mannitol in toluene-treated cells. After growth at 30°C on D-sorbitol, strains 75-88 (Sor⁺ non-O157:H7 strain) and 96336 (Sor⁺ O157:H7 strain) exhibited PEP-dependent phosphorylation of [¹⁴C]sorbitol and [¹⁴C]mannitol. Strain of Ec40 (wild type) showed a very low PTS activity for D-sorbitol and D-mannitol, whereas in the Sor⁺ variant the transport activities were detected. When the same experiments were done with cells grown at 40°C we observed that, in Sor⁺ O157:H7 strains (96336 and Ec40 variant), the sorbitol transport was lower than in cells grown at 30°C. However, in strain 75-88, transport activities were not inactivated at 40°C. D-Sorbitol- and D-mannitol-phosphotransferase systems were induced in cells grown on D-mannitol, whereas the sorbitol-PTS activity was induced to a low level. Both activities were present at both temperatures. The detection of an ATP-dependent phosphorylation activity of D-sorbitol and D-mannitol suggested the presence of kinases. It thus appears that O157:H7 strains demonstrate a temperature-sensitive D-sorbitol transport.

View this table: [in this window] [in a new window]   TABLE 1.   Activities of the PTS and sorbitol-6-phosphate dehydrogenase in Sor+ and Sor strains of E. coli growth anaerobically at 30 and 40°C

The effect of the temperature on the sorbitol-6-phosphate dehydrogenase activity was studied in cell extracts of strains grown at 30 and 40°C. The sorbitol-6-phosphate dehydrogenase activity was assayed as described previously (13). It was found that the dehydrogenase activity was lower in cell extracts of strains grown at 40°C than in cells grown at 30°C (Table 1). This dehydrogenase activity was inducible by D-sorbitol but not by D-mannitol and was stable at a high temperature. No appreciable loss of activity was found in cell extracts incubated 60 min at 40°C (data not shown).

Our results show that Sor⁺ O157:H7 strains express a temperature-sensitive D-sorbitol phenotype. D-Sorbitol transport and sorbitol-6-phosphate dehydrogenase activities were expressed in cells grown at 30°C but only at a low level at 40°C. Sorbitol-positive variants able to transport D-sorbitol were easily selected at 30°C from a culture of Sor E. coli O157:H7 in D-sorbitol-containing medium. Genetic analysis will be required to establish the type of mutation undergone by these variants.