Nonculturable State of Vibrio vulnificus

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1989, p /89/ $02.00/0 Copyright D 1989, American Society for Microbiology Vol. 55, No. 11 Membrane Fatty Acid and Virulence Changes in the Viable but Nonculturable State of Vibrio vulnificus KATHERINE LINDER AND JAMES D. OLIVER* Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina Received 5 July 1989/Accepted 22 August 1989 The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C.6 and C16.1) had decreased 57% from the To level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in sie and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence. Survival of bacteria in aquatic environments is known to be affected by a variety of parameters, including salt concentration, presence of heavy metals, nutrient levels, and temperature. The viability of such bacteria has routinely been estimated by plating techniques using solid media and enumeration of the resulting CFU. It is now evident that such plating methods may be misleading and underestimate the number of viable cells in marine and nonmarine habitats. This appears to be due to the presence of stressed cells or cells in a dormant state in these populations (9, 28). Injured bacteria show increased sensitivity to the surface-active compounds, organic acids, salts, and dyes generally present in selective media, leading to increased lag times and inability to multiply. This results in nondetection of cells by isolation techniques that use solid selective media. In contrast, many aquatic bacteria appear to enter a natural dormant state when subjected to certain environmental stresses, such as low temperature or nutrient deprivation. This response appears to be a survival strategy. These cells, termed nonrecoverable or viable but nonculturable, have become the subject of increasing investigation and speculation (for a review, see reference 24). Much of this interest is due to the inability of standard methods to detect the presence of specific waterborne pathogens or coliform indicators (4, 9). Among those organisms which have been reported to enter the state of nonrecoverability are Escherichia coli (4, 30), Shigella sonnei, S. flexneri (4), Salmonella enteriditis (25), Aeromonas salmonicida (1), and Vibrio cholerae (4, 30). V. vulnificus is a human pathogen which has been isolated from many marine and estuarine sources, including water, sediment, plankton, and shellfish on the Atlantic, Gulf, and West coasts (16). This bacterium is unusual in its ability to cause both fatal septicemia and serious wound infections. Patients who develop septicemia become infected by ingestion of contaminated seafoods, most often raw oysters. This form of the infection progresses rapidly and is lethal in approximately 60% of the cases (16). Wound infections may be severe and involve progressive cellulitis. The mortality for wound infections is approximately 20%, and tissue * Corresponding author. debridement or limb amputation is often required. During winter months, the occurrence of Vibrio-associated illnesses is lowest and V. vulnificus is not recoverable from cold estuarine environments (16). Thus, it was of interest to examine the possibility that this organism enters into a state of nonculturability in cold, nutrient-limited seawater. Such a finding would be of interest from both public health and ecological standpoints. To this end, artificial seawater (ASW) microcosms were used and cells were assayed for total bacterial numbers by acridine orange direct counts (AODC), for viability by the method of Kogure et al. (7), and for culturability on both selective and nonselective media. Because of its importance as an indicator organism, a parallel study was performed with E. coli. In an attempt to relate the population changes observed to the metabolic and biochemical status of these cells, membrane changes that occurred during incubation were monitored. Specifically, membrane fatty acid species of both organisms were characteried. Further, because these membrane studies might relate to the ultrastructure of the bacteria, an electron microscopic study was performed on V. vulnificus. Finally, because of the public health importance of V. vulnificus in waters and shellfish and the general lack of knowledge regarding the virulence of human pathogens on entrance into the state of nonculturability, the pathogenicity of nonculturable cells of V. vulnificus was compared with that of culturable cells. MATERIALS AND METHODS Bacterial cultures. A virulent isolate, C7184, of V. vulnificus and E. coli ATCC were chosen for this study. For the experiments described, seed cultures were incubated in 100 ml of heart infusion (HI) broth (Difco Laboratories) at 25 C overnight. Cells were harvested and washed twice with sterile ASW. Cells were transferred into acid (6 N HCI)- washed 1-liter flasks containing 750 ml of ASW at 25 C. These microcosms were then chilled to 5 C at 0.1 C/min and maintained at that temperature throughout incubation. Cell enumerations. To (inoculation time) and subsequent samples were taken for plate counts, AODC, and direct viable counts (DVC). For plate counts, V. vulnificus was 2837

2 2838 LINDER AND OLIVER spread onto HI agar, thiosulfate-citrate-bile salts-sucrose agar (BBL Microbiology Systems), and a nonselective agar, MSWYE (17). E. coli was plated onto HI agar and eosinmethylene blue agar (BBL). Dilution blanks were initially at 5 C but were warmed to 25 C at 4 C/min after addition of cells and before plating. Plates were incubated at 25 C, and CFUs were counted at 3 days. Samples were taken at various times for up to 24 days for V. vulnificus and up to 32 days for E. coli. To determine CFUs at concentrations of less than 1 cell per ml toward the end of incubation, 10.0 ml of the microcosm was filtered through a 0.2-pum-pore-sie sterile Nuclepore filter which was placed onto the various solid media and observed for growth. AODCs were performed as previously described (15) to determine total bacterial numbers. Samples were also taken for enumeration of DVCs as described by Kogure et al. (7). In this procedure, a sample is enriched to a final concentration of 0.25% yeast extract (Difco) in 0.02% nalidixic acid (Sigma) and incubated for 6 h at 25 C. Following observation by epifluorescence microscopy, cells with noticeable elongation were deemed to be viable. Fatty acid analysis. Characteriation of cell envelopes of both V. vulnificus and E. coli was performed by examining the fatty acid composition of cells taken at To with that obtained from cells as they entered the state of nonculturability. One hundred milliliters of each microcosm was aseptically removed and centrifuged at 10,000 x g at 5 C for 10 min to harvest the cells. Cells were then suspended in 1.6 ml of ASW (5 C) for extraction of lipids by the method of Bligh and Dyer (3). Fatty acids were hydrolyed from the extracted phospholipids with BF3 and analyed by gas chromatography as described previously (19). Electron microscopy. V. vulnificus cells from seed cultures, To microcosm cultures, and the state of nonculturability were harvested, stained with ruthenium red to detect acidic polysaccharides, and examined by electron microscopy as described previously (26). Virulence assays. By using the method of Reed and Muench (23), a 50% lethal dose (LD50) was calculated for V. vulnificus cells before stress and determined by intraperitoneal injections into randomly bred albino mice 6 to 8 weeks old. Cells in the state of nonculturability (including dead cells) were injected intraperitoneally into groups of 10 mice. Because the virulence of V. vulnificus has been shown to be markedly increased by elevating the levels of iron in serum (29), nonculturable cells of V. vulnificus were also injected concurrently with 0.2 ml of filter-sterilied ferric ammonium citrate (80,ug of iron per injection) in phosphate-buffered saline. RESULTS Survival curves. Figures 1 and 2 are survival curves for V. vulnificus and E. coli, respectively (a minimum of two additional growth studies indicated similar trends). AODCs remained extremely constant for both cultures from To throughout the experiment. V. vulnificus cells decreased in sie during the study from an average of 1.7,um in length at To to an average of 0.8,um by day 17 of incubation. E. coli cells remained consistent in cell sie throughout incubation at 1.5,um in length. DVCs of both bacteria were lower than AODCs yet higher than CFUs on any of the media. The DVCs of V. vulnificus after 5 days of incubation were at their lowest point, approximately 2 orders of magnitude lower than the AODCs but up to 5 orders of magnitude higher than the CFUs. Cells treated cc ' :)~~~ INCUBATION TIME I Days) FIG. 1. V. vulnificus survival at 5 C. Symbols: El, AODC; 0, DVCs; *, CFU on heart infusion agar; (3, CFUs on marine agar MSWYE; O, CFUs on thiosulfate-citrate-bile salts-sucrose agar. with nalidixic acid were easily visible at approximately 8 to 10 times the length of the cells observed in AODCs. DVCs of E. coli fluctuated but also remained higher than the CFUs. DVCs were only 0.5 order of magnitude lower than AODCs and 0.5 to 1 order of magnitude higher than CFUs for at least the first 21 days of incubation. The V. vulnificus population entered the state of nonculturability on all three media by day 24 of incubation. Plate counts on nonselective HI agar remained the highest of the media tested until after day 16 of incubation. Reduced CFUs were most apparent on selective and differential thiosulfatecitrate-bile salts-sucrose agar, and nonculturability was exhibited first on this medium. E. coli exhibited decreasing 8- CD 0J 3 2 APPL. ENVIRON. MICROBIOL INCUBATION TIME (Days) FIG. 2. E. coli survival curve at 5 C. Symbols: E, AODC; 0, DVCs; 0, CFUs on heart infusion agar; O, CFUs on eosinmethylene blue agar.

3 VOL. 55, 1989 CHANGES IN VIABLE BUT NONCULTURABLE V. VULNIFICUS CJ 0 S -c, FIG. at 5 C. I- CJ I- -LI cc CIO Icis C1: INCUBATION TIME (Days) 3. Fatty acid composition of V. vulnificus during incubation CFUs throughout incubation but, unlike V. vulnificus, remained culturable through day 32, at which time experimentation was stopped. While CFUs on HI and eosin-methylene blue agar were initially equal, CFUs on eosin-methylene blue agar decreased at a slightly more rapid rate and remained at a level below that of HI throughout incubation. Fatty acids. Figures 3 and 4 show results of fatty acid analyses of V. vulnificus and E. coli cultures, respectively. V. vulnificus at To contained palmitic (C16) and palmitoleic (C16:1) acids as the most predominant fatty acid species. Whereas little change in fatty acid distribution had occurred following 13 days of incubation, the composition reflected considerable change at the point of nonculturability (day 24). The percentage of saturated fatty acids increased from 49 to 69.5%, and unsaturated species decreased from 45 to 17%. At this point, C16 and C16:1 were no longer predominant. The percentage of fatty acid species with chain lengths less than 16 increased when the cells became nonculturable, while long-chain fatty acid species (C19, C20, and C22:1) which were not detected before day 13 were also found. E. coli showed a more gradual change in fatty acid composition from To to day 32 of incubation. The predominant fatty acid species at To were palmitic (C16), palmitoleic (C16:1), and oleic (C18l:) acids, and these remained so throughout the experiment. In a trend similar to that observed with V. vulnificus, however, the percentage of saturated fatty acids increased from 34 to 44%, whereas the percentage of unsaturated species decreased from 52 to 39%. Also as with V. vulnificus, the percentage of fatty acid species with chain lengths less than C16 increased from 7% at To to 23% by the end of incubation. Electron microscopy. Electron micrographs of To cells of V. vulnificus revealed an external layer of polysaccharide stained by ruthenium red (Fig. 5A). A normal concentration of ribosomes was evident, and the cell membrane appeared normal. Micrographs of cells in the state of nonculturability C.J CL INCUBATION TIME (Days) FIG. 4. Fatty acid composition of E. coli during incubation at S C. (Fig. 5B) showed only rounded cells, with sies only one-half that of To cells. Ribosomes at this point were much less dense and nucleic material was less distinguishable, but the external polysaccharide layer was retained. Virulence. The LD50 of V. vulnificus before stress was determined to be 2.3 x 105 cells per ml. Testing the virulence of cultures in the state of nonculturability, however, poses a problem in that cell enumeration by CFUs is impossible. The total bacterial number by AODCs was 9.1 x 106 cells per ml, and injection of 0.5 ml per mouse, on the basis of this assay, would equal 4.5 x 106 cells. The DVCs at this point were 105 cells, and injection of 0.5 ml per mouse would equate to a count of 5.0 x 104 cells. Thus, depending on the basis of enumeration, from <10 to 4.5 x 106 cells per ml would be injected. The most accurate figure seems to be 5 x 104 (DVC data), since this number was known to represent viable cells. Among the group of 10 mice injected intraperitoneally with 0.5 ml (5 x 104) of nonculturable cells, no deaths were observed. However, because 5 x 104 is less than the LD50 of this bacterium for healthy cells (20), iron was also injected. This elevates the iron level in serum, which we have shown to reduce the LD50 by a factor of 106 (29). A group of 10 mice injected intraperitoneally with 0.5 ml of undiluted, nonculturable V. vulnificus cells (5 x 104) concurrently with 80,ug of ferric ammonium citrate in phosphate-buffered saline also resulted in 100% survival. DISCUSSION The most obvious result of the survival data reported here is that cells of V. vulnificus, a marine organism, were found to become nonculturable on solid media after incubation for 23 to 24 days in ASW at 5 C. In contrast, E. coli, a nonmarine organism, still formed colonies on solid media after 32 days. However, DVCs for V. vulnificus remained at a level comparable to that of E. coli at this point. These data

4 2840 LINDER AND OLIVER APPL. ENVIRON. MICROBIOL..'N kl w. _L Downloaded from 1 '~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 FIG. 5. Electron micrographs of V. vulnificus cells stained for external polysaccharide. (A) Cells at To in an ASW microcosm. (B) Cells at the point of nonculturability. suggest that cells of V. vulnificus maintain viability and are capable of responding to nutrients, although the cells are stressed by low temperature and apparently exist in a dormant (28) or starved (10) state. Because V. vulnificus is naturally distributed in marine waters year round, it must have a survival strategy for the cold winter months until suitable conditions return to its aquatic environment or, possibly, through its presence in an animal host. A study, similar to that reported here, of E. coli in natural seawater at 25 C showed that CFUs dropped from 107 to 104 cells per ml in approximately 26 days (11). The survival of a nonmarine organism for a long time under such conditions is surprising. However, the numbers of culturable cells had dropped below the DVCs, indicating a subpopulation of injured cells of the entire population. A study by Dawe and Penrose (5) concluded that the rate of injured coliform cells in seawater is very high but survival is high as well. They speculated that while seawater injures coliforms, it actually protects debilitated cells from death. The culturability of both bacterial species examined in this study remained the highest on HI agar, a nonselective and nondifferential medium. With both bacteria, the most selective media produced the lowest CFUs. Stressed or injured cells have been shown in several studies to be sensitive to many selective agents at concentrations that do not affect uninjured populations (22). McFeters et al. (9) showed that sublethal and reversible injury caused by chemical and physical factors of drinking water systems inhibits coliforms from forming colonies on media such as m-endo. It is of interest that, despite this knowledge, selective and differential media remain those primarily used for specific isolations and evaluation of water safety. A reduction in the sie of V. vulnificus was observed during incubation. This is a phenomenon associated with survival of psychrophilic Vibrio species and well documented by Novitsky and Morita (12-14). Postgate (21) theoried that true aquatic bacteria have evolved resistance to starvation by reducing their sie and minimiing their maintenance requirements. This seems consistent with the V. vulnificus survival pattern observed in this study. Fatty acid analysis. The bacterial envelope plays a crucial role in facilitating response to environmental change. The inner membrane maintains the fluidity essential for membrane functions, with the phospholipids forming the bilayer of this structure composed primarily of straight-chain fatty acid esters linked to glycerol phosphates. These fluctuate in the proportion of saturated-to-unsaturated fatty acid species and in chain length, depending on environmental conditions. A study of the fatty acid composition of marine bacteria reported by Oliver and Colwell (18) indicated that the fatty acids of such microorganisms were very similar to those of nonmarine bacteria. Of the vibrios examined in that study, the major fatty acid species were found to be C16, C16:1, and C18:1. Similar results were found in this study. At To, the fatty acids extracted from V. vulnificus and E. coli were the same as those from cells grown in HI broth at 25 C. As on September 27, 2018 by guest

5 VOL. 55, 1989 CHANGES IN VIABLE BUT NONCULTURABLE V. VULNIFICUS 2841 expected, C16:1 and C16 were the most predominant fatty acid species in V. vulnificus, and the percentages of saturated and unsaturated fatty acid species were similar (49.1 and 45.4%, respectively). Only 5.4% of the fatty acids had chain lengths of less than C16. From To to day 24, the percentage of saturated fatty acid species of V. vulnificus increased from 49.1 to 69.1%. Saturated fatty acid species in E. coli increased in concentration from To to day 32 as well. As the incubation temperature decreased, the cell would be expected to increase the percentage of unsaturated fatty acid species of the membrane phospholipids in order to maintain fluidity (18). However, decreasing chain length has the same effect as addition of double bonds in achieving membrane fluidity at lower temperatures (19). The latter mechanism appeared to operate in the present studies, as V. vulnificus cells from To to day 24 increased their percentage of short-chain fatty acid species from 5.4 to 28.9%. E. coli exhibited the same response, with an increase of acids less than C16 from 7.4% at To to 24.4% at day 32 of incubation. Electron microscopy. Studies by Simpson et al. (26), Amako et al. (2), and Yoshida et al. (31) have demonstrated variations in colony opacity within single strains of V. vulnificus, and this is now realied to be due to the presence or absence of an acidic polysaccharide capsule. Presence of the capsule has also been correlated with virulence (8, 26). It is not known which cell type predominates in the natural environment (16). It is possible that the lower-virulence translucent cells lacking the capsule may predominate in the environment, and this could help explain the relatively small number of infections reported. Such a possibility is not indicated by the electron micrographs of nonculturable cells examined in this study, however, which showed retention of the polysaccharide layer associated with opacity and virulence. Electron micrographs further showed that the membrane integrity of the cells was maintained. Micrographs did show a visible reduction in the number of ribosomes in cells in the state of nonculturability, as might be expected of cells in a dormant or survival strategy state. A study by Hood et al. (6) of V. cholerae under starvation conditions showed a similar reduction in ribosomal numbers. The nuclear region of the nonculturable cells in our study was less distinguishable, which also correlates with the findings by Hood et al. (6). Additionally, these investigators described the cell wall as convoluted and pulled form the cell membrane. They attributed this to the continuous sie reduction and decreasing volume of the cells. A similar phenomenon was observed in the present study, with nonculturable V. vulnificus cells being rounded and reduced in sie by at least one-half compared with those at To. Although the polysaccharide external layer was retained, loss of virulence of V. vulnificus cells in the state of nonculturability was observed in the mouse model used. Concurrent injection of iron with nonculturable cells, which we have shown to reduce the LD50 to 1.0 x 100 cells per ml (29), did not result in reversion to the virulent state. Thus, these data suggest that even though virulent (encapsulated) cells were present, the virulence of V. vulnificus is greatly decreased for cells in the state of nonculturability. This suggests that loss of virulence is due to factors other than loss of the antiphagocytic capsule, although this point has yet to be investigated. Studies on the virulence of other bacterial species present in the state of nonculturability have only recently been conducted. Viable but nonculturable cultures of E. coli and V. cholerae have been recovered from ligated ileal rabbit loops in which enterotoxigenicity was exhibited (4). In a study of Singh and McFeters (27), chlorine-stressed cells of Yersinia enterocolitica had virulence for mice similar to that of control cultures. However, copper-stressed cells showed reduced virulence. Results further indicated that chlorinestressed cells were not affected by gastric ph, whereas copper-stressed cells were sensitive. Pathogenicity in a host organism is a complex and dynamic process. Variation in virulence factors between species and in the invasive processes definitely complicate investigations of virulence. However, the results of the present study indicate that nonculturable cells of V. vulnificus are not virulent compared with unstressed cells of this species, at least in the mouse model used. In conclusion, it is apparent that V. vulnificus cells may exist in the marine environment under adverse conditions in a dormant, viable state not detectable by standard plating techniques. These cells undergo metabolic and ultrastructural changes which are presumed to be a survival strategy mechanism. A polysaccharide external layer and gross membrane integrity are maintained during entry into the state of nonculturability, although changes in membrane components, specifically, fatty acids, do occur. Although no reversion from the encapsulated to the nonencapsulated cell type was observed, loss of virulence by nonculturable V. vulnificus cells was demonstrated. Under the same incubation conditions, less dramatic decreases in E. coli culturability were observed. A similar pattern of fatty acid changes was detected, however. ACKNOWLEDGMENT We thank Sandra Zane for conducting the electron microscopy reported here. LITERATURE CITED 1. Allen-Austin, D., B. Austin, and R. R. 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