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Resuscitation of Viable but Non-Culturable Vibrio Parahaemolyticus in a Minimum Salt Medium

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Resuscitation of Viable but Non-Culturable Vibrio Parahaemolyticus in a Minimum Salt Medium FEMS Microbiology Letters 233 (2004) 269–275 www.fems-microbiology.org Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium Hin-chung Wong *, Peily Wang, Shau-Yan Chen, Shen-Wen Chiu Department of Microbiology, Soochow University, Taipei 111, Taiwan, ROC Received 11 June 2003; received in revised form 12 February 2004; accepted 22 February 2004 First published online 3 March 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 de- termining 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. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Vibrio parahaemolyticus; Viable but non-culturable; Resuscitation; Temperature upshift 1. Introduction during the winter. VBNC V. parahaemolyticus has been developed by starving cells at low temperature Vibrio parahaemolyticus, a halophilic Gram negative [5], but has not been well characterized. They also bacterium, causes acute gastroenteritis in humans. reported that the resuscitation of VBNC cells was the This bacterium is a prevalent food-borne pathogen in results of cell regrowth of a limited amount of cul- Japan, Taiwan, and other coastal countries [1,2]. Most turable cells [5]. Mizunoe et al. [6] reported on the clinical isolates are hemolytic on Wagatsuma agar recovery of nonculturable V. parahaemolyticus cells in (Kanagawa-positive, KP+) and produce a major vir- agar medium containing catalase or pyruvate. How- ulence factor, thermostable direct hemolysin (TDH) ever, the cells were probably injured to become non- [1]. culturable by the low salinity used in the study, and V. parahaemolyticus inhabits seawater, but is seldom later recovered by the presence of these protective isolated when the temperature of the seawater is under agents instead of resuscitation of VBNC cells. In this 13–15 °C [3]. Like other vibrios that inhabit similar report, the nonculturable bacterial cells were still rod marine environments [4], V. parahaemolyticus may be shaped with blebs instead of the usual coccoid VBNC present in a viable but nonculturable (VBNC) state cells [6]. This investigation analyzes the resuscitation process of VBNC V. parahaemolyticus triggered by temperature upshift in a minimum salt medium which alone limited * Correspondence author. Tel.: +886-2-28819471x6852; fax: +886-2- multiplication of cells. The induction and resuscitation 28831193. of various strains of V. parahaemolyticus were also E-mail address: [email protected] (H.-c. Wong). compared. 0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.02.015 270 H. Wong et al. / FEMS Microbiology Letters 233 (2004) 269–275 2. Materials and methods and 1.21 g Tris buffer (pH 7.8) in one liter of deionized water to prevent the carry-over of nutrients [7]. The 2.1. Cultures and media bacterial cells were suspended in the 250-ml Erlenmeyer flask that contained 100 ml MMS-0.5% NaCl medium at V. parahaemolyticus ST550, a serotype O4:K13 and a concentration of about 107 cells mlÀ1, and incubated KP+ clinical strain isolated in Thailand, was used in this at 4 °C in a static state, to induce the VBNC state. study. A total of 20 clinical and 4 environmental strains Bacteria were counted every seven days. were used for comparison (Table 1). These strains were stocked in culture broth with 10% (v/v) glycerol at 2.3. Resuscitation )85 °C, and cultured in Tryptic Soy Broth Medium (TSB, Difco Laboratories, Detroit, Mich.)-3% (w/v) NaCl or The temperature upshift method was used to resus- Tryptic Soy Agar (TSA, Difco)-3% NaCl at 25 °C. citate VBNC V. parahaemolyticus [5]. The temperature of the VBNC culture with under one culturable cell per 2.2. Induction of VBNC state milliliter was changed from 4 to 25 or 37 °C and incu- bated for one to three days. Viable and culturable cells Bacteria were cultured in 50 ml TSB-3% NaCl me- were counted. Nalidixic acid at 5, 10, 20, 50, 100, or 200 dium in a 250-ml Erlenmeyer flask, and incubated at lgmlÀ1 was added to assay inhibition of bacterial cell 25 °C with shaking at 110 rpm for 4.5 h to reach the multiplication during the temperature upshift [5]. The mid-exponential phase. Bacterial cells were harvested by VBNC culture was also diluted 10-fold to assay the ef- centrifugation at 6000g for 15 min, washed twice in fect of dilution, and diluted 5-fold into TSB-3% NaCl or modified Morita mineral salt solution (MMS-0.5% MMS-0.5% NaCl media, to assay the effect of nutrients NaCl) which consisted of 5 g NaCl, 0.8 g KCl, 5.6 g on resuscitation. Chloramphenicol (100 lgmlÀ1) was MgCl2 Á 6H2O, 5.6 g, 7.6 g MgSO4 Á 7H2O, 0.9 mg added to the VBNC culture to determine the importance FeSO4 Á 7H2O, 1.54 g CaCl2 Á 2H2O, 0.1 g Na2HPO4 of protein synthesis in the resuscitation [8]. Table 1 Induction and resuscitation of VBNC state by different strains of Vibrio parahaemolyticus Strain no. Date of isolation Serotype KP VBNC by (day)a Resuscitable period (day)b Clinical ST550 1983 O4:K13 + 35–49 14 1264 1998 O5:K68 ND 28–35 14 1265 1998 O5:K68 ND 28–35 <7 1292 1999 O2:K3 + 35–42 <7 1079 1997 O3:K6 + 35–42 7 1089 1997 O3:K6 + 42–49 7 1091 1997 O3:K6 + 42–49 <7 1092 1997 O3:K6 + 35–42 14 1099 1997 O3:K6 + 42–49 14 1109 1997 O3:K6 + 42–49 7 1119 1997 O3:K6 + 35–42 7 1129 1997 O3:K6 + 35–42 7 1132 1997 O3:K6 + 14–28 7 1137 1997 O3:K6 + 28–35 <7 1147 1997 O3:K6 + 28–35 <7 1188 UN O3:K6 + 14–28 7 1288 1996 O3:K6 + 35–42 <7 1289 1996 O3:K6 + 28–35 7 1290 1996 O3:K6 + 28–35 7 1291 1996 O3:K6 + 35–42 <7 Environmental 1256 1999 ND ND 14–28 7 1257 1999 ND ND 28–35 <7 1258 1999 ND ND 28–35 <7 1266 1998 ND ND 28–35 7 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 phe- nomenon; UN, unknown. H. Wong et al. / FEMS Microbiology Letters 233 (2004) 269–275 271 2.4. Enumeration of bacteria 9 8 The culturability of bacteria was determined by the plate count method. Decimal dilutions were obtained in 7 -1 MMS-0.5% NaCl at 4 °C and plated on TSA-3% NaCl. 6 The plates were incubated at 37 °C for 16 h and the 5 number of colonies counted. Duplicate experiments were performed and the data were obtained as means of 4 three determinations in each experiment. 3 The total number of viable cells was counted using Log cell number ml 2 the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probe Inc., Eugene, OR, USA) following the 1 procedures of Mizunoe et al. [6]. This kit has been used 0 to determine the number of total and viable cells of 0 1020304050 several other bacteria [9–11]. Time, day Effect of catalase on the culturability was assayed Fig. 1. Induction and resuscitation of VBNC Vibrio parahaemolyticus according to the method of Mizunoe et al. [6]. Plate in MMS-0.5% NaCl medium. The cells were cultured at 4 or 25 °C. counts were performed using agar plates with or without The 4 °C culture was shifted to 25 °C (arrow) when all cells entered the the supplementation of filter-sterilized Aspergillus niger VBNC state. Viable cell counts and plate counts were determined. Plate counts are given in cfu mlÀ1. Vertical bars designate the standard catalase (Sigma Co., St. Louis, MO, USA), 2000 U per errors. d, plate count of the 25 °C culture; ,, viable count of the 25 °C plate. culture; j, viable count of the 4 °C culture; ., plate count of the 4 °C The resuscitating bacteria were also enumerated by culture without catalase; s, plate count of the 4 °C culture catalase. the most probable number method (MPN) [5]. Decimal dilutions were obtained in MMS-0.5% NaCl. One mil- liliter of the diluted samples was inoculated into MPN significantly enhance the culturability during the VBNC tubes containing 9-ml of MMS-0.5% NaCl and incu- induction period (Fig. 1). bated at 25 °C in a static state for three days. Equal volume of TSB-3% NaCl was added to the MPN tubes 3.2. Resuscitation of VBNC cells and incubated for one more day. Resuscitation of the VBNC cells was performed by 2.5. Determination of minimum inhibition concentration temperature upshift treatment to 25 °C in the MMS- 0.5% NaCl medium.
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