State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry.
BACKGROUND
Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans.


METHODS
The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damaged-cells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity.


RESULTS
Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure.


CONCLUSIONS
The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state.
Falcioni
Tania
T
Centro di Citometria e Citomorfologia, Università Carlo Bo, Urbino, Italy.
Papa
Stefano
S
Campana
Raffaella
R
Manti
Anita
A
Battistelli
Michela
M
Baffone
Wally
W
D000818:Animals; D000906:Antibodies; D004269:DNA, Bacterial / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D014736:Vibrio parahaemolyticus / Q000166:cytology* / Q000648:ultrastructure
D000906:Antibodies; D004269:DNA, Bacterial