Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co-culture with eukaryotic cells

Authors


  • A preliminary reporting of these findings was presented at the 43rd Annual Joint Panel Meeting of Cholera and Other Enteric Diseases, US-Japan Cooperative Medical Science Program, Fukuoka, Japan, 17–19 November, 2008.

Correspondence
Yoshifumi Takeda, Collaborative Research Center of Okayama University for Infectious Diseases in India, Dr. S. C. Banerjee Rd, ID Hospital Campus, Beliaghata, Kolkata 700010, India.
Tel: 91-33-2363-3373; fax: 91-33-2363-2398; email: takeda.yoshi@pharm.okayama-u.ac.jp

ABSTRACT

VBNC Vibrio cholerae O139 VC-280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co-cultured with CHO cells. Other eukaryotic cell lines, including HT-29, Caco-2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC-280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co-culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.

List of Abbreviations: 
APW

alkaline peptone water

FBS

fetal bovine serum

GFP

green fluorescent protein

IO

instant ocean

MEM

minimum essential medium

MEM-FBS

MEM supplemented with 10% fetal bovine serum, but without supplementation of streptomycin and penicillin

NA

nutrient agar

TCBS

thiosulfate citrate bile salts sucrose

VBNC

viable but nonculturable

The VBNC state in bacteria is defined as that in which the bacteria remain viable but the cells do not grow or divide on, or in, routinely used bacteriological media. VBNC state of Vibrio cholerae O1 was first reported by Xu et al. (1) and this finding was subsequently confirmed by many studies (2–7). Conversion of VBNC V. cholerae O1 to the culturable state was first demonstrated by Colwell et al. (8) who showed that inoculation of VBNC V. cholerae O1 into rabbit ileal loops resulted in fluid accumulation from which culturable V. cholerae O1 could be isolated. Furthermore, it was shown that VBNC V. cholerae O1 converted to the culturable state after ingestion during a human volunteer study (9).

Wai et al. (3) reported in vitro conversion of VBNC V. cholerae O1 to the culturable state by heat shock, but confirmation of this finding was not successful. In the present study, we report in vitro conversion of VBNC V. cholerae O139 and O1 to the culturable state by co-culture with eukaryotic cells.

MATERIALS AND METHODS

Bacterial strains and culture media

Vibrio cholerae O139 VC-280 isolated in Kolkata from a cholera patient, V. cholerae O1 N16961 which is a stock culture in our laboratory, and V. cholerae O139 VC-280/pG13, which is a transformed strain of V. cholerae O139 VC-280 by a plasmid pG13 encoding GFP, were used in the present study. Culture media used were NA (Difco, Franklin Lakes, NJ) supplemented with 1% NaCl (NA-NaCl) and APW, pH 8.8. TCBS agar (Eiken, Tokyo, Japan), a medium selective for V. cholerae, was also used.

Eukaryotic cells and culture conditions

CHO, Caco-2, T84, HeLa, and Intestine 407 cell lines, available as stock cultures in our laboratory, were used in the present study. The HT-29 cell line was kindly provided by Dr Y. Fujinaga, Research Institute for Microbial Diseases, Osaka University, Suita, Japan. MEM (catalogue number: 61100-061; Gibco Life Science, Paisley, Scotland) and DMEM (catalogue number: 12800-017; Gibco Life Science) were used as basic culture media. CHO and HeLa cells were cultured in MEM supplemented with 10% FBS (catalogue number: 10082-147; Gibco Life Science). HT-29 cells were cultured in DMEM supplemented with 1.5 g/l NaHCO3 (Sigma, St. Louis, MO), 3.56 g/l 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES; Sigma) and 10% FBS. Caco-2 cells were cultured in MEM supplemented with 20% FBS. The T84 and Intestine 407 cells were cultured in DMEM supplemented with 10% FBS. All media were supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin. When VBNC V. cholerae cells were co-cultured with all the eukaryotic cells mentioned above, the medium used was MEM supplemented with 10% FBS, but without supplementation of streptomycin and penicillin (this modified medium was further abbreviated as MEM-FBS), and incubated at 37°C in 5% CO2.

Preparation of VBNC V. cholerae

Vibrio cholerae was induced to the VBNC state as described by Chaiyanan et al. (10), with the following modification. V. cholerae was inoculated into APW and incubated at 37°C for 16 hr, after which the cells were collected by centrifugation at 5000 ×g for 10 min at 25°C (Heraeus Biofuges Stratos; Kendro, Langenselbold, Germany), washed twice with 1% sterile solution of artificial seawater IO (Aquarium Systems, Mentor, OH), and suspended in 200 ml of 1% IO in a l-liter flask to give a final concentration of V. cholerae of approximately 108 cells/ml. Microcosms thus prepared were incubated at 4°C in the dark, without shaking.

Viability of VBNC

Viability of the prepared VBNC was examined by a LIVE/DEAD BacLight bacterial viability kit (Invitrogen, Carlsbad, CA). Procedures were as instructed by the manufacturer.

Conversion of VBNC to the culturable state

Conversion of VBNC V. cholerae cells to the culturable state by co-culture with eukaryotic cells was determined as follows. The eukaryotic cells were grown in each well of a 24-well plate (Techno Plastic Products AG, Trasadingen, Switzerland) with 0.5 ml of the respective medium at 37°C in a CO2 incubator under an atmosphere of 5% CO2. After the cells became confluent, they were washed twice with sterile medium, the same as that used for growth, after which 0.5 ml MEM-FBS was added to each well. Then, 0.1 ml each of twofold serial dilutions of the microcosm containing VBNC cells was inoculated into each well. The cultures were observed for 4 days after inoculation with the VBNC cells and if the medium became turbid, growth was confirmed by plating on TCBS agar and the subsequent appearance of characteristic sucrose fermenting yellow colonies. The yellow colonies were plated on NA-NaCl and each colony was tested by slide agglutination for positive reaction with either anti-O139 or anti-O1 typing serum (Denka Seiken, Tokyo, Japan).

V. cholerae cells converting to the culturable state were expressed as the highest dilution of the initial microcosm containing VBNC cells at which turbidity of MEM-FBS in the well was observed. Each experiment was carried out by inoculation of the same sample into two independent wells of the 24-well plate (i.e. in duplicate).

Preparation of conditioned medium

Confluent CHO cells were prepared using MEM-FBS, and incubated for 96 hr at 37°C in a CO2 incubator under 5% CO2. The spent medium was collected and sterilized by filtration through a 0.2-μm filter (Sartorius AG, Göttingen, Germany) and this was defined as conditioned medium.

Examination of morphology

Vibrio cholerae O139 VC-280 was transformed by a plasmid pG13 (11) that encodes GFP. Transformation was carried out by electroporation as described elsewhere (12). VBNC of the transformed cells, V. cholerae O139 VC-280/pG13, were prepared as described above by incubating the microcosm for 84 days. VBNC and culturable V. cholerae O139 VC-280/pG13 were harvested, washed twice with chilled sterile PBS (Sigma) and suspended in 50 μl sterile PBS. A drop of the prepared sample was mixed with a drop of 96% glycerol on a glass slide with a cover glass carefully placed on the mixture to avoid formation of air bubbles. The cell preparations were viewed using a laser scanning confocal microscope (LSM 510, Metal; Carl Zeiss, Thornwood, NY), with laser excitation at 488 nm and emission at 545 nm. Photographic records were made and analyzed using LSM 510 software (Carl Zeiss).

RESULTS

Preparation of VBNC V. cholerae

A microcosm containing approximately 108 cells/ml V. cholerae O139 VC-280 in 1% IO solution was incubated at 4°C for 42 days, after which plating on NA-NaCl yielded no growth. However, at this point of time, growth was confirmed by enrichment in APW. After incubation for an additional 28 days (70 days after the initiation of the original incubation), no growth was observed in APW. Yet, growth was observed by enrichment in MEM-FBS. By further incubation of the microcosm for 4 more days (74 days after the initiation of the original incubation) in 1% IO, the microcosm did not yield growth even after MEM-FBS enrichment (i.e. on NA-NaCl or in both APW and MEM-FBS), and it was concluded that the cells in the microcosm were in the VBNC state. These culture data are summarized in Table 1.

Table 1.  Culturability of microcosm V. cholerae O139 VC-280 in various media
Duration (days) of incubation in 1% IONA-NaClAPWMEM-FBS
  1. +, growth of the cell was observed.

  2. –, growth of the cell was not observed.

<41+++
42 − 69++
70 − 73+
74

Similarly, VBNC state of V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 was obtained under the same conditions. However, the length of time for the cells to enter the VBNC state varied (74–98 days) from strain to strain, with the size of the initial inoculum into 1% IO, and even with a slight change in the temperature during incubation.

Morphology of VBNC V. cholerae

VBNC cells of V. cholerae O139 VC-280/pG13 expressing the GFP gene were prepared and photographed as described above. As shown in Figure 1a, the morphology of all VBNC cells was round in shape and appeared as coccoid cells, whereas culturable cells were a typical curved rod shape (Fig. 1b).

Figure 1.

Morphology of VBNC and culturable V. cholerae O139 VC-280/pG13. Preparation of VBNC V. cholerae O139 VC-280/pG13 was as described in Materials and Methods. The microcosm was kept at 4°C in the dark for 84 days and the VBNC state of the cells were confirmed by culturing an aliquot of the microcosm in NA-NaCl, APW and MEM-FBS, as described in Materials and Methods. (a) VBNC state, (b) a fresh, actively growing culture in APW. Photographs were taken as described in Materials and Methods. Bar represents 5 μm.

Conversion of VBNC cells of V. cholerae to the culturable state by co-culture with CHO cells

In an experiment shown in Table 2, it was demonstrated that VBNC V. cholerae O139 VC-280 converted to the culturable state when co-cultured with CHO cells. At day 74 after the initiation of the original incubation of the microcosm, 1:32 dilution of the microcosm yielded culturable cells. No conversion was observed without CHO cells, that is, MEM-FBS alone.

Table 2.  Conversion of VBNC V. cholerae O139 VC-280 to the culturable state by co-culture with CHO cells
 Duration (days) of incubation in 1% IO
747781848891
  1. †Highest dilution of the microcosm at which MEM-FBS became turbid.

  2. Each experiment carried out in duplicate gave the same results.

  3. ‡No conversion was observed with undiluted sample.

  4. §No conversion was observed with any dilutions of the sample.

With CHO cells in MEM-FBS1:32†1:161:41:21:1−‡
With MEM-FBS alone−§

When the same VBNC sample was incubated for a longer time in 1% IO at 4°C, the highest dilution of the microcosm that showed conversion to the culturable state by co-culture with CHO cells slowly went down and 91 days after the initiation of the original incubation in 1% IO further conversion to the culturable state was not observed. No conversion was observed with MEM-FBS alone throughout this study. At the time of the incubation, viability of the cells in the microcosm was examined by a LIVE/DEAD kit and it was found that a considerable number of the cells were still alive, suggesting that only the part of the VBNC V. cholerae cells converted to culturable by co-culture with CHO cells.

These observations were confirmed with the strains of V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13, although the length of incubation in 1% IO until conversion was first observed and until further conversion no longer occurred varied (91–110 days) from strain to strain.

Effect of eukaryotic cells on conversion to the culturable state

Table 3 shows that several eukaryotic cell lines supported conversion of VBNC V. cholerae O139 VC-280 to the culturable state. VBNC cells co-cultured with eukaryotic cells, including HT-29, Caco-2, T84, HeLa, and Intestine 407, all converted to the culturable state, although the highest dilution in which the conversion was observed differed with eukaryotic cell lines. In all experiments, VBNC cells cultured with MEM-FBS alone (controls) did not yield culturable cells.

Table 3.  Conversion of VBNC V. cholerae O139 VC-280 to the culturable state by co-culture with various eukaryotic cells†
 HT-29Caco-2T84HeLaIntestine 407CHO
  1. †The microcosm used in this experiment was incubated in 1% IO for 74 days.

  2. ‡Highest dilution of the microcosm at which MEM-FBS became turbid. Each experiment carried out in duplicate and repeated twice gave the same results.

  3. §No conversion was observed with any dilutions of the sample.

With eukaryotic cells in MEM-FBS1:32‡1:161:321:161:161:32
With MEM-FBS alone−§

Effect of conditioned medium on conversion

The ability of conditioned medium to convert VBNC cells to culturability was tested using V. cholerae O139 VC-280. As shown in Table 4, VBNC cells incubated in conditioned medium did not convert to the culturable state. Fresh MEM-FBS, with and without CHO cells, served as controls, yielding growth only with CHO cells.

Table 4.  Effect of conditioned medium on conversion of VBNC V. cholerae O139 VC-280 to the culturable state
 Highest dilution of the microcosm at which MEM-FBS became turbid†
  1. †Microcosm used in this experiment was incubated in 1% IO for 77 days. Each experiment carried out in duplicate and repeated twice gave the same results.

  2. ‡No conversion was observed with undiluted sample.

With conditioned medium−‡
With CHO cells in MEM-FBS1:16
With MEM-FBS alone

DISCUSSION

VBNC V. cholerae are defined as cells unable to grow either directly on NA-NaCl or after enrichment in APW and in MEM-FBS after prolonged incubation in 1% IO at 4°C. The morphology of induced VBNC V. cholerae O139 VC-280/pG13 was coccoid (Fig. 1a). The coccoid forms of VBNC cells of several bacterial species including V. cholerae have been reported by other investigators (2, 10, 13).

As shown in Table 2, when V. cholerae O139 VC-280 cells in the VBNC state, after incubation in 1% IO for 74 days or more, were co-cultured with CHO cells, conversion to the culturable state was observed. Number of VBNC cells converted to the culturable state by co-culture with CHO cells decreased with each day of incubation in 1% IO, while further conversion of cells was no longer detected after 91 days of incubation. The medium used to culture CHO cells, MEM-FBS, did not yield culturable cells. Conversion was also observed with VBNC cells in co-culture with other cell lines (Table 3). From these results, we conclude that co-culture of VBNC V. cholerae with selected eukaryotic cells converts VBNC cells to the culturable state.

Conditioned medium, prepared by growth of CHO cells in MEM-FBS and then removing the cells, did not support VBNC conversion to the culturable state (Table 4), suggesting that conversion is not caused by a factor(s) released from CHO cells into the medium. It is assumed that it is the proximity of VBNC cells with eukaryotic cells that is a prerequisite for conversion to the culturable state. This is an important finding, considering that V. cholerae is the causative agent of the enteric disease, cholera.

Conversion, or recovery, of VBNC to the culturable state has been reported for several other pathogenic bacteria, notably V. parahaemolyticus (14), V. vulnificus (15), Salmonella enteritica serovar Typhimurium (16–18), Escherichia coli (16, 19) and Legionella pneumophila (20). The conditions that have been associated with recovery of culturability include temperature upshift (3, 17), incubation in phosphate buffer (19), supplementation with H2O2-degrading compounds, such as catalase or sodium pyruvate (14), addition of heat-stable autoinducer of growth (16), addition of resuscitation promoting factor (18), and presence of Acanthamoeba castellanii (20).

The conditions for converting VBNC V. cholerae to the culturable state, as reported in the present paper, are unique and can easily be incorporated into a method for testing environmental, food, or other samples of public health concern for VBNC pathogenic bacteria, and the natural aquatic environment, which has also been shown to be a source of human pathogens (18). By demonstrating that VBNC V. cholerae can convert to active growth and multiplication in samples from those sources will allow some of the heretofore unanswered questions concerning the epidemiology of infectious diseases, notably cholera, which is characterized by sudden occurrence and an episodic nature, to be answered.

ACKNOWLEDGMENTS

We thank Dr S. Ganguly and Ms E. Ghosh of the National Institute of Cholera and Enteric Diseases, Kolkata, India for their assistance with confocal microscopy. We thank Dr Y. Fujinaga, Research Institute of Microbial Diseases, Osaka University, Suita, Japan and Dr R. Nandy, National Institute of Cholera and Enteric Diseases, Kolkata, India for providing HT-29 cells and plasmid pG13, respectively. We also thank Ms D. Ganguly for assistance in editing the manuscript. This study was supported by the Program of Founding Research Center for Emerging and Reemerging Infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology of Japan, and NIH Grant ‘Epidemiology and Ecology of Vibrio cholerae in Bangladesh’, No. 1RO1A139129-01 and NOAA Distinguished Scholar in Oceans and Human Health Grant No. SO660009.

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