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Applied and Environmental Microbiology, February 2007, p. 1349-1354, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.02243-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate{triangledown}

Meng Du,1 Jixiang Chen,1* Xiaohua Zhang,1 Aijuan Li,1 Yun Li,1 and Yingeng Wang2

Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, People's Republic of China,1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, People's Republic of China2

Received 22 September 2006/ Accepted 12 December 2006

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4°C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

* Corresponding author. Mailing address: Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, 5# Yushan Road, Qingdao 266003, People's Republic of China. Phone: 86-532-82032767. Fax: 86-532-82032266. E-mail: betcen{at}ouc.edu.cn.

{triangledown} Published ahead of print on 22 December 2006.

Applied and Environmental Microbiology, February 2007, p. 1349-1354, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.02243-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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