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Applied and Environmental Microbiology, July 2001, p. 2895-2902, Vol. 67, No. 7
Department of Food Science and Technology,
Mississippi Agricultural and Forestry Experiment Station, Mississippi
State University, Mississippi State, Mississippi
397621 and Gulf Coast Seafood
Laboratory, U.S. Food and Drug Administration, Dauphin Island,
Alabama 365282
Received 8 January 2001/Accepted 15 April 2001
Viable counts of three strains of Vibrio vulnificus and
its phage were determined during exposure to a mechanical
gastrointestinal model with or without antacid for 9 h at 37°C.
V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable
V. vulnificus cells delivered from the gastric compartment
during the first 30 min of exposure reached 106 to
108 CFU/ml in the intestinal compartment after 9 h (pH
7.0). Phages were eliminated within 45 min in the gastric compartment
(pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was
observed in the intestinal compartment after 9 h (pH 7.0).
When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of
exposure (pH decline from 7.7 to 6.0), while counts in the intestinal
compartment (pH 7.5) reached 107 to 109 CFU/ml.
Phage numbers decreased 1 log after 2 h in the gastric compartment
(pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in
the intestinal compartment (pH 7.6) after 9 h. Presence of antacid
in the gastric compartment of the model greatly increased the ability
of both V. vulnificus and its phage to survive simulated
gastrointestinal transit and may be a factor involved with
oyster-associated illness.
Vibrio vulnificus is a
virulent pathogen (3, 42). It is most commonly found in
estuarine and marine waters of the U.S. Gulf Coast and other temperate
regions (7, 33). Of all food-borne infectious diseases in
the United States, V. vulnificus has the highest (0.39) case
fatality rate (31). Disease can follow the ingestion of
raw Gulf Coast oysters and may result in primary septicemia or
gastroenteritis in individuals who have underlying chronic disease,
including liver disease (37). Wound infections are
associated with exposure of wounds to seawater or shellfish.
Most-probable numbers of V. vulnificus organisms in Gulf
Coast oysters range from 102 to 104/g from
April through October, while most-probable numbers of <10/g occur
during the winter (33). Bacteriophages lytic to Vibrio species also are prevalent throughout the Gulf of
Mexico (26); they are found in estuarine water samples
(35) and in a variety of oyster tissues (8).
V. vulnificus phage numbers range from 104 to
105 PFU/g in Gulf Coast oysters throughout the year
(9).
Microorganisms causing human gastroenteritis must survive the gastric
barrier, resist bile in the small intestine, and colonize the
intestinal lumen. The gastric barrier is lethal to most ingested bacteria (10, 19, 20), including V. vulnificus
(27, 28). Vibrio cholerae is an acid-sensitive
pathogen compared to other enteric pathogens such as Salmonella
enterica serovar Typhi, S. enterica serovar
Typhimurium, Shigella flexneri, and Escherichia coli O157:H7 (44). V. cholerae does not
survive in either pH 4.0 or 5.0 broth after 2 h of exposure.
Likewise, V. vulnificus also is acid sensitive in pH 4.0 broth (27). Simulated gastric fluid (SGF) is more
inhibitory to V. vulnificus than acidified broth
(28).
Antacids neutralize stomach acidity and are widely used for relief of
gastric ulcers, duodenal ulcers, heartburn, and acid indigestion
(13, 14, 16, 29, 38, 45). Antacids neutralize acid in the
human stomach for a short duration, while acid blockers reduce acid
secretion for a prolonged duration (14). Reduction of
gastric acidity results in a substantial increase in survival rates of
common food-borne pathogens (36). Reduced gastric acidity by medications may favor increased survival and subsequent growth of
V. vulnificus, which could increase the risk of infection. In human volunteers, the dose of V. cholerae required to
induce diarrhea was lowered from 108 to 104
organisms by neutralizing stomach acidity with the antacid
NaHCO3 (4). Despite these reports, an
epidemiological study of V. vulnificus reported that use of
antacids or cimetidine, an acid blocker, were not significant risk
factors for primary sepsis (42).
The ability to tolerate low gastric pH and to resist intestinal bile is
necessary for V. vulnificus to cause food-borne infections in humans. Previous reports also discussed the effectiveness of phage
therapy applications to treat enteropathogenic E. coli
diarrhea in calves (39) and S. enterica serovar
Typhimurium in chickens (2). Thus, phages may be an
important factor affecting Vibrio populations in estuarine
environments and in the gastrointestinal tract of humans. The
objectives of the present study were to assess survival of V. vulnificus and its phage in oysters during in vitro transit in a
gastrointestinal (GI) model (12, 30, 32, 34) and to
determine the influence of antacid on survival of V. vulnificus and its phage in the GI model.
Bacterial cultures and phages.
V. vulnificus
strains A-9 (environmental isolate), MO6-24 (clinical isolate), and 304 (oyster isolate) were maintained at room temperature on
T1N1 agar slants containing 10 g of
tryptone (Difco Laboratories, Detroit, Mich.), 10 g of NaCl,
20 g of Bacto Agar (Difco), and 1.0 liter of distilled water.
Cultures were transferred biweekly to maintain viability. Strains were
streaked on tryptic soy agar (Difco) plates containing 2% NaCl (TSA-2) and incubated overnight at 37°C. Several isolated colonies of each
strain were picked using sterile loops and suspended in
phosphate-buffered saline (43). The bacterial suspension
was adjusted to a turbidity of 3.5 to 3.8 nephelometric turbidity units
(Hach Turbidimeter; Hach Co., Loveland, Colo.) to achieve a
concentration of 107 CFU/ml. V. vulnificus phage
strains 154A-9, 153A-7, and 110A-7 (all podophages of Bradley group C-3
morphology from Gulf Coast oysters) were maintained and prepared for
inoculation as described previously (27, 28).
Preparation of GI model.
The following equipment was used
for the GI model (Fig. 1): a 250-ml Pyrex
beaker gastric compartment (GC) with a pH meter probe (Orion 290A;
Orion Research Inc., Boston, Mass.), a 250-ml Pyrex beaker small
intestine compartment (IC) with a pH meter probe (Fisher Accumet 1001;
Fisher Scientific, Pittsburgh, Pa.), a peristaltic pump (Wheaton
Unispense; Wheaton Instruments, Millville, N.J.), a cassette pump
(Manostat, New York, N.Y.), two magnetic stirrers (Fisher), and a
37°C Isotemp Immersion Circulator model 730 water bath (Fisher). The
two beakers with magnetic stirring bars were placed in the water bath
and were connected by the peristaltic pump for gastric emptying from
the GC to the IC. Throughout the experiment, magnetic stirrers mixed
samples in each compartment at low speed. SGF, simulated intestinal
fluid (SIF), and bile solution were introduced into the GC or the IC by
the cassette pump. The pH of the GC or the IC was monitored and
adjusted by adding IN HCl manually into the GC and 0.1 or 0.3 M
NaHCO3 via the cassette pump into the IC.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2895-2902.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Antacid Increases Survival of Vibrio
vulnificus and Vibrio vulnificus Phage in a
Gastrointestinal Model
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
View larger version (49K):
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FIG. 1.
Diagram of a simulated dynamic-GI model. Components: 1, GC; 2, IC; 3, cassette pump; 4, peristaltic pump; 5, pH meters; 6, circulator-heater; 7, stirrers; 8, water bath. Fluids: A, SGF; B, SIF;
C, NaHCO3; D, 2% bile; E, 4% bile.
20°C until used. Autoclaved (121°C for 15 min) oysters (approximately 70 g) were mixed (1:1, wt/wt) with sterile electrolyte solution (32)
containing 6.2 g of NaCl, 2.2 g of KCl, 0.22 g of
CaCl2, 1.2 g of NaHCO3, and 1.0 liter of
distilled water in a Waring blender (Dynamics Corporation Division, New
Hartford, Conn.) for 2 min at high speed to simulate mouth chewing with
saliva. This oyster homogenate was passed through a sterile filtering
funnel to remove large particles to facilitate pumping from the GC to
the IC. One hundred milliliters of oyster homogenate and 1 ml of
bacterial or phage suspension were added into the GC to achieve a final
concentration of approximately 105 CFU/ml or PFU/ml,
respectively. All pumps were started simultaneously immediately after inoculation.
SGF contained 0.1 g of pepsin (Sigma Chemical Co., St. Louis,
Mo.), 3.5 g of mucin (Sigma), 8.5 g of NaCl, and 1.0 liter of distilled water adjusted to pH 2.0 with 1 N HCl (23). SGF
was pumped into the GC via the cassette pump at a flow rate of 0.33 ml/min. A predetermined constant volume (approximately 1.2 ml) of the
chyme (mixture of food with SGF) was delivered from the GC to the IC
every minute for 2 h via the peristaltic pump. The emptied chyme
was collected continuously in the IC. Gastric in vivo pH values
(5, 32) were obtained by manually adjusting the pH of the
GC every 10 min using sterile 1 N HCl. GC pH values were measured every
10 min for 2 h. The half-emptying time of this GI model was 61 min, which was similar to normal gastric half-emptying time based on
data from Ghoos et al. (18). SIF contained 0.1 g of
trypsin (Sigma), 3.5 g of pancreatin (Sigma), and 1.0 liter of
distilled water (23) and was pumped into the IC at a flow
rate of 0.33 ml/min. Two different concentrations of NaHCO3
were used to maintain a constant pH value in the IC (21).
For the first 50 to 60 min, 0.1 M NaHCO3 was pumped into the IC followed by 0.3 M NaHCO3 for the rest of the
experiment, both at a flow rate of 0.33 ml/min. IC pH values were
measured for 3 of the 9 h of exposure because pH remained
unchanged after 3 h (results not shown). Seven milliliters of 4%
bile solution (ox gall; Sigma) was added into IC before the experiment
began. A 2 or 4% bile solution also was pumped into the IC at a flow rate of 0.5 ml/min (28, 29, 30). A 4% bile solution was pumped during the first 30 min, and then 2% bile solution was pumped
until the end of the experiment. Gastrointestinal fluids were pumped
into both GC and IC until the contents of GC were emptied.
Influence of antacid in GI model. Aluminum hydroxide hydrate (692 mg) (32 to 35% water of hydration) (Sigma) and 400 mg of magnesium hydroxide (Fisher) were used as the antacid active ingredients found in 10 ml (2 teaspoonfuls) of Maalox (Ciba Self-Medication, Inc., Woodbridge, N.I.). They were added into the GC when bacterium or phage inocula and oyster homogenates were introduced. Gastric pH for antacid treatment was controlled by addition of 1 N HCl to GC to approximate normal gastric production. Sterile electrolyte solution was added in the IC to control intestinal pH for antacid treatment.
Microbiological analysis. One-milliliter samples were taken from the GC at 0, 10, 20, and 30 min for V. vulnificus or at 0, 15, 30, and 45 min for V. vulnificus phage. In GC containing antacid, samples were taken every 30 min for 2 h for both bacterium and phage. Samples were taken from IC every 1.5 h for 9 h for V. vulnificus and its phage. Samples for V. vulnificus enumeration were serially diluted in tryptic soy broth (Difco) containing 2% NaCl, and 0.1-ml aliquots were plated onto TSA-2. Samples for phage enumeration were serially diluted in sterile seawater and plated onto CPM agar containing Casamino Acids (5 g/liter; Difco), peptone (5 g/liter; Difco), 1.5% Bacto Agar (Difco), and 1 liter of seawater (Sigma) by using the soft-agar overlay technique containing V. vulnificus strain MO6-24 as host the culture (27, 28). All plates were incubated anaerobically (BBL Gas Pak Plus; Becton Dickinson, Cockeysville, Md.) at 37°C for 18 h. Viable cell or phage counts were divided by the dilution factors for GC or IC to take into account the volumes of secretions and emptied or delivered volumes at each sampling time interval. Dilution factors (all amounts in milliliters) were calculated as follows: dilution factor for GC = remaining GC contents/(remaining GC contents + HCl + SGF); dilution factor for IC = cumulative IC contents/(cumulative IC contents + delivered contents + bile + SIF + NaHCO3). Microbial count data obtained from duplicate samples per analysis time from three replicate experiments were analyzed by ANOVA and means were separated by least-significant difference (SAS user's guide, 5th ed., SAS Institute Inc., Cary, N.C.)
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Survival of V. vulnificus.
Within 20 min, a 4-log
reduction in V. vulnificus counts was observed in the normal
GC at 37°C. No viable V. vulnificus cells (counts reduced
below the limit of detection of 10 CFU/ml) were recovered after 30 min
in the normal GC (Fig. 2A). No difference (P > 0.05) was detected among strains in the normal
GC. Delivered cells were 4 to 4.5 log10 CFU/ml in the IC
from the first 30 min of gastric emptying (Fig. 2B). Cell counts of
strains A-9 and 304 increased continuously after 1.5 h and reached
8 log10 CFU/ml after 9 h at 37°C in the IC. In
contrast, counts of V. vulnificus MO6-24 in the IC dropped
1.5 log after 3 h and then reached 6 log10 CFU/ml
after 9 h (Fig. 2B). Strains A-9 and 304 in the IC grew faster
(P < 0.05) than strain MO6-24. Gastric pH declined from 5.0 to 1.7 during 2 h of gastric emptying and intestinal pH
ranged from 7.3 to 6.8 for 3 h, with no significant difference (P > 0.05) observed among trials using the three
bacterial strains.
|
, V. vulnificus A-9;
, V. vulnificus MO6-24; 
, V. vulnificus
304; 
, pH). Error bars, standard deviations.
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Survival of V. vulnificus phage.
Phage counts
decreased only 1 log during the first 30 min but were not recovered
from the GC after 45 min at 37°C after gastric pH declined below 3.5 (Fig. 4A). Phage numbers delivered from the GC to the IC during the first 30 min of gastric emptying were 5 log10 PFU/ml, which was similar to the initial GC inoculum
level (Fig. 4). Less than a 1.5-log phage count reduction was observed in the IC after 9 h at 37°C (Fig. 4B). For the entire GI model, phage numbers decreased less than 2 logs from the initial number. Phage
counts were not significantly different (P > 0.05)
among strains in both the GC and the IC. In trials with phages, gastric pH declined from 5.1 to 1.7 (Fig. 4A) and intestinal pH was maintained between 7.4 to 6.7 (results not shown), with no differences
(P > 0.05) among strains tested.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Behavior of V. vulnificus. Several studies have reported that the bactericidal effect of gastric juice is reduced by the presence of food (5, 10, 36). Food particles protect bacteria in the stomach by enrobing cells and by buffering acid. In the present study with oysters, a 3-log reduction in V. vulnificus counts was observed between 10 and 20 min when pH of GC ranged between 4.5 and 4.1 (Fig. 2A), which was in agreement with previous observations with SGF without oysters (28). Thus, autoclaved oyster homogenate provided no apparent protection to the gastric barrier. Others have reported that V. vulnificus was more resistant to heat treatment in proteinaceous materials such as intermittently sterilized oyster and fish homogenates than in buffer (1).
V. vulnificus was not detected in the GC at 30 min when the pH value was below 3.5. A rapid gastric emptying rate may be an important factor for survival of V. vulnificus and risk of infection in the GI tract. The pylorus, the circular muscle of the end of the stomach, normally remains almost, but not completely, closed for partial discontinuity between the stomach and duodenum. The closing force is weak enough that water and other fluids empty from the stomach with ease (22). Davenport (6) reported that the rate of emptying for liquid meals is greatest when the volume is greatest so that emptying is fastest at the beginning of digestion of a meal. Consequently, the greatest bulk of gastric contents is delivered to the duodenum before much gastric digestion or acidification has occurred. The volume of liquid meals emptied into the duodenum is greatest during the first 10 to 20 min, with peak emptying rates occurring during the first hour after ingestion (30). Because oysters are semisolid and are frequently consumed as an appetizer, they would be expected to empty from the stomach easily and rapidly. In the present study, more than 80% of the inoculated V. vulnificus reached the IC as culturable cells during the first 30 min of gastric emptying when the gastric pH ranged from 5.0 to 3.5 (Fig. 2). While V. vulnificus reductions in the GC could be partially attributable to gastric emptying, acid inactivation likely accounted for at least a 4-log reduction (27, 28). Present results showed counts of V. vulnificus MO6-24, a clinical isolate, were 2 logs lower than those of the environmental strains 304 and A-9 after 9 h in the IC. Counts in the IC did not increase until low-pH gastric contents were emptied, which took 2 h (Fig. 2). The environmental strain A-9 caused diarrhea in rabbits without septicemia at 109 CFU/loop, while the clinical strain MO6-24 caused death at the same dose (41). Since the present model is based on an in vitro study, virulence factors such as acid tolerance or adaptation, intestinal colonization, and invasion were not evaluated. After passage through mice, the 50% lethal doses for oyster and environmental V. vulnificus isolates were reduced 100- and 1,000-fold, suggesting that strains passed through the GI tract may increase in potential pathogenicity (25). In the present study, the clinical isolate was less acid tolerant and grew less than the environmental isolate or the oyster isolate in the model. While the differences in acid tolerance and growth were not significant (P < 0.05), these observations suggest that the clinical strain has no selective advantage from human carriage or may have lost it during lengthy laboratory storage and numerous subcultures. The role of survival and/or growth during gastrointestinal transit in human infection should not be based on the performance of a single strain. Present results on acid tolerance may be less important in human infection than other virulence traits. The pH values obtained in the present GI model compare well to in vivo results reported elsewhere (21, 32). The overall pH means of GC and IC in the present study were 2.7 and 6.8, which compare with the overall median fasting gastric pH of 1.7 in young and healthy men and women (11). During a meal, gastric pH increases to a median value of 5.0 (11). The overall fasting duodenal pH is 6.1, which increases to 6.3 during a meal (32). In the present study, numbers of V. vulnificus organisms delivered from the GC increased continuously for 9 h in the IC (Fig. 2), presumably because bile does not greatly affect survival and growth of V. vulnificus (28). This finding suggests that viable V. vulnificus cells can be delivered into the small intestine if gastric emptying occurs soon after ingestion and that they will multiply rapidly in the intestine. Because all strains responded similarly in the gastric model, the risk of infection would appear to be proportional to dosage. Gastric pH increased to 7.7 instantly after addition of antacid and oyster homogenate and remained above 5.5 for 2 h in the present study. The duration of the antacid effect may last longer than 2 h (15). V. vulnificus numbers in the IC containing antacid peaked sooner (4.5 to 6 h) than in the IC without antacid (9 h), because higher numbers of V. vulnificus organisms were delivered from the GC, and more rapid growth occurred probably due to the absence of a lag phase caused by acid stress (Fig. 3).Behavior of V. vulnificus phage. Present results showing that V. vulnificus phage was more acid resistant in the GC than its host agrees with previous studies using acidified broth and SGF (27, 28). These findings suggest that V. vulnificus phage surviving the gastric barrier might subsequently affect populations of susceptible V. vulnificus in the GI tract as they enter log phase growth. The ecological role of coliphage lytic to E. coli in the human intestine has been studied (17). Fecal samples with low coliphage titers from healthy subjects contained mainly temperate phages, while those with high titers from patients under medical treatment contained mainly virulent phages. Survival time of phages lytic to enteropathogenic E. coli in pH 2.0 milk whey at 37°C ranged from 0.5 to 5 min, compared to 5 to 60 min for that of their host cells, suggesting that E. coli phages were less acid resistant than their host cells (40). While we could not find studies regarding survival of bacteriophage in the human GI tract containing antacid, Smith et al. (40) showed that adding CaCO3 to milk whey inoculated with E. coli phages enhanced their survival as their numbers in the small intestines of calves 5 or 10 h after feeding were approximately four to five times higher than those without CaCO3.
Conclusion. V. vulnificus numbers were reduced by 5 logs within 30 min in the GC, but surviving cells grew well in the IC, reaching 106 to 109 CFU/ml within 9 h. Differences in acid tolerance among the three strains were minor; in fact the one clinical strain did not perform as well as either of the two environmental strains with regard to acid tolerance in GC or growth in IC. This observation suggests that gastric emptying rate may be more important in V. vulnificus infections than differences in acid tolerance. Unpublished U.S. Food and Drug Administration data indicate that V. vulnificus levels in Gulf Coast oysters at the point of consumption often exceed 104/g (D. W. Cook, personal communication). Thus, a meal of a single oyster could contain more than 105 V. vulnificus organisms, indicating that any gastric emptying within 30 min of consumption would readily deliver viable cells to the intestine where they could multiply. The use of antacids and probably acid blockers would substantially increase the chances of gastric survival and probably influence subsequent infection. Since V. vulnificus phages were more acid tolerant than their hosts they would likely be introduced into the small intestine simultaneously with V. vulnificus. However, the ability of the phages used in this study to lyse V. vulnificus in the small intestine is questionable as their culturability is greatly reduced in the absence of seawater in the media used for their propagation, but other phages may be less dependent on seawater. Certainly, the intriguing possibility that phages indigenous to oysters may help protect against V. vulnificus infections cannot be discounted and may help explain the low attack rate (<0.0001) in individuals with preexisting liver disease that consume raw Gulf Coast oysters (24).
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ACKNOWLEDGMENTS |
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We thank M.F.A. Bal'a for technical assistance.
This work was supported in part by a USDA-CSREES special grant (98-34231-6002) and by the Mississippi Agricultural and Forestry Experiment Station under project MIS-081040.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Food Science and Technology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Box 9805, Mississippi State, MS 39762-9805. Phone: (662) 325-8722. Fax: (662) 325-8728. E-mail: microman{at}ra.msstate.edu.
Approved for publication as journal article no. J9772 of the
Mississippi Agricultural and Forestry Experiment Station.
Present address: Virginia Seafood Agricultural Research and
Extension Center, Hampton, VA 23669.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, July 2001, p. 2895-2902, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2895-2902.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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