A tentative direct microscopic method for counting living marine bacteria

-The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 °C and Scott A strain at 4 °C. No culturable bacteria were detected in the VBNC state, although 10 4 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state. VBNC / virulence / Listeria monocytogenes / plaque forming assay

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“…A modified technique of Kogure et al [17] was used [5] to enumerate VBNC cells of Listeria monocytogenes.…”

Section: Characterization Of Metabolic Activitymentioning

confidence: 99%

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

et al. 2005

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“…Consequently, viable cells will be elongated and can be recognized and counted by microscopy. The addition of nutrients, usually a small amount of yeast extract, is generally required (61). This direct viable count (DVC) method has been applied for detection of viable Vibrio vulnificus (91,105), estimation of viable counts of several gramnegative bacteria in an aquatic environment (15,20,28,29,112,115,127,128,129,150), and detection of viable bacteria in biofilms (151,152).…”

Section: Flow Cytometrymentioning

confidence: 99%

Assessment of viability of microorganisms employing fluorescence techniques

Breeuwer

Abee

2000

International Journal of Food Microbiology

262

181

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“…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 state may retain their pathogenicity (8,34,35,37,44).…”

mentioning

confidence: 99%

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Chen

Zhang

et al. 2007

Appl Environ Microbiol

100

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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 ...

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A tentative direct microscopic method for counting living marine bacteria

Cited by 888 publications

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Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

Assessment of viability of microorganisms employing fluorescence techniques

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

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