Document Detail

Viability of the nonculturable Vibrio cholerae O1 and O139.
MedLine Citation:
PMID:  11822667     Owner:  NLM     Status:  MEDLINE    
Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.
S Chaiyanan; S Chaiyanan; A Huq; T Maugel; R R Colwell
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Systematic and applied microbiology     Volume:  24     ISSN:  0723-2020     ISO Abbreviation:  Syst. Appl. Microbiol.     Publication Date:  2001 Nov 
Date Detail:
Created Date:  2002-02-01     Completed Date:  2002-07-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8306133     Medline TA:  Syst Appl Microbiol     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  331-41     Citation Subset:  IM    
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
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MeSH Terms
Disinfectants / pharmacology
Microscopy, Electron
Polymerase Chain Reaction
Vibrio cholerae / genetics,  physiology*,  ultrastructure
Grant Support
1R01A139129-01//PHS HHS; R01 NR04527-01A1/NR/NINR NIH HHS
Reg. No./Substance:

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