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Applied and Environmental Microbiology, July 1999, p. 3015-3020, Vol. 65, No. 7
U.S. Environmental Protection
Agency1 and National Research
Council,2 National Health and Environmental
Effects Research Laboratory and Gulf Ecology Division, Gulf
Breeze, Florida 32561
Received 22 January 1999/Accepted 21 April 1999
A tetrazolium dye reduction assay was used to study factors
governing the killing of bacteria by oyster hemocytes. In vitro tests
were performed on bacterial strains by using hemocytes from oysters
collected from the same location in winter and summer. Vibrio
parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria
monocytogenes mutants lacking catalase, superoxide dismutase,
hemolysin, and phospholipase activities were examined in winter and
summer. Vibrio vulnificus strains, opaque and translucent
(with and without capsules), were examined only in summer. Among
V. parahaemolyticus and L. monocytogenes,
significantly (P < 0.05) higher levels of killing by
hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal
activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the
wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly
(P < 0.05) higher susceptibility to killing by
hemocytes than did the wild-type opaque strain. No significant
differences (P > 0.05) in killing by hemocytes were
observed between opaque (encapsulated) and translucent
(nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in
winter and summer. Only one enzyme, esterase (C4), showed a seasonal
difference in activity (higher in winter than in summer). These results
suggest that differences existed between bacterial genera in their
ability to evade killing by oyster hemocytes, that a trait(s)
associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular
defense, and that bactericidal activity of hemocytes was greater in
summer than in winter.
Eastern oysters, Crassostrea
virginica, are found in coastal waters of the Atlantic and Gulf
coasts of the United States. Of economic importance, the value of the
oyster harvest from United States waters was over $100 million in 1995 (24). Ecologically important as well, oysters possess the
capacity of filtering up to 34 liters of water per h, thereby removing
particulates and pollutants (20). This filter feeding
behavior exposes these benthic invertebrates to a constant challenge by
invasive and pathogenic microbes. To cope with this challenge, oysters
possess humoral (9) and cellular (13, 14) defense
mechanisms. Hemocytes form the cellular defense arsenal against
infectious microbes by their ability to phagocytize, encapsulate, and
kill microbes (43). Microbial killing may result from the
combined action of humoral defense factors such as agglutinins and
lysosomal enzymes plus toxic reactive oxygen intermediates formed
during a respiratory burst associated with the hemocyte phagocytic
process (2, 13, 43). Oyster agglutinins are thought to
enhance phagocytosis (opsonization) by facilitating bacterial
aggregation or binding of bacteria to hemocytes (15, 36).
Lysosomal enzymes may act on the surface of microbes, contributing to
their recognition and/or destruction by hemocytes (2).
Frequently eaten raw, oysters are often implicated as the source of
human vibrio and other food-borne diseases (33). Some oyster-associated human pathogens, such as enteric bacteria, are transients whose presence is largely due to contamination of the water
by human fecal wastes. Others, like Vibrio vulnificus and Vibrio parahaemolyticus, are of nonfecal origin and occur
naturally in most shellfish harvesting areas of the United States
(38). The effect of environmental salinity and temperature
on the occurrence of vibrios in oysters has been studied in an effort
to predict their densities in shellfish (32, 42). These
vibrios, which persist and replicate in oysters (17, 41),
were selected for the current study to identify additional factors that
modulate their densities in oysters.
Although reports of the isolation of Listeria monocytogenes
from oysters are lacking, this facultative, gram-positive bacterial pathogen was isolated from blue crab (11) and shrimp
(23). Since microscopic studies have revealed a fascinating
relationship between L. monocytogenes and its host
(25) and since mutants with defects in their primary
virulence factors were available (34), this bacterium was
selected as an additional test microbe.
Characterizing bacterial interactions with cellular defenses may help
explain the persistence of bacteria in oyster tissues. Few studies have
examined the direct interaction of hemocytes and bacteria (18,
21). Comprehensive analyses of the colonization potential of
specific bacteria in oysters are also lacking. In this study,
bactericidal activity of oyster hemocytes was measured seasonally.
Different bacterial mutants were used to identify potential strategies
used by bacteria to reduce susceptibility to hemocyte killing. In
mammalian systems, these strategies, aimed at blocking one or more
steps in phagocytosis, include avoiding contact with phagocytes,
inhibition of engulfment, and survival inside of phagocytes
(12). Test bacteria that included parental strains and
corresponding mutants of V. parahaemolyticus, V. vulnificus, and L. monocytogenes, lacking various
putative virulence factors, were chosen with these avoidance strategies
in mind. To quantify the bactericidal activity of oyster hemocytes, a
recently developed in vitro colorimetric method was used
(44).
Oyster harvesting and handling.
Oysters (C. virginica) were collected in winter (30 January 1998) and again in
summer (9 June 1998) from Bayou Texar, an inlet of Escambia Bay, Fla.
Temperatures and ambient salinities at the collection site were 14°C
and 5 Collecting hemolymph.
Hemolymph was withdrawn from the sinus
of the adductor muscle through a notch in the oyster shell by using a
syringe fitted with a 23-gauge needle. To reduce cell clumping,
hemolymph samples were placed on ice. For each trial, an equal volume
of hemolymph collected from each of 10 oysters was pooled to yield
sufficient numbers of hemocytes for multiple simultaneous tests. The
killing index (KI) of each bacterial species was assessed by using
three separate pools of hemolymph.
Bacteria and culture conditions.
Strains used are listed in
Table 1. The V. parahaemolyticus strains included the type strain (American Type
Culture Collection), wild-type opaque (OP) and translucent (Tra)
strains, a transposon mutant unable to switch to the OP parent (Fix
Tra), and three motility mutants. One of the motility mutants was
defective in swarming (Laf
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Factors Influencing In Vitro Killing of Bacteria by
Hemocytes of the Eastern Oyster (Crassostrea
virginica)
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
in January and 30°C and 16 in June, respectively. Oysters
were immediately transported to the U.S. Environmental Protection
Agency's Gulf Ecology Division laboratory and were held in a
1,099-liter holding tank equipped with a flow-through unfiltered
seawater delivery system for 2 to 14 days prior to experimentation. The
flow rate of seawater in this holding tank was approximately 90 liters/h.
), and two were defective in
both swimming and swarming (Fla Laf and
ParaFla Laf). Three different isogenic
pairs of V. vulnificus strains were also tested. One member
of each pair produced opaque colonies (O) and possessed capsules, and
the other member produced translucent colonies (T) and lacked capsules.
The L. monocytogenes strains included a parental wild-type
(Wild) strain and mutants defective in the hemolysin, listeriolysin O,
phospholipase C, superoxide dismutase activity, and catalase activity.
TABLE 1.
Bacterial strains used in this study
, sterilized by
filtration (pore size, 0.22 µm), and maintained at 25°C.
Bacteria were grown to late-logarithmic or early-stationary phase by
incubation for 18 h (25°C) with shaking (200 rpm) in 125-ml
Erlenmeyer flasks containing 10 ml of culture broth. Bacteria were
harvested by centrifugation (12,000 × g; 10 min) and
suspended in an equal volume of FSW. Appropriate dilutions were made in FSW for use in the killing assay. Numbers of culturable bacteria were
determined by spreading 0.1-ml aliquots of diluted bacterial suspension
in triplicate on NBS or TSB agar plates and counting colonies arising
after 18 to 36 h at 25°C.
Killing assay. The percentage of bacteria killed (KI) was determined in flat-bottomed 96-well microtiter plates as described by Volety et al. (44), except streptomycin was not used.
Essential steps in the procedure of Volety et al. (44) were performed as follows. First, bacteria suspended in FSW were incubated in the presence and absence of plasma-free hemocytes at a bacteria/hemocyte ratio of approximately 10:1. To prepare plasma-free hemocytes, aliquots of hemolymph containing 105 hemocytes were placed in two sets of wells containing 100 µl of FSW. Microtiter plates containing hemolymph were centrifuged (160 × g; 10 min) to promote hemocyte adhesion. The plasma-FSW supernatant was removed with a multichannel pipette. The first two sets of wells contained the hemocytes. One of these sets of wells received FSW, and the other set of wells received FSW plus bacteria. The last two sets of wells did not contain hemocytes. One of these sets of wells contained FSW only (blank control), and the other set of wells received FSW plus bacteria. Microtiter plates were then centrifuged (160 × g; 10 min) to encourage bacterial contact with hemocyte monolayers and were incubated in a moist chamber (3 h at 17°C) to allow hemocyte killing. In the second step, a recovery-growout period for surviving bacteria was started by adding the appropriate culture broth and incubating for 2 h at 25°C for the vibrios and 2.5 h at 37°C for L. monocytogenes strains. In the final step, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and phenylmethasulfazone (MTS-PMS) reagents (20 µl) (Promega Corporation, Madison, Wis., and Sigma Chemical Company, St. Louis, Mo., respectively) were added, and incubation was continued for an additional 30 min. Numbers of viable bacteria were determined colorimetrically by measurement of formazan, the soluble reduction product of MTS-PMS, at 490 nm with an enzyme-linked immunosorbent assay microplate reader (model 311-SX; Bio-Tek Instruments, Inc.). Eight replicate wells were used for each treatment. Absorbance (A) values were corrected by subtracting the background absorbances of the FSW only. The percentage of bacteria killed (KI) was calculated from the A values of the reduced MTS-PMS as follows:
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Enzyme assays.
Activities of 19 hydrolytic enzymes were
measured in hemolymph by using the API ZYM system (BioMerieux Vitek,
Inc., Hazelwood, Mo.). In six replicate determinations conducted in
both winter and summer sampling periods, 65 µl of freshly collected,
pooled hemolymph (3 × 106 cells · ml1) was
added to each microtube in the API ZYM test strip. Strips were
incubated for 4 h at 37°C and developed, and their color reactions were recorded according to the manufacturer's instructions.
Statistical analyses.
Two-factor analysis of variance
(ANOVA) was performed separately on V. parahaemolyticus and
L. monocytogenes KI data from all sampling periods to
elucidate differences in mean KI due to the main effects, strain and
season. No significant interaction between bacterial strain and season
was found for either V. parahaemolyticus or L. monocytogenes, so one-way ANOVA was employed for each species at
each sampling period to test for differences in KI due to the effect of
strain. Data collected in summer from V. vulnificus were
also analyzed by one-way ANOVA to test for differences in KI due to
strain. Where significant differences were found, Tukey's multiple
comparison test was employed to resolve significant differences between
means. A Student's t test was used to assess differences in
the profiles of the measured hemocytic enzymes in summer versus winter.
Results were deemed significant at P 
0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The potential of oyster hemocytes to kill V. parahaemolyticus was significantly greater in summer than winter. In summer, the mean KI ± standard deviation for all V. parahaemolyticus strains was 49 ± 16 compared to 19 ± 16 in winter.
Mean ± standard error (SE) KIs of hemocytes from oysters collected in summer and winter and incubated with seven strains of V. parahaemolyticus varied significantly (Fig. 1). Translucent strains of V. parahaemolyticus, the Tra and Fix Tra strains, were more susceptible to killing by oyster hemocytes than opaque strains. In both summer and winter, these two strains exhibited the highest KIs of all V. parahaemolyticus strains tested. In winter, both the Tra and Fix Tra strains yielded significantly higher KIs than the wild-type opaque strain, BB220P (Wild). In summer, the KI obtained with strain BB22TR (Tra) was significantly higher than any of the opaque V. parahaemolyticus strains tested. The KI ± SE of the fixed translucent strain LM4462 (Fix Tra) (58 ± 11) was again higher than that of the wild-type opaque strain (46 ± 10), but the difference was not statistically significant. Contrary to the responses of V. parahaemolyticus strains, the translucent phenotype (corresponding to lack of a capsule) did not appear to render strains of V. vulnificus more susceptible to killing by oyster hemocytes. No significant differences in KIs of any of the three wild-type V. vulnificus strains and their corresponding acapsular derivatives were obtained (Fig. 2).
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Defects in motility did not appear to affect the ability of V. parahaemolyticus to avoid killing by oyster hemocytes. In winter, all strains, including those defective in motility, yielded significantly higher KIs than the wild-type opaque strain (OP). This trend was not repeated in summer, when none of the strains defective in motility yielded KIs significantly higher than that of the OP strain (Fig. 1).
Colonies produced on NBS after 24 h at 25°C by translucent and opaque strains of V. vulnificus were more similar to each other than colonies produced by translucent and opaque strains of V. parahaemolyticus. Opaque strains of both V. parahaemolyticus and V. vulnificus produced circular colonies between 0.5 and 1 mm in diameter that had entire edges and were convex in elevation. In contrast, the translucent colonies of V. parahaemolyticus strains were much larger (3 to 6 mm in diameter) and irregular in shape, possessing lobate edges and raised elevations. No differences in colonial morphologies among L. monocytogenes strains were observed.
As with V. parahaemolyticus, the mean KI ± SE of all L. monocytogenes strains was higher in summer (23 ± 22) than in winter (18 ± 14); this difference, however, was not significant. L. monocytogenes was, on average, more resistant to killing by oyster hemocytes than either V. parahaemolyticus or V. vulnificus (Fig. 1, 2, and 3). In summer, the mean KIs ± SEs derived from all V. parahaemolyticus and V. vulnificus strains were 49.0 ± 16 and 44.3 ± 9.3, respectively. Both KI averages were significantly higher than the mean KI derived from L. monocytogenes strains in summer, 23.4 ± 6.5. In addition, L. monocytogenes strains lacking the potential virulence factors catalase (strain 1370), superoxide dismutase (strain DHL1), listeriolysin O (strain DP-L2161), and two secreted phospholipases C (strain DP-L1936) were not significantly more susceptible to killing by oyster hemocytes than their parental wild-type strains in either the winter or summer trials. In the summer trial, however, there was a nonsignificant trend that showed all L. monocytogenes strains lacking a putative virulence factor more susceptible to killing than their parental wild-type strain (Fig. 3).
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Using the API ZYM strips, activities of individual hydrolytic enzymes
were measured in 65-µl aliquots of each pooled hemolymph sample. As
shown in Table 2, only 6 of 19 enzymes
(C4 esterase, C8 esterase lipase, leucine arylamidase, phosphatase
acid, naphthol-AS-BI-phosphohydrolase, and 
-galactosidase) assayed
in hemolymph showed activities that averaged 
5 nmol of cleaved
substrate. Of these six, C4 esterase was the only enzyme that showed a
significant seasonal difference in activity (higher in winter than
summer). In both summer and winter, the aminopeptidase leucine
arylamidase displayed the highest activity (22 nmol of cleaved
substrate from 65 µl of hemolymph) (Table 2).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Research on cellular immune response in oysters has relied mainly on indirect measurements (e.g., chemiluminescence, agglutination, cell migration, and association of bacteria with hemocytes) to quantify different phases of the phagocytic process (2). Although these approaches have yielded a great deal of information regarding oyster immune function, direct methods, like dye reduction (used here) or plate count, not only yield comparable results (44) but also may better represent the cellular defense capability by directly measuring bacterial death.
Using the plate count to measure phagocytic activity and bacterial degradation by oyster hemocytes, Harris-Young et al. (22) found less killing of an opaque than a translucent morphotype of V. vulnificus biotype 1. In agreement with the results of Harris-Young et al. (22), we observed higher, although not significantly higher, KIs with translucent than with opaque V. vulnificus variants in two of the three isogenic pairs examined. We obtained contrary results with the only biotype 2 pair, 938 (O) and 938 (T), tested (Fig. 2). Strains belonging to this biotype are eel pathogens (1) and, unlike biotype 1 strains, the presence of a capsule is not required for the development of vibriosis in its host (4). It is possible that biotype 1 translucent variants did not completely lack capsules but rather possessed incomplete capsular materials (46), rendering them slightly more resistant to killing than a translucent variant completely lacking capsular material. Also in agreement with Harris-Young et al. (22) was our observation of higher KIs with Tra and Fix Tra V. parahaemolyticus strains than with the wild-type opaque variant (Fig. 1).
Colonial morphologies between the wild-type opaque (OP) and translucent variants (Tra and Fix Tra strains) of V. parahaemolyticus were more strikingly different than those between the opaque and translucent variants of V. vulnificus. Although the difference between the V. vulnificus forms was shown to be due to encapsulation (46), this has not been documented for V. parahaemolyticus. Moreover, OP and Tra phenotypes of V. parahaemolyticus involve multiple traits and may not be as simple as the presence or absence of capsules (28). Electron micrographs revealed a dense ruthenium red staining layer on the surface of plate-grown OP cells and a thin layer on plate-grown Tra cells (27).
Motility may play a role in the ability of some bacteria to evade killing by oyster hemocytes. Cells of V. parahaemolyticus possess two types of locomotion, swimming and swarming (30). Swimming cells, adapted for locomotion in liquid medium, are propelled by a single flagellum (Fla) located at one pole. When a swimming cell contacts and adheres to a surface, it becomes a swarmer cell through a series of changes that includes the production of numerous lateral flagella (Laf). This differentiation is an adaption for colonization of surfaces. From our results, it appears that motility may not be important in the cell's ability to evade killing by oyster hemocytes. However, it must be noted that in our tests, V. parahaemolyticus strains were cultured in broth where the production of lateral flagella (swarming motility) was repressed.
L. monocytogenes is a human, intracellular pathogen (35) known to be associated with shellfish (23). The KIs for all L. monocytogenes variants in both winter and summer were low, with a high degree of variability among hemocyte pools. This suggests that this pathogen was not easily killed by oyster hemocytes, and no single virulence factor tested rendered the cells more susceptible to hemocyte killing.
Both L. monocytogenes and V. parahaemolyticus KIs were higher in summer than in winter. Reasons for this are not entirely clear. Phagocytic killing is partially mediated through the action of digestive enzymes (9). Various hydrolase activities were identified in hemolymph of C. virginica (8); no seasonal differences in enzymatic activity were studied. Chu and La Peyre (10) found higher levels of hemolymph lysozyme in winter than summer months in Chesapeake Bay oyster hemolymph, but Fisher et al. (16) found the opposite for oysters from Apalachicola Bay, Fla. It is likely that one or more of the four phases of phagocytosis (attraction, attachment, internalization, and intracellular degradation) is more active because of a higher metabolic rate during the warmer summer months.
This study utilized several bacterial mutants defective in a variety of potential virulence factors and their corresponding wild-type parental strains to determine whether microbial factors were protective against hemocyte killing. It appeared the opaque phenotype of V. parahaemolyticus offered some protection against killing by oyster hemocytes. Although the absence of multiple traits characterizing the translucent phenotype of V. parahaemolyticus rendered this microbe more susceptible to killing by oyster hemocytes, the absence of single enzymatic virulence factors known to enable L. monocytogenes to become an intracellular human pathogen (34) did not increase oyster hemocyte killing of this bacterium. Perhaps this bacterium, like Staphylococcus aureus, is resistant to the action of oyster lysosomal hydrolases (37) and not reliant on other virulence factors to protect from oyster hemocyte killing.
It is not fully understood why certain bacteria were more susceptible to killing by oyster hemocytes. Cheng (7) hypothesized that surfaces of resistant cells lack substrates susceptible to humoral response molecules, including lysosomal enzymes, and that resistant cells lack a triggering mechanism for the release of lysosomal enzymes from hemocytes. Cheng (6) also postulated that substances that inactivate hydrolases are elaborated by resistant cells. Any combination of these and other factors may govern the capacity of a bacterium to evade killing by oyster hemocytes. Even without knowing the specific mechanisms, the results from this study clearly demonstrate a differential ability of oyster hemocytes to kill various bacteria.
Although the potential ramifications of these results for human health and ecological conditions remain to be explored, the significance of this work is multifaceted. Consumption of raw oysters has been implicated in numerous food poisoning outbreaks. Thus, their microbial flora is of great concern to public health. The ability of oysters to eliminate pathogenic bacteria from their systems may partially be determined by the ability of hemocytes to recognize, bind, and phagocytose these microbes. Virulence factors may also play a role in the ability of bacteria to survive molluscan cellular defense mechanisms. Thus, an understanding of the interactions between pathogenic bacteria and oyster hemocytes is important in elucidating mechanisms responsible for bacterial persistence in oyster tissues. Second, the ability of the oyster to defend against invasive bacteria may be an inherent requirement of a healthy oyster population, and a healthy oyster population is important to most estuarine ecosystems.
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ACKNOWLEDGMENTS |
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We thank A. DePaola, S. E. Martin, L. L. McCarter, D. A. Portnoy, and A. B. Zuppardo for providing bacterial strains, and J. T. Winstead for collecting oysters. We appreciate very helpful comments from L. L. McCarter.
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FOOTNOTES |
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* Corresponding author. Mailing address: U.S. EPA, 1 Sabine Island Dr., Gulf Breeze, FL 32561-5299. Phone: (850) 934-9342. Fax: (850) 934-2402. E-mail: genthner.fred{at}epamail.epa.gov.
Contribution no. 1063 of the U.S. EPA's National Health and
Environmental Effects Research Laboratory and Gulf Ecology Division, Gulf Breeze, Fla.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, July 1999, p. 3015-3020, Vol. 65, No. 7
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