Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor

Abstract

Escherichia coli O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of E. coli O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction of E. coli O157:H7 Tn7gfp transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), both E. coli O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10(9) and 10(6) E. coli O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log(10) cells but did not detect culturable cells. These findings indicate that E. coli O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

Fig. 1.

Culturability of E. coli O157:H7 plated on TSA (filled symbols) and SMAC (open symbols) media. E. coli O157:H7 ATCC 43895 (diamonds) and BRMSID 188 (squares) were incubated at 4°C in sterile deionized distilled water for up to 120 days. The data are mean values (n = 4) from two independent experiments with errors bars representing the standard deviations.

Fig. 2.

Fate of E. coli O157:H7 ATCC 43895 Tn7gfp (filled symbols) and BRMSID 188 Tn7gfp (open symbols) on lettuce plants incubated at 8°C (A) and 16°C (B). Culturable populations on TSA (diamonds) and SMAC (squares) media and viable counts (circles) are indicated. The data are mean values (n = 6) from two independent experiments with different bacterial cultures and different sets of plants. Error bars represent the standard deviations.

Fig. 3.

Effect of inoculum size on the VBNC response of E. coli O157:H7 ATCC 43895 Tn7gfp (A) and BRMSID 188 Tn7gfp (B). Lettuce leaves were inoculated with 10⁹ (filled symbols) and 10⁶ (open symbols) E. coli O157:H7 bacteria per g of leaf and incubated at 8°C. Culturable populations plated on TSA (diamonds) and SMAC (squares) media and viable counts (circles) are indicated. The data are mean values (n = 6) from two independent experiments with different bacterial cultures and different sets of plants. Error bars represent the standard deviations.

Fig. 4.

(A) Confocal microscopy showing the presence of VBNC forms of E. coli O157:H7 BRMSID 188 Tn7gfp in large clusters at 16 days postinoculation on the surface of lettuce leaves. (B) Optical cross-section of the same image that indicates the localization of E. coli O157:H7 cells on the surface of plant tissue. Scale bar, 20 μm.