Survivability and long-term preservation of bacteria in water and in phosphate-buffered saline*
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Ching-Hsing Liao, Eastern Regional Research Center, USDA, ARS, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA (e-mail: firstname.lastname@example.org).
Aims: To evaluate the suitability of using sterile water and phosphate-buffered saline (PBS) for preservation of bacteria pathogenic to plants or humans.
Methods and Results: The stationary-phase bacterial cells collected from rich agar media were transferred to 10 ml of sterile water or PBS (pH 7·2) containing KH2PO4, 15·44 μm; NaCl, 1·55 mm; Na2HPO4, 27·09 μm in a screw-cap tube. The tubes were sealed with parafilm membranes and stored in the dark and at room temperature. Almost all the bacteria tested (148 strains), including Pseudomonas fluorescens, P. viridiflava, Erwinia spp., Xanthomonas campestris, Cytophaga johnsonae, Salmonella spp., Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus, survived in water for at least several months and up to 16 years. A vast majority of the Gram-negative bacteria tested survived equally well in water and in PBS for at least 30 weeks. However, the populations of two Gram-positive bacteria [G(+)], L. monocytogenes and Staph. aureus, declined more rapidly in water than in PBS.
Conclusions: Plant- and human-pathogenic bacteria can be preserved in pure water or PBS for several years. G(+) bacteria appear to survive better in PBS than in water.
Significance and Impact of the Study: The method described here is a simple and economical means for preservation of bacterial cultures, which is especially useful for laboratories not equipped with the lyophilizer or ultra-low freezer. Long-term survival of food-borne pathogens in water underlines the importance of water as a potential vehicle for transmitting the diseases.
Preservation of bacterial cultures is a routine and an important task for most microbiological laboratories engaged in research, teaching or industrial application. For short-term storage, bacterial cultures may be preserved by immersing in mineral oil, by ordinary freezing or by drying on a suitable menstruum (Heckly 1978). For long-term storage, cultures are usually preserved by lyophilization or by ultra-freezing (Gherna 1994). Kelman (1956) reported that the viability of a soil-borne bacterium, Ralstonia solanacearum, can be maintained in tap water for several months. Two studies conducted later by Wakimoto et al. (1982) and by Van Elsas et al. (2001) showed that R. solanacearum not only survived but also multiplied for several generations in pure water. Iacobellis and DeVay (1986) reported that other phytopathogenic bacteria including Agrobacterium tumefaciens and Pseudomonas syringae ssp. syringae could survive in sterile water for over 20 years.
Despite the simplicity, preservation of bacterial cultures in water has not been widely adopted in microbiology laboratories, possibly because of the lack of experimental data from multiple-year studies to confirm the reliability of this method. Additionally, it has not been investigated if water preservation method can be applied to store bacteria which are of clinical, industrial or environmental importance. The objectives of this study were to: (a) examine the viability of plant-associated bacteria that were stored in water 16 years ago, (b) determine if water method could be extended for preservation of human-pathogenic bacteria and (c) compare the survivability of G(+) and Gram-negative [G(−)] bacteria stored in water, and in phosphate-buffered saline (PBS).
Bacteria, media and culture conditions
A total of 148 bacterial strains were tested during the study. The majority of them originated from the previous investigations of postharvest quality and safety of fresh vegetables and fruits (Liao and Wells 1986; Liao and Wells 1987a,b; Liao 1989; Liao et al. 1999). They included P. fluorescens (41 strains), P. viridiflava (16 strains), P. putida (one strain), Erwinia spp. (eight strains), Xanthomonas campestris (19 strains) and Cytophaga johnsonae (nine strains). Human-pathogenic bacteria included in the study were Salmonella spp. (14 strains), Listeria spp. (35 strains), Yersinia enterocolitica (two strains), P. aeruginosa (one strain), Escherichia coli O157:H7 (one strain) and Staphylococcus aureus (one strain). For preparation of stock cultures for long-term storage, plant-associated bacteria were grown on Pseudomonas agar F (PAF; Difco Lab., Detroit, MI, USA) at 28°C for 48 h and human-pathogenic bacteria were grown on brain heart infusion agar (BHIA; Difco) at 37°C for 24 h. When a liquid medium was needed, brain heart infusion broth (BHIB; Difco) was used.
Multiple-year preservation and storage conditions
Four to five loops of each bacterial strain grown on PAF or BHIA were transferred to 10 ml of sterile distilled water in a screw-cap tube (16 × 125 mm), normally two to three tubes per strain. The screw-cap of the inoculated tube was then sealed with a parafilm (3M Co., St Paul, MN, USA) membrane to minimize evaporation. The tubes were then stored in the dark and at room temperature (20–25°C). Plant-associated bacteria were stored under this condition for 8–16 years and human-pathogenic bacteria stored under the same condition for 3–5 years. The viability of the culture was determined by placing 250 μl aliquot of the bacterial suspension from each tube on PAF or BHIA and allowing the agar plate to incubate at 28 or 37°C for 2–3 days.
Thirty-week storage study
To examine the population changes of bacteria during the early stage of storage, nine representative strains of bacteria were preserved in sterile distilled water and in PBS (pH 7·2; KH2PO4, 15·44 μm; NaCl, 1·55 mm; Na2HPO4, 27·09 μm; Life Technologies, Rockville, MD, USA) and the numbers of viable bacteria in the preservation tubes were determined at 5-week intervals for 30 weeks. The nine representative strains of bacteria tested were P. aeruginosa PAO1, Salmonella Mbandaka S14, L. monocytogenes Scott A, P. fluorescens CYO91 (Biovar II), P. fluorescens LU-04-1B (Biovar IV), P. fluorescens BC-05-1B (Biovar V), P. putida ATCC 12633, Erw. carotovora ssp. carotovora SR319, X. campestris pv. campestris XC11. For preparation of the stock cultures, bacterial cells grown in BHIB were collected by centrifugation (12 000 g, 10 min), washed twice with water or PBS, and resuspended in water or PBS in half the volume as the original broth culture. One millilitre of the bacterial suspension was transferred to 9 ml of water or PBS depending on the wash fluid used. Six tubes were prepared for each bacterial strain in each preservation fluid, water or PBS, for each time point. The inoculated tubes were then sealed with parafilm membrane and stored in the dark at room temperature. The populations for each bacterial strain in six replicate tubes in water or PBS were determined at each 5-week interval.
Four-week storage study
To compare the population changes of G(+) and G(−) bacteria stored in water and in PBS, one G(−) pathogen (E. coli O157:H7) and two G(+) pathogens (L. monocytogenes and Staph. aureus) were preserved in sterile distilled water and in PBS according to the procedures described above. The changes in the viable cell counts of each strain during storage were determined at 1 week interval for 4 weeks. Six replicate tubes for each bacterial strain in water or PBS sample were examined every week.
All statistical analyses were performed using statistical analysis system software to a significance level of P < 0·05. For the 30 week study, the results were compared by anova to determine any significant interactions between organism and treatments for log reductions after storage for 30 weeks. The mean for the organism and treatment combinations were compared using the Bonferroni LSD technique. For the 4 week study, the results were compared by anova to determine any significant differences in the regressions of the log CFU vs time.
Recovery of plant- and human-pathogenic bacteria from water suspensions stored at room temperature for 3–16 years
After storage in sterile distilled water for 12–16 years, almost all the bacteria associated with postharvest rot of fresh produce including P. fluorescens, P. viridiflava, Erw. spp. and X. campestris (Table 1) remained viable and could be recovered by plating the preserved culture on rich agar media, BHIA or PAF. The recovered bacteria retained the ability to degrade pectic substances and to induce soft-rot disease in plants. However, only three of nine (or 33%) C. johnsonae strains were recovered after storage in water for 13 years. G(−) human-pathogenic bacteria, including Salmonella spp. (14 strains) and Y. enterocolitica (two strains), could also survive in water for at least 5 years. However, the G(+) Listeria spp. appeared to survive in water less favourably than G(−) bacteria. Only 27 of 35 strains of Listeria spp. were recovered after storage in water for 3 years. With a few exceptions (for example Cytophaga and Listeria), the majority of plant- and human-pathogenic bacteria tested remained viable in sterile distilled water for at least 5 years.
Table 1. Recovery of plant- and human-pathogenic bacteria from cultures preserved in sterile distilled water for different number of years*
The population changes of nine representative bacteria stored in water and PBS for 30 weeks
To further evaluate the feasibility of using water for preservation of pathogenic bacteria, three human pathogens (P. aeruginosa, Salm. Mbandaka and L. monocytogenes) in addition to six plant-associated bacteria were included in the study. The population change of each strain during the 30-week storage in water and in PBS was determined and the dynamics of the changes of three human pathogens and P. fluorescens LU-04-1B in water and in PBS were illustrated in Fig. 1. The results show that all three G(−) bacteria pathogenic to plants and humans (e.g. P. fluorescens LU-04-1B, P. aeruginosa and Salm. Mbandaka) appeared to survive equally well in water and in PBS. However, the G(+) bacterium, L. monocytogenes, appeared to survive better in PBS, than in water. The decline in the bacterial population was observed largely during the first 5 or 10 weeks of storage for all of the G(−) bacteria tested. (Fig. 1). By comparison, the viable cells of L. monocytogenes continued to decline after storage in water for 10 weeks, but at a relatively slower rate. However, when stored in PBS, the declines in the populations of both G(−) and G(+) bacteria occurred mainly during the first 5 weeks of storage. These results suggest that, although pure water is, in general, suitable for preservation of G(−) bacteria, PBS should be a better choice for G(+) bacteria such as L. monocytogenes. Nevertheless, only a small proportion of bacteria (1% or less) survived after storage in water or PBS for 30 weeks.
Table 2 compares the reduction in the population of plant- and human-pathogenic bacteria after storage in water and in PBS for 30 weeks. As noted above, there was no significant difference in the reduction in the population of G(−) bacteria that were stored in water and PBS for 30 weeks. The difference in the reduction in viable cell counts of G(−) bacteria after storage in water or in PBS for 30 weeks was in the range of 0·08–0·55 log10 CFU ml−1. However, the difference in the reduction in viable cell counts of L. monocytogenes was 4·55 log10 CFU ml−1 (Table 2). Thus, the difference in the log reduction for L. monocytogenes stored in water and in PBS was approximately six to 10 times larger than that observed for G(−) bacteria stored under similar conditions (Table 2).
Table 2. Reduction in the population of plant- and human-pathogenic bacteria after storage in water and phosphate-buffered saline (PBS) for 30 weeks
|Pseudomonas aeruginosa PAO1||2·67 (0·21)||2·27 (0·54)||0·40|
|Salmonella Mbandaka S14||2·56 (0·28)||2·82 (0·15)||−0·26|
|Listeria monocytogenes Scott A†||6·18 (< 0·01)||1·63 (2·34)||4·55|
|Pseudomonas fluorescens CY091 (Biovar II)||0·91 (12·35)||0·99 (10·24)||−0·08|
|Pseudomonas fluorescens LU-04-1B (Biovar IV)||1·82 (1·51)||1·94 (1·15)||0·12|
|Pseudomonas fluorescens BC-05-1B (Biovar V)||1·98 (1·04)||1·43 (3·71)||0·55|
|Pseudomonas putida ATCC 12633||2·22 (0·60)||1·92 (1·21)||0·30|
|Erwinia carotovora ssp. carotovora SR319||2·97 (0·11)||2·60 (0·25)||0·37|
|Xanthomonas campestris pv·campestris XC11||2·42 (0·38)||2·28 (0·52)||0·14|
Comparison of the population changes of two strains of G (+) and one strain of G(−) pathogen stored in water and PBS for 4 weeks
To further confirm that G(+) bacteria survived better in PBS than in water, the population changes of two G(+) (L. monocytogenes and Staph. aureus) and one G(−) pathogen (E. coli O157:H7) that were stored in water and PBS were monitored weekly for a period of 4 weeks. The results (Fig. 2, left) show that, while the population of E. coli O157:H7 declined only slightly during the 4 week storage in water (reduction in 0·61 log unit), the populations of two G(+) bacteria that were stored in water decreased more sharply, reduction in 2·32 and 1·55 log units for L. monocytogenes and Staph. aureus, respectively. However, when three strains of human pathogens were stored in PBS, the results (Fig. 2, right) show that the patterns of population changes are not significantly different (P < 0·05) among three strains of bacteria tested. The results from the 4-week study further demonstrated the improved survival of G(+) bacteria in PBS than in pure water.
Previously, it has been reported that the viability of phytopathogenic bacteria including R. solanacearum (Kelman 1956; Wakimoto et al. 1982; Van Elsas et al. 2001), A. tumefaciens (Iacobellis and DeVay 1986) and P. syringae (Iacobellis and DeVay 1986) can be maintained in pure water for several years. The data presented here further demonstrate that pure water is also suitable for preservation of a large number of G(−) bacteria pathogenic to plants or humans, which include P. fluorescens, P. viridiflava, X. campestris, Salmonella spp., Y. enterocolitica and possibly E. coli O157:H7. Nevertheless, this study also shows that water preservation does not appear to be suitable for G(+) bacteria. First, when stored in water, the viable cell counts of two G(+) bacteria included in this study (Staph. aureus and L. monocytogenes) declined very rapidly during the first 4 or 30 weeks of storage. Secondly, eight of the 35 strains of Listeria spp. included in this study were no longer recoverable after storage in water for 3 years. However, the data from 4 and 30 week studies with L. monocytogenes and Staph. aureus suggested that PBS may be used to replace pure water for preservation of non-spore forming G(+) bacteria. As only a small proportion (1% or less) of bacteria survived after storage in water or PBS for 30 weeks, it is not clear if the surviving cells represent the selection of a special segment of stress-tolerant cells or variants in the population.
Inspite of the simplicity, preservation of bacteria in pure water has not been widely adopted in most of the microbiological laboratories possibly because of the lack of experimental data from multiple-year studies to validate the reliability of the method. Furthermore, only a small number of bacteria, mainly phytopathogens have been tested in the past. It was not known prior to this study, if this method might be applied to store bacteria which are of clinical, environmental or industrial importance. The data presented here affirm the dependability of using water or PBS for preservation of the majority of G(−) bacteria included in this study. Cytophaga strains are the only group found not to survive well in water and C. johnsonae is unique among the G(−) bacteria for its ability to form spherical resting bodies in aged cultures (Liao and Wells 1986). It is not clear if formation of the resting bodies is required for Cytophaga, to survive in extreme environments including hypotonic conditions. In addition, more studies are needed to determine if non-spore forming G(+) bacteria other than L. monocytogenes and Staph. aureus can be preserved in PBS for years.
Food-borne pathogen, Salm. typhi, have been shown to survive in tap water for up to 7 days (Mitscherlich and Marth 1984) and E. coli strains in groundwater for at least 132 days (Banning et al. 2002). In this study, 14 strains of Salmonella, two strains of Yersinia and one strain of E. coli O157:H7 have been shown to survive in sterile water for several months. Apart from its potential application for culture preservation, demonstration of the long-term survival of food-borne pathogens in water as described in this study further emphasizes the importance of sanitization treatments for water to be used for drinking or for cleaning of fresh and ready-to-eat produce.
We wish to thank the following colleagues for providing human pathogens included in this study: Virginia Miller, Mike Corby, Jeff Call, William Fett, Bassam Annous and George Somkuti. We would also like to thank Melvin Suárez and Gabriel Hoffman for technical assistance, Jim Smith for review of this manuscript and John Phillips for invaluable assistance with statistical analyses.