Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Abstract

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. 10 9 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.Edwardsiella tarda, an enteric gram-negative bacterium of the Enterobacteriaceae, is the causative agent of the systemic disease edwardsiellosis in freshwater and marine fish worldwide. The bacterium causes septicemia with extensive skin lesions and affects internal organs, such as the liver, kidney, spleen, and muscle (33, 52). It has been isolated from a variety of animals, including fish, birds, mammals, and reptiles (12,27,31,46,47), and environmental water (32, 50). Several potential virulence properties have been suggested to contribute to the pathogenesis of E. tarda, namely, the production of dermatotoxin (45) and hemolysin (14) and the ability to resist phagocyte-mediated destruction and to invade epithelial cells (19,22,43). However, little is known about the pathogenic mechanism of E. tarda, and the causes of disease occurrence are still elusive.Many bacteria have developed strategies for metamorphosis into more or less sophisticated survival forms in response to harsh environmental conditions, such as a temperature change, high salinity, or nutrient deprivation (4, 5). The formation of viable but nonculturable (VBNC) cells of bacteria has been proposed as a strategy to survive adverse conditions (10). The first clear evidence of the existence of the VBNC state in pathogens was provided by Xu et al. It has been demonstrated that VBNC cells are active in metabolism (16,20,53) and are able to recover from their dormant state, becoming metabolically active and fully culturable (26,40,48). Some pathogens in the VBNC ...

Nutrient-dependent, rapid transition of Vibrio cholerae to coccoid morphology and expression of the toxin co-regulated pilus in this form

“…GbpA, a characterized GlcNAc-binding protein, is an example of a V. cholerae colonization factor utilized in both the host and the aquatic environment . In addition, pathogenic strains of a variety of bacterial species have been characterized to retain and express virulence factors when in the VBNC state (Du et al, 2007;Fischer-Le Saux et al, 2002;Rahman et al, 1996;Smith & Oliver, 2006b), and also maintain antibiotic resistance (Kamruzzaman et al, 2010;Lleò et al, 2003). In the host environment, virulence factors such as TCP are required for colonization and persistence in the small intestine.…”

Survival behaviour and virulence of the fish pathogen Vibrio ordalii in seawater microcosms

“…In the present study, a visual light microscopy analysis of V. ordalii cells indicated that these were reduced in size after the third day of incubation in the sterile microcosm; however, future analysis through scanning electronic microscopy is needed to confirm this observation. Such a drastic reduction in cell size may be a survival strategy to confer resistance to environmental stress, as has been observed during the starvation survival process for several bacterial species in the aquatic environment (Amy & Morita 1983, Kjelleberg & Hermansson 1984, Jiang & Chai 1996, Vatsos et al 2003, Avendaño-Herrera et al 2006, Du et al 2007, Arias et al 2012. Also in the sterile microcosm, V. ordalii cells changed from a rod to a cocci shape.…”

Challenges in Biofilm Inactivation: The Use of Cold Plasma as a New Approach

“…This approach relies only on the presence of culturable cells but does not take into account that cells might still be alive, although non-culturable, after plasma treatment. This may have catastrophic consequences if microorganisms that are assumed dead, are pathogenic ones who may retain virulence even when they are nonculturable [44]. Plasma-mediated biofilm inactivation may proceed through a first step in which bacterial cells might enter a Viable-ButNon-Culturable (VBNC) state.…”