Susceptibility of Vibrio parahaemolyticus to disinfectants after prior exposure to sublethal stress

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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2009, p. 870–871 0095-1137/09/$08.00⫹0 doi:10.1128/JCM.02306-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 47, No. 3

Susceptibility of Vibrio parahaemolyticus to Tris-Dependent DNA Degradation during Pulsed-Field Gel Electrophoresis䌤 This study reports a characteristic of some Vibrio parahaemolyticus strains which are susceptible to electrophoresis-related DNA-degradation (ERDD) by pulsed-field gel electrophoresis (PFGE) in Tris-borate EDTA (TBE) buffer. V. parahaemolyticus is a halophilic bacterium naturally inhabiting coastal environments worldwide, with reported pandemic strains, warranting the need for surveillance and epidemiological research on clinical and food isolates for source tracking and risk assessment (10). PFGE has been widely used as the “gold standard” for molecular typing of bacterial isolates, to which other molecular typing techniques are compared (3–5). DNA degradation yielding a smeared profile and affecting 100% typeability of strains has been observed in other bacteria (3, 4, 7). However, the proportion and impact of V. parahaemolyticus strains prone to similar degradation are not known. The strains of V. parahaemolyticus used in this study are shown in Table 1. All of the characterized isolates were obtained from our culture collection (1). PFGE was performed according to the standard protocol for nontyphoidal Salmonella (Pulse-Net, Centers for Disease Control and Prevention, Atlanta, GA) (2, 6, 9) with modifications. Briefly, organisms were grown on tryptic soy agar with 2% NaCl (pH 8.5) at 35°C overnight and suspended in buffer (100 mM Tris, 100 mM EDTA [pH 8.5]) to a density of 0.6 ⫾ 0.02 in a Dade Microscan turbidity meter (Dade Behring, Inc., West Sacramento, CA). Cell suspensions were treated with 4% formaldehyde to arrest the endogenous DNase activity or were untreated and immobilized into low-melting-point, SeaKem Gold (SKG) agarose plugs, lysed, washed, and digested with NotI or SfiI, as described elsewhere (6). Restriction fragments were resolved in 1% SKG agarose by PFGE using a CHEF DRIII aparatus (Bio-Rad Laboratories, Hercules, CA) containing 0.5⫻ TBE buffer with or without 50 ␮M thiourea. Restricted DNAs were electrophoresed at 6 V/cm for 22 h at 14°C, with pulse times ramping from 2 s to 40 s. Gels were stained, destained, and

photographed using a Gel-Doc 1000 system (Bio-Rad) as described earlier (4). The ERDD phenomenon was observed in 29 out of 164 (⬃18%) of the isolates from seafood using NotI digestion (figures not shown). Among the representative strains (Table 1), ERDD was observed irrespective of whether NotI or SfiI was used for digestion (figures not shown). Both clinical (tdh⫹ tlh⫹) and environmental (tdh⫺ tlh⫹) strains (Fig. 1) were susceptible. The effectiveness of thiourea in preventing smearing of NotI-digested V. parahaemolyticus DNA can be seen in Fig. 1. The use of formaldehyde before DNA isolation did not prevent ERDD in the selected strains, but addition of thiourea (50 ␮M) to the running buffer resolved the problem without altering the macrorestriction profiles of the typeable strains (Fig. 1). We recommend doing electrophoresis using the regular buffer (TBE) to detect the ERDD types of V. parahaemolyticus, which can then be run separately by using thiourea in the running buffer to obtain macrorestriction patterns. Using this protocol, the ERDD subtypes can be identified and their banding patterns revealed with minimum exposure, use, and disposal of the toxic compound thiourea.

Acknowledgments and appreciation are expressed to the Listeriosis Reference Service for Canada, particularly Franco Pagotto and Kevin Tyler, for expertise and generosity in sharing their laboratory equipment for PFGE analysis.

REFERENCES 1. Banerjee, S. K., S. Pandian, E. C. Todd, and J. M. Farber. 2002. A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters. J. Food Prot. 65:1049–1053. 2. Centers for Disease Control and Prevention. 2002. Standardized molecular subtyping of foodborne bacterial pathogens by pulsed-field gel electrophoresis. Centers for Disease Control and Prevention, Atlanta, GA. 3. Inglis, T. J. J., L. O’Reilly, N. Foster, A. Clair, and J. Sampson. 2002. Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei. J. Clin. Microbiol. 40:3198–3203. 4. Leclair, D., F. Pagotto, J. M. Farber, B. Cadieux, and J. Austin. 2006. Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic. J. Clin. Microbiol. 44:1635– 1644. 5. Marshall, S., C. G. Clark, G. Wang, M. Mulvey, M. T. Kelly, and W. M. Johnson. 1999. Comparison of molecular methods for typing Vibrio parahaemolyticus. J. Clin. Microbiol. 37:2473–2478. 6. Martinez-Urtaza, J., A. Lozano-Leon, A. DePaola, M. Ishibashi, K. Shimada, M. Nishibuchi, and E. Liebana. 2004. Characterization of pathogenic Vibrio parahaemolyticus isolates from clinical sources in Spain and comparison with Asian and North American pandemic isolates. J. Clin. Microbiol. 42:4672–4678. 7. Silbert, S., L. Boyken, R. J. Hollis, and M. A. Pfaller. 2003. Improving typeability of multiple bacterial species using pulsed-field gel electrophoresis and thiourea. Diagn. Microbiol. Infect. Dis. 47:619–621. 8. Spite, G. T., D. P. Brown, and R. M. Twedt. 1978. Isolation of an enteropathogenic, Kanagawa-positive strain of Vibrio parahaemolyticus from seafood implicated in acute gastroenteritis. Appl. Environ. Microbiol. 35:1226– 1227. 9. Wagley, S., K. Koofhethile, J. B. Wing, and R. Rangdale. 2008. Comparison

TABLE 1. Strains of V. parahaemolyticus used in this study Result from PCR analysis targeting: Strain

Thermolabile hemolysin (tlh)

Thermostable hemolysin (tdh)


ATCC 17802

NY477 S107-2

⫹ ⫹

⫹ ⫺




American Type Culture Collection 1, 8 Ladysmith Harbor, British Columbia, Canada, May 2005 Ladysmith Harbor, British Columbia, Canada, May 2005 Ship’s Point, British Columbia, Canada, July 2005 Ladysmith Harbor, British Columbia, Canada, August 2005 870

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FIG. 1. PFGE patterns of V. parahaemolyticus strains digested with NotI and run with regular TBE buffer (A) and TBE buffer to which thiourea was added (B). Four seafood isolates and two standard strains (Table 1) were processed without (lanes 1 to 6) as well as with (lanes 7 to 12) formaldehyde fixation and run separately with the two buffers. Lanes M, standard DNA size markers, consisting of DNA from Salmonella enterica serovar Braenderup H9812 digested with 50 U of XbaI at 37°C for 2 h.

of Vibrio parahaemolyticus isolated from seafoods and cases of gastrointestinal disease in the UK. Int. J. Environ. Health Res. 18:283–293. 10. Wong, H., S. Liu, C. Chiou, M. Nishibuchi, B. Lee, O. Suthienkul, G. B. Nair,

C. A. Kaysner, and H. Taniguchi. 2007. A pulsed-field gel electrophoresis typing scheme for Vibrio parahaemolyticus isolates from fifteen countries. Int. J. Food Microbiol. 114:280–287.

Swapan K. Banerjee* Jeffrey M. Farber Bureau of Microbial Hazards Food Directorate, Health Canada Tunney’s Pasture Ottawa, Ontario K1A 0K9, Canada *Phone: 613-941-9141 Fax: 613-941-0280 E-mail: [email protected]

Published ahead of print on 7 January 2009.

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