Abstract

Vibrio harveyi has been reported to enter into a viable but nonculturable (VBNC) state. Two clinical V. harveyi strains, SF1 and CW2, and the type strain, VIB295T, were incubated in sterilized seawater at 4 °C. Plate counts in these strains declined to undetectable levels (<0.1 CFU mL−1) within 69, 67 and 65 days, respectively. The direct viable count (DVC) declined from 106 to 104 active cells mL−1 and remained constant at this level by a DVC. VBNC cell numbers could be restored via a temperature upshift when grown in yeast extract with the addition of Tween 20 or compound vitamin B. Reverse transcriptase-PCR was used to monitor virulence gene expression within VBNC cells. No expression of the hemolysin gene was detected in VBNC cells. VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the groups inoculated with VBNC cells. The fish inoculated with the resuscitative cells died within 7 days, the lethal dose 50% (LD50) being 2.85 × 104 CFU mL−1, a value similar to that for groups inoculated with normal cells (2.28 × 104 CFU mL−1). This suggested that VBNC V. harveyi might retain pathogenic potential.

Introduction

Vibrio harveyi is a Gram-negative, luminous, marine bacterium occurring either free living or in association with marine forms of life such as fish and crustaceans (O'Brien & Sizemore, 1979; Orndorff & Colwell, 1980; Ramesh & Venugopalan, 1989). The organism has been reported as a major causal agent of luminous vibriosis, which affects a diverse range of marine invertebrates. Mass mortalities of shrimp larvae due to V. harveyi infection have been reported from a number of countries, especially in South America and south-east Asia (Liu et al., 1996; Alvarez et al., 1998; Vandenberghe et al., 1998). Additionally, V. harveyi has been associated with diseases in fish (Sunaryanto & Mariam, 1986; Saeed, 1995; Hispano et al., 1997). The pathogenicity of V. harveyi can be attributed to the presence of extracellular virulence factors such as proteases, hemolysins, chitinases, lipases and phospholipases (Liu et al., 1996). Vibrio harveyi luminescence has been related to protease toxin activity (Nakayama et al., 2006). Quorum sensing in V. harveyi has also been reported to be related to regulation of virulence genes (Lowery et al., 2005). However, the mechanisms of pathogenicity of V. harveyi are not completely understood and the causes of disease occurrence remain elusive.

It has been reported that many Vibrio species can enter into the viable but nonculturable (VBNC) state as a response to natural stresses (Oliver & Bockian, 1995). The characterization of these pathogenic bacteria in the VBNC state has been well documented (Xu et al., 1982; Oliver & Wanucha, 1989; Wong et al., 2004). Cells entering the VBNC state change from short rods to coccoids and decrease in size, and some metabolic changes occur, including reductions in nutrient transport, respiration rates and macromolecular synthesis (Porter et al., 1995; Oliver, 2000). It has also been reported that under appropriate conditions these microorganisms can nevertheless recover from this dormant state, becoming metabolically active and fully culturable (Roth et al., 1988; Nilsson et al., 1991; Whitesides & Oliver, 1997; Mizunoe et al., 2000). Some pathogenic bacteria may retain pathogenicity when in the VBNC state (Colwell et al., 1985; Oliver & Bockian, 1995; Baffone et al., 2003). Recent studies have demonstrated gene expression by cells in the VBNC state (Lleò, 2000; Yaron & Matthews, 2002). Saux & Dominique (2002) reported continued production of mRNA for several genes of Vibrio vulnificus for as long as 4.5 months after the cells entered into the VBNC state. Ramaiah et al. (2002) reported that luminous V. harveyi ATCC 14126 could enter the VBNC state in response to nutrient-limited and low-salinity incubation at 22 °C; recovery of culturability of cells in the VBNC state was achieved by addition of nutrient broth (Ramaiah et al., 2002).

The aim of the present study was to characterize the experimentally induced VBNC forms of clinical V. harveyi isolates and their virulence retention.

Materials and methods

Bacterial strain and growth conditions

Vibrio harveyi VIB295T (LMG4044T) was obtained from the bacteria collection in the School of Life Sciences (Heriot-Watt University, Edinburgh, UK). Strains SF1 and CW2 were isolated from diseased seabass (Lateolabraxj aponicus) and turbot (Scophthalmus maximus) in China and were identified as representing V. harveyi by the Vitek 32 microbial analysis system (bioMérieux, Marcy l'Etoile, France) and 16S rRNA gene sequence analysis. Authenticity was then verified based on Holt et al. (1994). The strains were stocked in cultured broth with 10% (v/v) glycerol at −85 °C, and cultured in 2216E agar at 26 °C.

Preparation of microcosms of V. harveyi

Microcosms were prepared by filtering seawater through a 0.22-μm pore-size filter (Millipore) and sterilized by autoclaving. Vibrio harveyi cells were cultured in 2216E agar overnight at 26 °C to the logarithmic phase of growth, and the cells were washed with sterile 0.22-μm pore-size filtered seawater. The washed cells were then inoculated into the seawater microcosms at a final concentration of c. 106 cells mL−1 and maintained at 4 °C without shaking to induce the VBNC state as described (Lleò, 2000; Du et al., 2007).

Culturability and viability assays

Culturability was determined every 6 days via a spread plate count (PC) with 2216E agar plates. Samples from the microcosms were serially diluted in sterilized seawater, spread in triplicate on 2216E agar and incubated at 26 °C for 48 h. When the culturable cell populations were <10 CFU mL−1, a 10-mL aliquot of the microcosm was filtered onto a 0.22-μm pore-size filter and the filter placed on 2216E agar plates. The bacteria were considered to be nonculturable when <0.1 CFU mL−1 of the culturable cells could be detected in the microcosms (Baffone et al., 2003).

To determine the total number of cells, samples from the microcosm were serially diluted, fixed with formalin (2%, v/v), stained with acridine orange (0.01%, w/v) for 2 min, and filtered onto 0.22-μm pore-size black polycarbonate filters (Millipore). The filters were then examined under an Olympus BH-2 epifluorescence microscope (Japan).

The number of viable cells was measured by a direct viable count (DVC) method, as described by Kogure et al. (1979). Yeast extract (0.025%, w/v) and nalidixic acid (0.002%, w/v) were added to the serially diluted samples, the samples were incubated at 26 °C for 14 h, cells were stained with acridine orange and observed as described above. Viable cells that responded to this treatment in the presence of yeast extract and nalidixic acid became elongated to at least twice the length of cells treated with acridine orange direct count (AODC).

Each method was tested in three replicates with reproducible results. Statistical analysis was performed using the commercial software program spss10.0.

Resuscitation of VBNC cells

For resuscitation of VBNC cells with increasing temperature, 5 mL of the VBNC cells was removed from the microcosms and allowed to incubate at 26 °C with or without addition of yeast extract (0.025%, w/v). Culturability was determined by plating 0.1-mL samples on 2216E agar.

To determine the effects of different chemicals on resuscitation of the VBNC V. harveyi, Tween 20 and compound vitamin B were added to samples of the VBNC cells containing yeast extract at final concentrations of 6% (v/v) and 1 mg mL−1, respectively. The samples were then incubated at 26 °C for 48 h to determine culturability. All experiments were performed in triplicate.

Scanning electron microscopy

Vibrio harveyi cells in different states were fixed with 3% (v/v) glutaraldehyde at room temperature for 4 h, and filtered onto 0.22-μm pore-size nucleopore polycarbonate filters. The samples were then dehydrated and coated as described by Du et al. (2007). The coated samples were then observed under a JSM-840 scanning electron microscope (JEOL, Japan).

Reverse transcriptase (RT)-PCR detection of the virulence gene of VBNC cells

Total RNA extraction

For the culturable samples, 5 mL of cells at the logarithmic phase were centrifuged at 11 200 g for 5 min at 4 °C and the cell pellets were collected. Cells in the VBNC state (104 active cells mL−1) were harvested by aseptic filtration (25-mL volumes) using a 25-mm-diameter autoclaved polycarbonate filter (0.2-μm pore-size Nuclepore) placed on a Millipore membrane. The polycarbonate filter was rolled up and transferred, using sterile forceps, into a 1.5-mL polypropylene tube containing 1 mL of TRIzol reagent; 0.2 mL of chloroform was added to the TRIzol Reagent. The tubes were vortexed vigorously for 30 s and incubated for 2 min, then centrifuged at 10 000 r.p.m. for 15 min at 4 °C. The aqueous phase was transferred to a fresh tube, 0.5 mL of isopropyl alcohol was added to the tuber containing 1 mL of TRIzol reagent and incubated for 10 min, then centrifuged at 10 000 r.p.m. for 15 min at 4 °C. The RNA pellets were washed twice with 1 mL of 70% ethanol and dissolved in 25 μL of diethylpyrocarbonate-treated water and stored at −80 °C.

Oligonucleotide primers

The two primers (forward, 5′-TCACCTTCTTTGCAGCCGATAACC-3′; reverse, 5′-CAAGGCTTCCGCGTAGTCTGATTT-3′) delineate a 455-bp region within the ORF of the hemolysin gene unique to V. harveyi.

RT-PCR amplification

DNAse-treated RNA (4 μL) was reverse transcribed in a 20-μL volume with 5 mM MgCl2, 1 × PCR buffer II [50 mM KCl, 10 mM Tris-HCl (pH 8.3)], 1 mM each dNTP, 1 U of RNAse inhibitor per microliter, 2.5 U of murine leukemia virus reverse transcriptase per microliter, and 1 μM reverse primer. The tubes were incubated at 42 °C for 15 min, heated at 96 °C for 5 min and cooled to 5 °C for at least 5 min. Half of the RT mixture was added to 40 μL of the PCR mixture. The final concentrations in the PCR mixture were as follows: 2 mM MgCl2, 1 × PCR buffer II, 200 μM each dNTP, 1.25 U of AmpliTaq DNA polymerase per 50 μL, and 0.5 μM each primer. The thermocycling program was optimized as follows: a first denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. After a final extension at 72 °C for 10 min, the tubes were cooled to 4 °C.

Virulence of VBNC and resuscitative cells

Zebra fish were used to investigate the virulence retention of VBNC and resuscitative cells. One-hundred zebra fish were divided into 10 groups, each comprising 10 fish. The fish were kept in 2-L tanks with aerated water at 16–20 °C and fed with commercial pellets. The 10 groups were injected intraperitoneally with 20 μL of the VBNC cells (104 CFU mL−1), resuscitative cells (103, 104, 105 and 106 CFU mL−1), normal cells (103, 104 and 105 106 CFU mL−1) and autoclaved saline respectively. The injected fish were then kept at 16–20 °C for 30 days and the mortality was recorded. The lethal dose 50% (LD50) was calculated by the method of Reed & Munench (1938).

Biochemical characterization of resuscitative cells – API 20E test

Biochemical characterization of resuscitative cells of V. harveyi strains SF1 and CW2 was made by API 20E test strips (API system, Montalieu-Vercieu, France) according to the manufacturer's instructions.

Results

Entry into the VBNC state of V. harveyi

Figure 1 shows the changes in cell numbers of V. harveyi VIB295T, SF1 and CW2 during incubation in seawater microcosms at 4 °C. Total cell counts remained constant over 80 days, but PCs rapidly declined from 106 CFU mL−1 to undetectable levels (<0.1 CFU mL−1) by 65, 69 and 67 days, respectively. Active cells, by contrast, declined to c. 104 CFU mL−1 and remained fairly constant at this level, as demonstrated by DVC staining. These results indicated that a large population of cells existed in the VBNC state.

1

Entry into the VBNC state in seawater microcosms at 4°C of Vibrio harveyi strains SF1, VIB295T and CW2.

1

Entry into the VBNC state in seawater microcosms at 4°C of Vibrio harveyi strains SF1, VIB295T and CW2.

Resuscitation of VBNC cells

When the VBNC cells of V. harveyi were subjected to a temperature increase (from 4 to 26 °C) in the presence or absence of yeast extract, no culturable population was observed on 2216E agar. When the VBNC cell solutions containing yeast extract were supplemented by Tween 20 or compound vitamin B and subjected to a temperature increase (from 4 to 26 °C), cell numbers were restored. The culturable cell counts rose to about 106 CFU mL−1 on days 8 and 9 on 2216E agar (Fig. 2).

2

Resuscitation of VBNC Vibrio harveyi SF1, VIB295T and CW2 via a temperature increase with addition of Tween 20 (filled squares) and compound vitamin B (open squares).

2

Resuscitation of VBNC Vibrio harveyi SF1, VIB295T and CW2 via a temperature increase with addition of Tween 20 (filled squares) and compound vitamin B (open squares).

Morphological changes of VBNC V. harveyi cells

The VBNC cells exhibited several morphological changes under the epifluorescence microscope via the AODC method. Compared with normal cells, the VBNC cells changed from short rods to coccoid cells and decreased in size. Red, elongated cells were observed via DVC (not shown). Similar changes could be observed under the scanning electron microscope (data not shown). The average size of the normal cells was 1.5 × 0.5±0.1 μm, but the average radius of the coccoid VBNC cells was 0.2±0.1 μm. The normal cells and restored cells showed no morphological difference.

Detection of mRNA for the hemolysin gene in VBNC V. harveyi cells

We attempted to monitor expression of the virulence gene from the VBNC cultures by RT-PCR. The mRNA for the hemolysin gene was amplified in the normal cells and resuscitative cells, but expression of the gene was not detectable in the VBNC cells (Fig. 3).

3

Detection of hemolysin mRNA in VBNC and resuscitative populations of Vibrio harveyi by RT-PCR. M, DNA ladder. 1, Normal V. harveyi SF1 as positive control; 2, V. paraheamolytics as negative control; 3, VBNC V. harveyi SF1; 4, resuscitative V. harveyi SF1; 5, VBNC V. harveyi VIB295T; 6, resuscitative V. harveyi VIB295T; 7, VBNC V. harveyi CW2; 8, resuscitative V. harveyi CW2.

3

Detection of hemolysin mRNA in VBNC and resuscitative populations of Vibrio harveyi by RT-PCR. M, DNA ladder. 1, Normal V. harveyi SF1 as positive control; 2, V. paraheamolytics as negative control; 3, VBNC V. harveyi SF1; 4, resuscitative V. harveyi SF1; 5, VBNC V. harveyi VIB295T; 6, resuscitative V. harveyi VIB295T; 7, VBNC V. harveyi CW2; 8, resuscitative V. harveyi CW2.

Virulence of the VBNC and the resuscitative cells

Some of the zebra fish in groups inoculated with normal and resuscitative V. harveyi died over a period of 7 days. LD50 for the resuscitative cells was 2.85 × 104 CFU mL−1, which was close to that for normal cells (2.28 × 104 CFU mL−1). The dead fish all showed similar symptoms, such as ascites and congestion of the focus. Culturable cells of V. harveyi could be isolated from ascites fluid and organs. The bacteria were identified by PCR amplification and analysis of the 16S rRNA gene. The fish inoculated with the VBNC cells and autoclaved saline remained alive during the experimental period, and V. harveyi was not isolated from these animals.

Discussion

A number of bacteria have been reported to be capable of entering into a VBNC state as a response to environmental stresses, such as starvation, incubation outside the temperature range of growth and elevated osmotic concentrations (Oliver, 2005; Chattopadhyay, 2006). The time entering into such state varies greatly in different bacteria and their physiological state. Wong et al. (2004) reported that 24 strains of Vibrio parahaemolyticus from clinical and environmental sources entered the VBNC state in 14–49 days when incubated in artificial seawater at 4.5 °C. Vibrio vulnificus CVD713 and C7184 entered the VBNC state in artificial seawater at 5 °C within 7 days (Oliver & Bockian, 1995), while Linder & Oliver (1989) reported that V. vulnificus entered the VBNC state after incubation for 24 days.

Duncan et al. (1994) observed that V. harveyi 1280 entered into the VBNC state within 11 days at 4 °C. Ramaiah et al. (2002) suggested that the adaptation of V. harveyi ATCC 14126 to the VBNC state was influenced by salinity. In the present study, we used two clinical V. harveyi strains, SF1 and CW2, and the type strain, VIB295T. These three strains became nonculturable when inoculated in seawater microcosms at 4 °C within 65–69 days. These data were in agreement with those of V. harveyi ATCC 14126 incubated in microcosms of 35g kg−1 artificial seawater at 22 °C (Ramaiah et al., 2002).

An important characteristic of the VBNC cells is their ability to be resuscitated in vitro and in natural estuarine environments (Oliver & Bockian, 1995). Resuscitation of the VBNC cells was achieved by a temperature increase in the presence of yeast extract by addition of either Tween 20 or compound vitamin B, but their exact mechanisms in this process remain to be clarified. Ramaiah and Ravel reported that V. harveyi ATCC 14126 cell numbers could be restored through a temperature increase without nutritients, and they also obtained an increase in luminescence and cell length (indicating viability) on an increase from 4 to 30 °C. In the present study, a temperature increase with and without the presence of nutrition did not result in recovery from nonculturability.

When entering into the VBNC state, V. harveyi cells decreased in size and became coccoidal, which was in agreement with the results reported by other authors (Oliver & Wanucha, 1989; Morita, 1993; Benjamin & Datta, 1995; Federighi et al., 1998). Several studies have demonstrated that the VBNC cells showed greater resistance to various stress conditions (Jiang & Chai, 1996; Chaiyanan et al., 2001). There were no significant differences in antibiotic resistance and main biochemical characterizations between the restored cells and normal cells of V. harveyi SF1 and CW2 (data not shown).

It has been reported that some pathogens are able to express the virulence determinant and toxin genes in the VBNC state. Vibrio cholerae entering the VBNC state showed a different reduction in the number of copies of 16S rRNA and mRNA for the tuf, rpoS and relA genes, but the mRNA for the relA gene was selectively increased in VBNC cells (González-Escalona et al., 2006). Lleò (2000) reported the expression of a gene involved in peptidoglycan synthesis in VBNC forms of Enterococcus faecalis. Cytotoxin–hemolysin mRNA was detected in VBNC populations of environmental and clinical V. vulnificus strains maintained in artificial seawater (Saux & Dominique, 2002). Coutard et al. (2005) reported that the 16S–23S rRNA and rpoS genes of V. parahaemolyticus Vp4 were expressed in VBNC cells whereas no expression of the tdh1 and tdh2 genes were observed in the same populations. We could not detect hemolysin mRNA of the VBNC cells of V. harveyi by RT-PCR, but the mRNA for the 16S rRNA gene was detectable in the same VBNC cells. The mRNA for the hemolysin gene could be detected in the normal and resuscitative cells.

It has been known that some Vibrio species can initiate infections from the VBNC state. Vibrio cholerae cells in the VBNC state were recovered from rabbit ileal loops in which enterotoxigenicity was exhibited (Kogure et al., 1979; Duncan et al., 1994). Baffone et al. (2003) reported that VBNC V. parahaemolyticus and Vibrio alginolyticus strains lost the virulence characteristics after resuscitation in mouse and were subsequently reactivated by means of two consecutive passages of the strains in a rat ileal loop model. Fish injected with the VBNC V. harveyi SF1 remained alive and did not show any visible signs. The resuscitative V. harveyi SF1 cells showed pathogenicity when they were intraperitoneally inoculated into fish. The fish inoculated with the resuscitative cells died within 7 days, showing similar symptoms with those inoculated with the normal cells. Vibrio harveyi was isolated from these organs. This result was in agreement with the analysis of hemolysin mRNA in the VBNC cells, normal cells and resuscitative cells. Because hemolysin has been reported to be the major virulence factor of V harveyi (Sun et al., 2007), it could be assumed that the loss of the pathogenicity in VBNC cells of V. harveyi SF1 was due to a temporary inability to express its virulence characteristics, such as hemolysin genes. The resuscitative V. harveyi SF1 cells expressed the hemolysin gene and showed pathogenicity to the fish. These results indicated that VBNC V. harveyi might retain its pathogenic potential and cause disease.

Acknowledgements

This work was supported by a grant from the National Natural Science Foundation of China (30371108), Special Nonprofit Research Projects from the Ministry of Agriculture of the People's Republic of China (nyhyzx 07-046) and Student Research Training Program of Ocean University of China (0711010401).

References

Alvarez
JD
Austin
B
Alvarez
AM
Reyes
H
(
1998
)
Vibrio harveyi a pathogen of penaeid shrimps and fish in Venezuela
.
J Fish Dis
 
21
:
313
316
.
Baffone
W
Citterio
B
Vittoria
E
Casaroli
A
Campana
R
Falzano
L
Donelli
G
(
2003
)
Retention of virulence in viable but non-culturable halophilic Vibrio spp
.
Int J Food Microbiol
 
89
:
31
39
.
Benjamin
MM
Datta
AR
(
1995
)
Acid tolerance of enterohemorrhagic Escherichia coli
.
Appl Environ Microbiol
 
61
:
1669
1672
.
Chaiyanan
S
Chaiyanan
S
Huq
A
Maugel
T
Colwell
RR
(
2001
)
Viability of the nonculturable Vibrio cholerae O1 and O139
.
Syst Appl Microbiol
 
24
:
331
341
.
Chattopadhyay
MK
(
2006
)
Mechanism of bacterial adaptation to low temperature
.
J Biosci
 
31
:
157
165
.
Colwell
RR
Brayton
PR
Grimes
DJ
Roszak
DB
Huq
SA
Palmer
LM
(
1985
)
Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms
.
Biotechnology
 
3
:
817
820
.
Coutard
F
Pommepuy
M
Loaec
S
Hervio-Heath
D
(
2005
)
mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state
.
J Appl Microbiol
 
98
:
951
961
.
Du
M
Chen
JX
Zhang
XH
Li
AJ
Li
Y
Wang
YG
(
2007
)
Retention of virulence in a viable-but-nonculturable Edwardsiella tarda isolate
.
Appl Environ Microbiol
 
73
:
1349
1354
.
Duncan
S
Grover
LA
Killham
K
Prosser
JI
(
1994
)
Luminescence-based detection of activity of starved and viable but non-culturable bacteria
.
Appl Environ Microbial
 
60
:
1308
1316
.
Federighi
M
Tholozan
JL
Cappelier
JM
Tissier
JP
Jouve
JL
(
1998
)
Evidence of non-coccoid viable but non-culturable Campylobacter jejuni cells in microcosm water by direct viable count, CTC-DAPI double staining, and scanning electron microscopy
.
Food Microbiol
 
15
:
539
550
.
González-Escalona
N
Fey
A
Höfle
MG
Espejo
RT
Guzmán
CA
(
2006
)
Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock
.
Environ Microbiol
 
8
:
658
666
.
Hispano
C
Nebra
Y
Blanch
AR
(
1997
)
Isolation of Vibrio harveyi from an ocular lesion in the short sunfish (Mola mola)
.
Bull Eur Assoc Fish Pathol
 
17
:
104
107
.
Holt
JG
Krieg
NR
Sneath
PHA
Staley
JT
Williams
ST
(
1994
)
Bergey's Manual of Determinative Bacteriology
 ,
9th edn
.
Williams & Wilkins
,
Baltimore
.
Jiang
X
Chai
TJ
(
1996
)
Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells
.
Appl Eviron Microbiol
 
62
:
1300
1305
.
Kogure
K
Simidu
U
Taga
N
(
1979
)
A tentative direct microscopic method for counting living marine bacteria
.
Can J Microbiol
 
25
:
415
420
.
Lowery
CA
McKenzie
KM
Qi
L
Meijler
MM
Janda
KD
(
2005
)
Quorum sensing in Vibrio harveyi: probing the specificity of the LuxP binding site
.
Bioorg Med Chem Lett
 
15
:
2395
2398
.
Linder
K
Oliver
JD
(
1989
)
Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus
.
Appl Environ Microbiol
 
55
:
2837
2842
.
Liu
PC
Lee
KK
Chen
SN
(
1996
)
Pathogenicity of different isolates of Vibrio harveyi in tiger prawn Penaeus monodon
.
Lett Appl Microbiol
 
22
:
413
416
.
Lleò
MM
Pierobon
S
Tafi
MC
Signoreto
C
Canepari
P
(
2000
)
mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm
.
Appl Environ Microbiol
 
66
:
4564
4567
.
Mizunoe
Y
Wai
SN
Ishikawa
T
Takade
A
Yoshida
S
(
2000
)
Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation
.
FEMS Microbiol Lett
 
186
:
115
120
.
Morita
RY
(
1993
)
Bioavailability of energy and the starvation state
.
Starvation in Bacteria
  (
Kjelleberg
S
, ed), pp.
81
102
.
Plenum Press
,
New York, NY
.
Nakayama
T
Nomura
N
Matsumura
M
(
2006
)
Study on the relationship of protease production and luminescence in Vibrio harveyi
.
J Appl Microbiol
 
101
:
200
205
.
Nilsson
L
Oliver
JD
Kjelleberg
S
(
1991
)
Resuscitation of Vibrio vulnificus from the viable but nonculturable state
.
J Bacteriol
 
173
:
5054
5059
.
O'Brien
O
Sizemore
RK
(
1979
)
Distribution of the luminous bacterium Beneckea harveyi in the semi-tropical estuarine environment
.
Appl Environ Microbiol
 
38
:
928
933
.
Oliver
JD
(
2000
)
Problems in detecting dormant (VBNC) cells and the role of DNA elements in this response
.
Tracking Genetically-Engineered Microorganisms
  (
Jansson
JK
Van Elsas
JD
Bailey
MJ
, eds), pp.
1
15
.
Landes Biosciences
,
Georgetown, TX
.
Oliver
JD
(
2005
)
The viable but nonculturable state in bacteria
.
J Appl Microbiol
 
43
:
93
100
.
Oliver
JD
Bockian
R
(
1995
)
In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus
.
Appl Environ Microbiol
 
61
:
2620
2623
.
Oliver
JD
Wanucha
D
(
1989
)
Survival of Vibrio vulnificus at reduced temperatures and elevated nutrient
.
J Food Saf
 
10
:
79
86
.
Orndorff
SA
Colwell
RR
(
1980
)
Distribution and identification of luminous bacteria from the Sargasso Sea
.
Appl Environ Microbiol
 
39
:
983
987
.
Porter
J
Edwards
C
Pickup
RW
(
1995
)
Rapid assessment of physiological status in Escherichia coli using fluorescent probes
.
J Appl Bacteriol
 
4
:
399
408
.
Ramaiah
N
Ravel
J
Straube
WL
(
2002
)
Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state
.
Syst Appl Microbiol
 
93
:
108
116
.
Ramesh
A
Venugopalan
VK
(
1989
)
Response of enteric luminous bacteria to environmental conditions in the gut of the fish
.
J Appl Bacteriol
 
66
:
529
533
.
Reed
LJ
Munench
H
(
1938
)
A simple method of estimating fifty percent endpoints
.
Am J Hyg
 
27
:
493
497
.
Roth
WG
Leckie
MP
Dietzler
DN
(
1988
)
Restoration of colony forming activity in osmotically stressed Escherichia coli by betaine
.
Appl Environ Microbiol
 
54
:
3142
3146
.
Saeed
MO
(
1995
)
Association of Vibrio harveyi with mortalities in cultured marine fish in Kuwait
.
Aquaculture
 
136
:
21
29
.
Saux
MF-L
Dominique
H-H
(
2002
)
Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater
.
Appl Environ Microbiol
 
68
:
5641
5646
.
Sun
BG
Zhang
XH
Tang
XX
Wang
SS
Zhong
YB
Chen
JX
Austin
B
(
2007
)
A single residue change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus)
.
J Bacteriol
 
189
:
2575
2579
.
Sunaryanto
A
Mariam
A
(
1986
)
Occurrence of a pathogenic bacteria causing luminescence in penaeid larvae in Indonesian hatcheries
.
Bull Brackishwater Aquac Dev Cent
 
8
:
105
112
.
Vandenberghe
J
Li
Y
Verdonk
L
Li
J
Sorgeloos
P
Xu
HS
Swings
J
(
1998
)
Vibrios associated with Penaeus chinensis (Crustacea: Decapoda) larvae in Chinese shrimp hatcheries
.
Aquaculture
 
169
:
121
132
.
Whitesides
MD
Oliver
JD
(
1997
)
Resuscitation of Vibrio vulnificus from the viable but nonculturable state
.
Appl Environ Microbiol
 
63
:
1002
1005
.
Wong
HC
Wang
P
Chen
SY
Chiu
SW
(
2004
)
Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium
.
FEMS Microbiol Lett
 
233
:
269
275
.
Xu
HS
Roberts
N
Singleton
FL
Attwell
RW
Grimes
DJ
Colwell
RR
(
1982
)
Survival and viability of non-culturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment
.
Microb Ecol
 
8
:
313
323
.
Yaron
S
Matthews
KR
(
2002
)
A reverse transcriptasepolymerase chain reaction assay for detection of viable Escherichia coli O157: H7: investigation of specific target genes
.
J Appl Microbiol
 
92
:
633
640
.

Author notes

Editor: Julian Marchesi