Volume 4, Number 2—June 1998
Resistance to Dryness of Escherichia coli O157:H7 Strains from Outbreak in Sakai City, Japan, 1996
To the Editor: A large outbreak of Escherichia coli O157:H7 with more than 6,000 cases occurred in Sakai City, Osaka Prefecture, Japan, in July 1996 (1); after the outbreak, more than 1,000 secondary infections occurred in the families of the patients (2). We studied the resistance of E. coli O157:H7 to dryness because the survival on surfaces of inert materials under dry conditions may be related to the transmissibility of the strains.
E. coli O157:H7 strains grown on 3.0% nutrient broth with 1.5% agar for 20 to 22 hours at 37°C were suspended at a concentration of approximately 5 x 108 cfu/ml in a 10% skim milk, 0.5% NaCl solution. Aliquots (10 µl) of bacterial suspensions were spread to approximately 10 cm2 on plastic petri plates for bacterial culture and dried under the air flow of a clean bench until no aliquots were evident. After storage in the dark at room temperature, bacteria were harvested with saline and gauze and the viable number was counted (n = 3).
The log reductions 12 hours after drying were employed to show the resistance levels of E. coli O157:H7 to dry stress. The log reductions of the three strains from the Sakai City outbreak (RIMD0509950, RIMD0509894, and RIMD0509951) were 0.04 ± 0.34, 0.14 ± 0.06, and 0.20 ± 0.60, respectively (mean = 0.13), whereas those of the E. coli O157:H7 strains from the other cases varied from 0.71 ± 0.18 to 4.57 ± 1.02. (RIMD0509861, 0.71 ± 0.18; ATCC35150, 1.15 ± 0.35; RIMD0509826, 1.23 ± 0.60; RIMD0509742, 1.28 ± 0.18; ATCC43890, 1.31 ± 0.17; RIMD0509933, 1.48 ± 0.19; RIMD0509932, 1.48 ± 0.22; RIMD0509765, 3.60 ± 0.24; RIMD0509764, 4.57 ± 1.02; mean = 1.87). Although the strain from the Sakai City outbreak (RIMD0509950) survived for at least 35 days under the dry conditions, the strains from the other cases had no viable cells after 7 days. The log reductions of E. coli JM109 and DH10B strains were more than 10, and no viable cells were detected in 12 hours.
The strains from the Sakai City outbreak also showed marked acid resistance. Acid resistance of E. coli O157:H7 has been reported to depend on rpoS, which is induced in a stationary phase (3). Since the E. coli O157:H7 strains in a stationary phase were more resistant to dry and acid stresses than those in a log phase, rpoS may also be associated with resistance to dry stress. However, no deletions of rpoS were detected by polymerase chain reaction analysis in the E. coli O157:H7 strains used in this experiment. Further study on the mechanism of resistance will be needed to establish new strategies for eradicating the bacteria.
A case-control study by the Ministry of Health and Welfare of Japan showed that uncooked radish sprouts were the vehicle of the largest outbreak in Sakai City (4). In two small outbreaks of E. coli O157:H7 in March 1997, the vehicle of infection might have been radish sprouts; therefore, the possible contamination of white radish seeds with E. coli O157:H7 has been discussed (5). If such contamination was present, dry resistance might be involved in the survival on or in white radish seeds because the bacteria were exposed to dry conditions for a long period before sprouting. We propose that dry resistance be considered an important factor in infection.
- National Institute of Health and Infectious Diseases Control Division, Ministry of Health and Welfare of Japan. Verocytotoxin-producing Escherichia coli (Enterohemorrhagic E. coli) infections, Japan, 1996-June, 1997. Infectious Agent Surveillance Report. 1997;18:153–4.
- Takatorige T. Study on the patients of the diarrheal disease outbreak among schoolchildren in Sakai City; oc-currence of infections among the families of the school-children. In: Report of Osaka Prefecture Research Society for Enterohemorrhagic E. coli Infectious Disease, Osaka; 1997. p. 85-96. (in Japanese)
- Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl Environ Microbiol. 1996;62:3094–100.
- Michino H, Araki K, Minami S, Takaya S, Sakai N. Investigation of large-scale outbreak of Escherichia coli O157:H7 infection among schoolchildren in Sakai City, 1996. In: Proceedings of the 32nd Joint Conference on Cholera and Related Diarrheal Diseases, U.S.-Japan Cooperative Medical Science Program(USJCMSP); 14-16 Nov 1996; Nagasaki University, Nagasaki, Japan. USJCMSP: 1996. p. 84-9.
- Nathan R. American seeds suspected in Japanese food poisoning epidemic. Nat Med. 1997;3:705–6.