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Journal of Clinical Microbiology, March 2009, p. 870-871, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.02306-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Susceptibility of Vibrio parahaemolyticus to Tris-Dependent DNA Degradation during Pulsed-Field Gel Electrophoresis{triangledown}


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LETTER
 
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 0.5x TBE buffer with or without 50 µM 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).


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  		TABLE 1. Strains of V. parahaemolyticus used in this study

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.


Figure 1
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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.

The use of formaldehyde before DNA isolation did not prevent ERDD in the selected strains, but addition of thiourea (50 µM) 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.


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ACKNOWLEDGMENTS
 
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.


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FOOTNOTES
 
{triangledown} Published ahead of print on 7 January 2009. Back


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REFERENCES
 
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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: swapan_banerjee{at}hc-sc.gc.ca

Journal of Clinical Microbiology, March 2009, p. 870-871, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.02306-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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