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Resuscitation of viable but non-;culturable Vibrio parahaemolyticus in a minimum salt medium

Hin-chung Wong , Peily Wang , Shau-Yan Chen , Shen-Wen Chiu
DOI: http://dx.doi.org/10.1111/j.1574-6968.2004.tb09491.x 269-275 First published online: 1 April 2004

Abstract

Vibrio parahaemolyticus is food-;borne pathogen prevalent in Asian countries. This work analyzes factors that influence the resuscitation of the viable but nonculturable (VBNC) state in this bacterium. The MMS-;0.5% NaCl medium alone limited cell multiplication, and in this medium, resuscitation was successful when the temperature was upshifted to 25 °C but not 37 °C. Chloramphenicol inhibition experiments revealed that protein synthesis in the first 24 h of temperature upshift was critical in determining the success of the three-;day resuscitation period. The VBNC state induction period and the age of the VBNC cells for successful resuscitation were strain-;dependent. Results of this work facilitate further physiological and pathological study of the VBNC state in this pathogen.

Keywords
  • Vibrio parahaemolyticus
  • Viable but non-;culturable
  • Resuscitation
  • Temperature upshift

1 Introduction

Vibrio parahaemolyticus, a halophilic Gram negative bacterium, causes acute gastroenteritis in humans. This bacterium is a prevalent food-;borne pathogen in Japan, Taiwan, and other coastal countries [1,2]. Most clinical isolates are hemolytic on Wagatsuma agar (Kanagawa-;positive, KP+) and produce a major virulence factor, thermostable direct hemolysin (TDH) [1].

V. parahaemolyticus inhabits seawater, but is seldom isolated when the temperature of the seawater is under 13–15 °C [3]. Like other vibrios that inhabit similar marine environments [4], V. parahaemolyticus may be present in a viable but nonculturable (VBNC) state during the winter. VBNC V. parahaemolyticus has been developed by starving cells at low temperature [5], but has not been well characterized. They also reported that the resuscitation of VBNC cells was the results of cell regrowth of a limited amount of culturable cells [5]. Mizunoe et al. [6] reported on the recovery of nonculturable V. parahaemolyticus cells in agar medium containing catalase or pyruvate. However, the cells were probably injured to become nonculturable by the low salinity used in the study, and later recovered by the presence of these protective agents instead of resuscitation of VBNC cells. In this report, the nonculturable bacterial cells were still rod shaped with blebs instead of the usual coccoid VBNC cells [6].

This investigation analyzes the resuscitation process of VBNC V. parahaemolyticus triggered by temperature upshift in a minimum salt medium which alone limited multiplication of cells. The induction and resuscitation of various strains of V. parahaemolyticus were also compared.

2 Materials and methods

2.1 Cultures and media

V. parahaemolyticus ST550, a serotype O4:K13 and KP+ clinical strain isolated in Thailand, was used in this study. A total of 20 clinical and 4 environmental strains were used for comparison (Table 1). These strains were stocked in culture broth with 10% (v/v) glycerol at −85 °C, and cultured in Tryptic Soy Broth Medium (TSB, Difco Laboratories, Detroit, Mich.)-;3% (w/v) NaCl or Tryptic Soy Agar (TSA, Difco)-;3% NaCl at 25 °C.

View this table:
1

Induction and resuscitation of VBNC state by different strains of Vibrio parahaemolyticus

Strain no.Date of isolationSerotypeKPVBNC by (day)aResuscitable period (day)b
Clinical
ST5501983O4:K13+35–4914
12641998O5:K68ND28–3514
12651998O5:K68ND28–35<7
12921999O2:K3+35–42<7
10791997O3:K6+35–427
10891997O3:K6+42–497
10911997O3:K6+42–49<7
10921997O3:K6+35–4214
10991997O3:K6+42–4914
11091997O3:K6+42–497
11191997O3:K6+35–427
11291997O3:K6+35–427
11321997O3:K6+14–287
11371997O3:K6+28–35<7
11471997O3:K6+28–35<7
1188UNO3:K6+14–287
12881996O3:K6+35–42<7
12891996O3:K6+28–357
12901996O3:K6+28–357
12911996O3:K6+35–42<7
Environmental
12561999NDND14–287
12571999NDND28–35<7
12581999NDND28–35<7
12661998NDND28–357
  • All of the strains except no. ST550 (Thailand), 1264 and 1265 (Singapore) were isolated from clinical or seawater samples from different locations in Taiwan. Induction of VBNC state and resuscitation of these strains were determined.

  • a Time in day required to induce VBNC state.

  • b Time in day for the cells in VBNC state successfully resuscitated by temperature upshift method; ND, not determined; KP, Kanagawa phenomenon; UN, unknown.

2.2 Induction of VBNC state

Bacteria were cultured in 50 ml TSB-;3% NaCl medium in a 250-;ml Erlenmeyer flask, and incubated at 25 °C with shaking at 110 rpm for 4.5 h to reach the mid-;exponential phase. Bacterial cells were harvested by centrifugation at 6000g for 15 min, washed twice in modified Morita mineral salt solution (MMS-;0.5% NaCl) which consisted of 5 g NaCl, 0.8 g KCl, 5.6 g MgCl2· 6H2O, 5.6 g, 7.6 g MgSO4· 7H2O, 0.9 mg FeSO4· 7H2O, 1.54 g CaCl2· 2H2O, 0.1 g Na2HPO4 and 1.21 g Tris buffer (pH 7.8) in one liter of deionized water to prevent the carry-;over of nutrients [7]. The bacterial cells were suspended in the 250-;ml Erlenmeyer flask that contained 100 ml MMS-;0.5% NaCl medium at a concentration of about 107 cells ml−1, and incubated at 4 °C in a static state, to induce the VBNC state. Bacteria were counted every seven days.

2.3 Resuscitation

The temperature upshift method was used to resuscitate VBNC V. parahaemolyticus[5]. The temperature of the VBNC culture with under one culturable cell per milliliter was changed from 4 to 25 or 37 °C and incubated for one to three days. Viable and culturable cells were counted. Nalidixic acid at 5, 10, 20, 50, 100, or 200 μg ml−1 was added to assay inhibition of bacterial cell multiplication during the temperature upshift [5]. The VBNC culture was also diluted 10-;fold to assay the effect of dilution, and diluted 5-;fold into TSB-;3% NaCl or MMS-;0.5% NaCl media, to assay the effect of nutrients on resuscitation. Chloramphenicol (100 μg ml−1) was added to the VBNC culture to determine the importance of protein synthesis in the resuscitation [8].

2.4 Enumeration of bacteria

The culturability of bacteria was determined by the plate count method. Decimal dilutions were obtained in MMS-;0.5% NaCl at 4 °C and plated on TSA-;3% NaCl. The plates were incubated at 37 °C for 16 h and the number of colonies counted. Duplicate experiments were performed and the data were obtained as means of three determinations in each experiment.

The total number of viable cells was counted using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probe Inc., Eugene, OR, USA) following the procedures of Mizunoe et al. [6]. This kit has been used to determine the number of total and viable cells of several other bacteria [911].

Effect of catalase on the culturability was assayed according to the method of Mizunoe et al. [6]. Plate counts were performed using agar plates with or without the supplementation of filter-;sterilized Aspergillus niger catalase (Sigma Co., St. Louis, MO, USA), 2000 U per plate.

The resuscitating bacteria were also enumerated by the most probable number method (MPN) [5]. Decimal dilutions were obtained in MMS-;0.5% NaCl. One milliliter of the diluted samples was inoculated into MPN tubes containing 9-;ml of MMS-;0.5% NaCl and incubated at 25 °C in a static state for three days. Equal volume of TSB-;3% NaCl was added to the MPN tubes and incubated for one more day.

2.5 Determination of minimum inhibition concentration

An aliquot of 5 μl of V. parahaemolyticus ST550 culture was inoculated into 0.1 ml of TSB-;3% NaCl in microplate wells supplemented with different concentrations of nalidixic acid (5–1000 μg ml−1). The microplate was incubated at 37 °C for 16 h and bacterial growth was determined by a microplate reader (MRXII, Dynex Technologies, Inc., Chantilly, VA, USA).

3 Results

3.1 Induction of VBNC state

When the bacterial cells at exponential phase were incubated at 25 °C in MMS-;0.5% NaCl medium (pH 7.8) in a static state, the viable and culturability counts were similar and only dropped by about one log cell number per milliliter at the end of the incubation period (Fig. 1). When the bacterial cells were incubated at 4 °C, culturability counts declined while the viable counts were at high level. Zero culturability was reached after incubating at low temperature for about 35–49 days in repeated experiments (Fig. 1). Plate counts with the supplementation of 2000 U catalase per plate did not significantly enhance the culturability during the VBNC induction period (Fig. 1).

1

Induction and resuscitation of VBNC Vibrio parahaemolyticus in MMS-;0.5% NaCl medium. The cells were cultured at 4 or 25 °C. The 4 °C culture was shifted to 25 °C (arrow) when all cells entered the VBNC state. Viable cell counts and plate counts were determined. Plate counts are given in cfu ml−1. Vertical bars designate the standard errors. ●, plate count of the 25 °C culture; ∇, viable count of the 25 °C culture; ▪, viable count of the 4 °C culture; ▾, plate count of the 4 °C culture without catalase; ○, plate count of the 4 °C culture catalase.

3.2 Resuscitation of VBNC cells

Resuscitation of the VBNC cells was performed by temperature upshift treatment to 25 °C in the MMS-;0.5% NaCl medium. In this minimal medium, proliferation of cells of V. parahaemolyticus was not possible due to low salinity and nutrient limitation (Fig. 1).

When the temperature was increased to 25 °C in the resuscitation of VBNC cells, the number of viable cells remained unchanged, while the culturability resumed rapidly, reaching a maximum level in two days, which value was near to the initial cell level. No significant increment was observed on the third day (Figs. 1 and 2). When the VBNC cells were treated at 37 °C, the viable count remained high and constant, while no culturable cell was recovered on the TSA-;3% NaCl medium (data not shown).

2

Resuscitation of VBNC Vibrio parahaemolyticus cells by temperature upshift treatment in diluted sample. When all cells entered the VBNC state, the cells suspended at the original density (●, ○) or in a 10-;fold diluted sample (▾, ∇) were incubated at 25 °C for three days. Viable cell counts (open symbols) and plate counts (solid symbols) were determined. Plate counts are given in cfu ml−1.

The resuscitated cells were also enumerated by a MPN method. The VBNC culture was diluted into MPN tubes containing MMS-;0.5% NaCl medium and incubated at 25 °C for three days. A level of 108 cells per ml was observed close to the initial inoculation level (Fig. 1).

Nalidixic acid inhibited the multiplication of cells and thereby prevented the regrowth of culturable cells during the temperature upshift [5]. The minimum inhibition concentration (MIC) of nalidixic acid against V. parahaemolyticus was determined to be less than 5 μg ml−1. Nalidixic acid (5–200 μg ml−1) was added to the resusctitation medium, MMS-;0.5% NaCl, before the temperature upshift treatment. However, the rate of culturable cell recovery and the maximum number of recovered cells were the same regardless of the presence of nalidixic acid (data not shown).

If all VBNC cells were resuscitated, the plate count in the 10-;fold diluted sample would be expected to recover to 10% of the level in the undiluted sample. The VBNC cultures were diluted 10-;fold and then subjected to temperature upshift. Plate counts reached a maximum of 106 cells ml−1 in two days and remained constant thereafter (Fig. 2).

Nutrition in the resuscitation medium may influence the resuscitation of VBNC cells. The VBNC V. parahaemolyticus cells were harvested and resuspended in TSB-;3% NaCl or MMS-;3% NaCl and incubated at 25 °C for 48 h. In the MMS-;3% NaCl medium, the number of culturable cells increased immediately and reached the original cell density, remaining constant thereafter (Figs. 2 and 3). In the TSB-;3% NaCl medium broth, a lag period of 12 h was observed and then the culturability and the viability increased, reaching 1010 cells ml−1, far in excess of the initial cell density (Fig. 3).

3

Resuscitation of VBNC Vibrio parahaemolyticus cells in nutrient-;rich medium. When all cells entered the VBNC state, the cell suspension was 5-;fold diluted in MMS-;0.5% NaCl (●, ○) or TSB-;3% NaCl (▾, ∇) and incubated at 25 °C for 48 h. Viable cell counts (open symbols) and plate counts (solid symbols) were determined. Plate counts are given in cfu ml−1.

When chloramphenicol was added at the beginning or the first day of the temperature upshift process, no culturable cells were recovered. During the second day or third day of the temperature upshift, adding chloramphenicol only reduced the culturable cells by about one log level or less cells ml−1 (Fig. 4).

4

Inhibition of resuscitation of VBNC Vibrio parahaemolyticus cells by chloramphenicol. When all cells entered the VBNC state, they were moved to MMS-;0.5% NaCl and incubated at 25 °C for three days. Chloramphenicol was not added (●), or was added at 100 μg ml−1 to the VBNC cells on the zeroth (○), first (▾), second (∇) or third (▪) day during the temperature upshift. Plate counts were determined and given in cfu ml−1.

3.3 Induction and resuscitation of VBNC state in different strains

The cultures were separately suspended in MMS-;0.5% NaCl medium and incubated at 4 °C. Culturability was examined by plate counts on TSA-;3% NaCl after 0, 14, 28, 35, 42, and 49 days to induce the VBNC state. After 35, 42, 49, 56 and 63 days, the cultures underwent temperature upshift treatment, and the number of culturable cells was determined. The length of time for the induction of the VBNC state (14–49 days) was strain-;dependent. After certain period of time in VBNC state, the cells could not be resuscitated by the temperature upshift treatment. The VBNC cells of strains ST550, 1264, 1092 and 1099 could be recovered in approximately two weeks. Several strains like 1265, 1292, 1137, and others could be recovered in less than a week (Table 1). The categories of strains, including the clinical, environmental, or serotypical, did not appear to differ in the induction and resuscitation of VBNC state. Even in most of the O3:K6 strains with very similar genotypes [12], the induction and resuscitation of the VBNC state also depended on strain (Table 1).

4 Discussion

The VBNC cell is a metabolically active one although cells are incapable of undergoing the sustained cellular division required to form a colony on regular agar media. This state has been demonstrated in numerous bacteria [13], although the existence of the VBNC state remains somewhat controversial. Application of catalase to the plate count medium did not significantly increase the culturable cells of V. parahaemolyticus (Fig. 1). Additionally, results of the MPN and other resuscitation experiments suggested that the VBNC state was induced in this species in this study and this state was different from the peroxide sensitive nonculturable cells which could be recovered by adding catalase or pyruvate to the medium as described by Bogosian et al. [7] for V. vulnificus.

The VBNC state is typically resuscitated by temperature upshift treatment [8,14]. However, strong arguments concerning the recovery of VBNC cells as the results of real resuscitation or just the regrowth of a few culturable cells apply during the temperature upshift treatment. Ravel et al. [15] reported that the recovery of VBNC cells of V. cholerae after a temperature upshift to 30 °C follows the regrowth of surviving cells, but not the resuscitation of all VBNC cells. Jiang and Chai [5] showed that the recovery of VBNC V. parahaemolyticus cells in a MMS-;2.6% NaCl medium involves the multiplication of cells during the temperature upshift period. However, in this study, we used MMS-;0.5% NaCl medium and this medium alone did not allow multiplication of growing cells of this halophilic bacterium (Fig. 1). The MPN result also demonstrated that the number of resuscitated cells observed was not likely to be due to the presence of growing cells in the culture as these would be below the level of detection. In the dilution experiments, the density of culturable cells should increase if multiplication occurs [15], however, in our study, the cells density only restored to those of the original or diluted samples and remained constant (Fig. 2).

Nalidixic acid inhibits proliferation of bacterial cells, so addition of this antibiotic could rule out the effect of multiplication of cells in the resuscitation experiment. The level of nalidixic acid added (5–200 μg ml−1) in this study was above the MIC. However, addition of nalidixic acid at these concentrations did not affect the number of resuscitated cells. It may be due to the existing of extremely low number of growing cells in the VBNC culture, or the multiplication of bacterial cells already held by the low salinity medium. Therefore, these results together support the hypothesis that resuscitation rather than regrowth of VBNC cells of V. parahaemolyticus occurs during the temperature upshift in the MMS-;0.5% NaCl medium.

In V. vulnificus, the resuscitation is almost completely inhibited by a adding protein synthesis inhibitor, chloramphenicol, during the first 24 h of temperature upshift, and is slightly affected when the inhibitor is added later [8]. Adding chloramphenicol at the beginning of or after 24 h of temperature upshift completely suppressed the resuscitation of nonculturable V. parahaemolyticus, but only slightly reduced the culturable cell density, when this inhibitor was added after 24 h (Fig. 4). The temperature upshift may trigger the expression of certain genes during resuscitation [16]. The expression of particular genes during the first 24 h of temperature upshift may be critical and generate enough components for the recovery of the nonculturable cells on the second or third day of the temperature upshift process [17].

During the early stage of resuscitation, proteins or enzymes for building peptidoglycan or other cellular components may be synthesized [8]. Other protective agents, including catalase and superoxide dismutase, may also be synthesized and protect the recovering cells from the adverse effect of increased metabolic activities [18,19]. According to the authors’ unpublished study, a significant quantity of superoxide dismutase is present in the exponential phase cells, but the quantity becomes undetectable in the VBNC V. parahaemolyticus. In the present work, the temperature upshift to 37 °C failed to recover any VBNC V. parahaemolyticus cells, although this temperature was optimal for bacterial growth as well as inducing VBNC cells (Fig. 3). Metabolic activities may have been initiated when the VBNC cells are temperature upshifted to either 25 or 37 °C. However, more peroxide or other harmful free radicals may be generated at 37 °C than at 25 °C. The presence of these harmful radicals may be fatal or cause sublethal injury to the sensitive resuscitating cells and inhibits the recovery of colony-;formation ability at 37 °C.

Resuscitation in a rich medium may be harmful. Incubating VBNC V. vulnificus in a rich heart infusion medium at room temperature has been proven not to recover the culturability for 24 h [14]. In this study, the nutrients in TSB-;3% NaCl medium were also responsible for the 12 h delay in the recovery of the culturability of the VBNC V. parahaemolyticus (Fig. 3). The viability and the culturability increased to around 1010 cells ml−1 after the temperature upshift (Fig. 3), a thousand times more than those of the initial population. Therefore, a few of the culturable cells were probably multiplied and the final population may have been a mixture of regrown and resuscitated cells, since VBNC cells, indistinguishable from those regrown cells, may have been resuscitated after a delay. When the old VBNC cells were suspended in this rich medium and shifted to 25 °C for resuscitation, the microscopic viable count revealed that these cells had multiplied. However, these viable cells failed to form colonies on agar medium (data not shown). The reason for the nutrients’ inhibition on the recovery of culturability of VBNC cells is unclear, but may be attributable to the presence of harmful radicals in the medium. During the preparation of rich medium, a certain amount of peroxide or harmful free radicals are generated and suppress the growth of Campylobacter[20] and other sensitive resuscitating VBNC cells. They also constrain the recovery of injured cells [6].

Under the optimal conditions reported in here, the VBNC state of 24 V. parahaemolyticus strains was induced over a period of 14–49 days, depending on the strain. VBNC induction for the clinical and environmental strains was unrelated to the source or virulence of these strains (Table 1). The O3:K6 strains considered are the first pandemic strains of this pathogen that spread rapidly throughout many Asian countries since 1996. These O3:K6 strains have a very close clonality, as determined by pulsed-;field gel electrophoresis or other molecular methods [12,21]. However, the induction time of the VBNC state was also strain-;dependent (Table 1). These strains may respond differently to the low and high temperature cycles of the natural marine environment [4]. Jiang and Chai [5] reported that a non-;pathological Kanagawa negative strain lost culturability more slowly than a Kanagawa positive strain. Their results concern only the characteristics of specific strains but not the general properties of different categories of V. parahaemolyticus strains.

In conclusion, recovery of cells by temperature upshift in the MMS-;0.5% NaCl medium which alone limited cell multiplication allowed only resuscitation of VBNC cells. The induction period and the age of the VBNC cells for successful resuscitation were strain-;dependent.

Acknowledgements

The authors thank the National Science Council of the Republic of China for financially supporting this research under Contract Nos. NSC 89-;2313-;B-;031-;001 and NSC90-;2313-;B-;031-;004.

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