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Keywords:

  • morphology;
  • resuscitation;
  • VBNC;
  • Vibrio cincinnatiensis;
  • virulence retention

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation.

Methods and Results: Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4°C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4°C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells.

Conclusions: Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity.

Significance and Impact of the Study:  The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Vibrio cincinnatiensis is a halophilic Gram-negative bacterium, which presents in freshwater and brackish waters. The bacterium was first recognized as human being pathogen (Brayton and Bode 1986). It was found to be widely distributed in the estuarine and coastal waters of the Chesapeake Bay and its viable counts changed in seasonality and reached to the highest in July and September (Heidelberg et al. 2002). Vibrio cincinnatiensis was detected in harvested mussels (Mytilus galloprovincialis) (Ripabelli et al. 1999). Vibrio cincinnatiensis has been reported to be one of the important bacterial pathogens of mud crab, which caused crab mortalities and serious economic losses in China (Mao et al. 2001). Recently, several virulence-related genes of Vibrio species such as haemolysin, toxS, flaB and flaC were detected in V. cincinnatiensis by PCR amplification (Bai et al. 2008). Vibrio cincinnatiensis was also reported to resistant to complement mediated lysis of humoral fluids of amphioxus (Branchiostoma belcheri), and its LPS profile was suggested to be correlated with humoral fluid resistance (Li et al. 2008). However, the pathogenic mechanisms of V. cincinnatiensis are far from understood and the causes of disease occurrence are still elusive.

Viable but nonculturable (VBNC) denotes a state in which bacterial cells can not be cultured on conventional media, but remain metabolic activities in response to harsh environmental conditions, such as low temperature, visible light, nutrient-limited, salinities and aeration (Xu et al. 1982; Barer and Harwood 1999; Lleòet al. 2001). More than 20 Vibrio species have been reported to enter into VBNC state (Gourmelon et al. 1994; Ramaiah et al. 2002; Randa et al. 2004). It has also been reported that under appropriate conditions these microorganisms could recover from this dormant state, becoming metabolically active and fully culturable (Whitesides and Oliver 1997; Mizunoe et al. 1999). The time for entry into VBNC state and resuscitation was dependant on strains (Grimes and Colwell 1986; Wolf and Oliver 1992; Wong et al. 2004). Some bacterial pathogens in VBNC state may retain their pathogenicity (Oliver and Bockian 1995).

In this study, we investigated the characteristics of V. cincinnatiensis in VBNC state in seawater microcosms at low temperature, their resuscitation and virulence retention.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

Vibrio cincinnatiensis VIB287 (LMG 7891T, ATCC 35912) was obtained from the bacteria collection in the School of Life Sciences (Heriot-Watt University, Edinburgh). The strain was stocked in 2216E broth supplemented with 10% (v/v) glycerol at −85°C, and grown in 2216E agar at 26°C.

Preparation of microcosms of Vibrio cincinnatiensis

Microcosms were prepared by filtering seawater through a 0·22-μm pore-size filter (Millipore) and sterilized by autoclaving at 121°C for 15 min. Vibrio cincinnatiensis VIB287 was cultured in 2216E agar at 26°C overnight. The cells were harvested by centrifugation at 6000 g for 10 min at 4 °C, washed twice with 0·22-μm pore-size filtered sterile seawater to avoid any carryover of nutrients. The washed cells were then inoculated into the seawater microcosms at a final concentration of approx. 107 cells ml−1 and maintained at 4°C without shaking to induce the VBNC state as described (Lleòet al. 2001).

Culturability and viability assays

Culturability was determined by spread plate count (PC) with 2216E agar plates every 5 days. One milliter of the microcosms was removed and serially diluted in sterilized seawater, 0·1 ml of the diluted microcosms was spread in triplicate on 2216E agar and incubated at 26°C for 48 h. When the culturable cell populations were <10 CFU ml−1, 10 ml of the microcosm was filtered onto a 0·22-μm pore-size filter, the filter was then placed on 2216E agar plate and incubated at 26°C for 48 h. The bacterial cells were considered to be nonculturable when <0·1 CFU ml−1 of culturable cells could be detected in the microcosms (Baffone et al. 2003).

The number of total cells was determined by acridine orange direct count (AODC) method as described by Hobbie et al. (1977). Samples from the microcosms were 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 (Olympus, Japan).

The number of viable cells was measured by direct viable count (DVC) method as described by Kogure et al. (1979). Samples were withdrawn from the microcosms every 5 days and serially diluted in sterilized seawater. The diluted samples were added with yeast extract (0·025% w/v) and nalidixic acid (0·002% w/v) and incubated at 26°C for 14 h. The cells were stained with acridine orange and observed under the fluorescence microscope. Cells which were elongated to at least twice the length of AODC control cells were recorded as viable cells. Each method was tested in three replicates with reproducible results.

Resuscitation of the VBNC cells

Resuscitation of the VBNC cells with upshifting temperature: 5 ml of the VBNC cells was removed from the microcosms and allowed to incubate at 26°C. Culturability was determined by plating 0·1 ml of the samples on 2216E agar.

To determine effects of some chemicals on resuscitation of the VBNC cells, 10 ml of the samples were aseptically removed from the seawater microcosms at 4°C and placed into steriled test tubes respectively. Tween 20, compound vitamin B and catalase were added to the samples of the VBNC cells containing yeast extract (0·25% w/v) and peptone (0·5% w/v) at final concentrations of 6% (v/v), 1·5 mg ml−1 and 1000 U ml−1 respectively. The samples were then incubated at 26°C for 48 h, 0·1 ml of the sample was removed and spread on 2216E −0·2% sodium pyruvate to determine culturability at time intervals. The test was performed in triplicate.

When the VBNC cells were resuscitated, the chromosomal DNA was extracted and the 16S rRNA gene was amplified with PCR amplification, the amplified products were digested with Mbo I, analysed by electrophoresis for 30 min in 1·5% (w/v) agarose gels. The amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA) profiles were analysed to confirm that the resuscitative cells were the same as initial inoculated strain.

Scanning electron microscopy

Vibrio cincinnatiensis cells in different states were fixed with 3% (v/v) glutaraldehyde at room temperature for 4 h, 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).

Transmission electron microscopy

The VBNC cells were harvested by centrifugation at 12 000 g for 15 min at 4°C. The harvested cells were fixed with 2% glutaraldehyde at room temperature for 24 h, suspended with phosphate buffered saline and postfixed using 1% osmium tetraoxide in phosphate buffer for 1 h, dehydrated by series of a graded ethanol series, embedded in Epon 812 resin. Ultrathin sections were stained with lead citrate and uranyl acetate. Overnight grown cells on 2216E agar were used as controls. The samples were observed under a JEOL 1200EX transmission electron microscope (JEOL).

Virulence of the VBNC and the resuscitative cells

Eighty zebra fish were divided into eight groups, each comprising of 10 fish. The fish were kept in 2 L tanks with aerated water at 16–20°C and fed with commercial pellets. The eight groups were intraperitoneally injected with 50 μl of the VBNC cells (105 cell ml−1), resuscitative cells (105, 106, 107 CFU ml−1), normal cells (105, 106, 107 CFU ml−1) and autoclaved saline respectively. The injected fish was then kept at 16–20°C for 10 days and the mortality was recorded. The LD50 was calculated by the method of Reed and Muench (1938).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Entry into the VBNC state of Vibrio cincinnatiensis

Figure 1 showed the changes in cell numbers of V. cincinnatiensis during incubation in seawater microcosms at 4°C. Total cell counts remained constant, on the other hand, the PCs rapidly declined from 106 CFU ml−1 to undetectable levels (<0·1 CFU ml−1) in 50 days. The number of active cells declined from 106 to 105 CFU ml−1 and remained constant at this level as determined by DVC staining. These results indicated that a large population of cells entered into VBNC state.

image

Figure 1.  The viable but nonculturable response of Vibrio cincinnatiensis VIB 287. PC (bsl00066), DVC (bsl00001), AODC (◆).

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Resuscitation of the VBNC cells

Resuscitation was achieved by addition of catalase or compound vitamin B into the VBNC cells supplemented with yeast extract and peptone separately. The culturable colony counts rose to 4·5 × 104 and 2·3 × 103 CFU ml−1 2 days after they were incubated at 26°C respectively. The VBNC cells maintained their abilities to resuscitate after entry into VBNC state at least 60 days, the viable counts increased from zero to 2·7 × 104 CFU ml−1 and to 1·5 × 103 CFU ml−1 respectively (Fig. 2). The bacterium was confirmed to be V. cincinnatiensis by comparison of ARDRA profiles of the resuscitative cells and the normal cells (data not shown). No resuscitation was observed when the VBNC cells were subjected to a direct temperature upshift (from 4 to 26°C) in the presence or absence of yeast extract and peptone. Addition of Tween 20 to the VBNC cell solutions containing yeast extract and peptone did not induce recovery of the dormant cells.

image

Figure 2.  Resuscitation of Vibrio cincinnatiensis VIB 287 from the viable but nonculturable (VBNC) state by temperature upshift with addition of catalase (bsl00001) and compound vitamin B (◆) in the presence of yeast extract and peptone. For analysis, the first day of nonculturability was designated as time zero VBNC (Tvbnc 0) and was used as the time reference for subsequent analysis.

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Morphological changes of the VBNC cells

Scanning electron microscopy of the normal cells showed that they were mostly curved rods that were 1·8 ± 0·2 μm long (Fig. 3a), while the VBNC cells gradually changed from curved rods to coccoid (Fig. 3b).Transmission electron microscopy images of the normal cells indicated that intact structure of the membranes and out membranes (Fig. 3c).VBNC cells of V. cincinnatiensis exhibited several changes when observed by TEM. Compared with the normal cells, the average radius of the coccoid cells was 0·2 μm ± 0·1. The periplasmic spaces increased greatly and nuclear region (electron clear area) was compressed into the center of the VBNC cells under TEM. Membrane curling was observed in some of the VBNC cells (Fig. 3d). The resuscitative cells showed similar morphology with the normal cells.

image

Figure 3.  Electron micrographs of Vibrio cincinnatiensis VIB 287. Scanning electron micrograph of the normal cells (a) and the viable but nonculturable (VBNC) cells (b). Transmission electron microscope of the normal cells (c) and the VBNC cells (d).

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Virulence of the VBNC and the resuscitative cells

Zebra fish inoculated with normal cells and resuscitative cells of V. cincinnatiensis died during 7 days. LD50 for the resuscitative cells was 2·77 × 105 CFU ml−1, which was similar to that of the normal cells (3·87 × 105 CFU ml−1). Vibrio cincinnatiensis could be isolated from the ascites fluid and the organs of the diseased fish and identified by PCR amplification and ARDRA profile analysis of 16S rRNA gene. The fish inoculated with the VBNC cells and autoclaved saline remained alive during the experimental time and V. cincinnatiensis was not isolated from these animals Table 1.

Table 1.   Pathogenicity to zebra fish of Vibrio cincinnatiensis VIB 287 in different states*
Inoculum Dose (no. of CFU ml−1)Total no. fishNo. dead fish LD50 (CFU ml−1)
  1. *Ten zebra fish were used in each group.

  2. †Determined by DVC counting.

Normal cells1051043·87 × 105
106106
107109
Resuscitative cells1051052·77 × 105
106107
107109
VBNC cells†1051000
Sterile saline01000

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Low temperatures have been shown to be important to induce formation of VBNC cells (Oliver et al. 1991; Wolf and Oliver 1992). In the present study, we confirmed that V. cincinnatiensis VIB 287 could enter VBNC state in nutrition-deficient seawater microcosms at low temperature. The bacterial cells reduced size and changed their morphology from rods to coccoid. Morphological changes such as large periplasmic spaces, the compressed nuclear region and membrane curling were observed in the VBNC cells by TEM, which was in agreement with Chaiyanan who reported that the VBNC cells V. cholerae O1 and O139 decreased in size and became coccoid in morphology and the cytoplasm of the cells condensed, resulting in a significant space between the cytoplasmic membrane and the cell wall (Chaiyanan et al. 2001). Reduction of cell size during starvation is suggested to be a survival strategy for minimizing cell maintenance requirements and increasing substrate uptake because of a high surface-to-volume ratio (Morita 1993).

Resuscitation of bacteria in VBNC state has attempted by temperature upshift (Whitesides and Oliver 1997) and addition of nutrient broth to the VBNC cells (Ramaiah et al. 2002). Stevenson reported that there is a state of dormancy in which aquatic bacteria can survive until nutrients are again available (Stevenson 1978). In the present study, recovery of culturability of V. cincinnatiensis in the VBNC state was achieved by addition of compound vitamin B or catalase to the VBNC cells containing the yeast extract and peptone. It has been reported that addition of catalase or peroxide-degrading compounds such as sodium pyruvate to the media can improve recovery of heat- and freeze-stressed Escherichia coli, for they degrade H2O2 and protect the cells from oxidative stress in the media (MacDonald et al. 1983; Calabrese and Bissonnette 1990; Mizunoe et al. 1999). Ordax et al. reported the recovery of long-term copper induced VBNC cells could resuscitate till 9 months (Ordax et al. 2006). Vibrio cincinnatiensis cells maintained the ability to resuscitate at least 60 days after they entered into VBNC state, these results supported the hypothesis that VBNC state is part of the life cycle of pathogens (McDougald et al. 1998). Some bacteria maintained their culturability for a few days, and they can hardly resuscitate from the nonculturable state (Eguchi et al. 2000). Whitesides and Oliver (1997) reported that nonculturable cells of Vibrio vulnificus in steriled seawater could be returned to culturability by a temperature upshift in the absence of any added nutrient. However, temperature upshift from 4 to 26°C did not result in recovery from the VBNC state in this study. Our results are in accordance with those of Bogosian who reported that a temperature upshift had no effect on nonculturable cells of five species of enteric bacteria (Bogosian et al. 1998).

Many pathogenic bacteria retained their virulence in the VBNC state (Morgan et al. 1993; Ravel et al. 1995; Pruzzo et al. 2003). The VBNC cells of Shigella dysenteriae type 1 retained several virulence factors and remained potentially virulence (Rahman et al. 1996). The resusciative V. cincinnatiensis cells showed pathogenicity to zebra fish, which suggested their pathogenic potential to human being and marine animals.

In conclusion, V. cincinnatiensis VIB287 entered into the VBNC state in seawater at low temperature. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient for at least 60 days after entered into VBNC state and retained their pathogenic potential.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grants of Special Non-profit Research Projects from Ministry of Agriculture of China (nyhyzx 07-046) and National High-Tech. R & D Program (2007AA09Z416).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References