bacteria associated with crabs from cold waters with emphasis on the occurrence of potential

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1984, p. 1054-1061 Vol. 47, No. 50099-2240/84/051054-08$02.00/0Copyright © 1984, American Society for Microbiology

Bacteria Associated with Crabs from Cold Waters with Emphasis onthe Occurrence of Potential Human Pathogens

MOHAMMAD A. FAGHRI, CHARLES L. PENNINGTON, LOIS S. CRONHOLM, AND RONALD M. ATLAS*Department of Biology, University of Louisville, Louisville, Kentucky 40292

Received 21 November 1983/Accepted 27 February 1984

A diverse array of bacterial species, including several potential human pathogens, was isolated fromedible crabs collected in cold waters. Crabs collected near Kodiak Island, Alaska, contained higherlevels of bacteria than crabs collected away from regions of human habitation. The bacteria associated withthe crabs collected near Kodiak included Yersinia enterocolitica, Klebsiella pneumoniae, and coagulase-negative Staphylococcus species; the pathogenicity of these isolates was demonstrated in mice. Althoughcoliforms were not found, the bacterial species associated with the tissues of crabs collected near Kodiakindicate possible fecal contamination that may have occurred through contact with sewage. Compared withsurrounding waters and sediments, the crab tissues contained much higher proportions of gram-positivecocci. As revealed by indirect plate counts and direct scanning electron microscopic observations, muscleand hemolymph tissues contained much lower levels of bacteria than shell and gill tissues. After the death ofa crab, however, the numbers of bacteria associated with hemolymph and muscle tissues increasedsignificantly. Microcosm studies showed that certain bacterial populations, e.g., Vibrio cholerae, can bebioaccumulated in crab gill tissues. The results of this study indicate the need for careful review of wastedisposal practices where edible crabs may be contaminated with microorganisms that are potential humanpathogens and the need for surveillance of shellfish for pathogenic microorganisms that naturally occur inmarine ecosystems.

The ability of shellfish to concentrate pollutants andpotential human pathogens has led to the establishment oftest procedures and standards aimed at assuring the safety ofshellfish as a human food source (1). Indicator organisms,most frequently coliforms, have been used to determine thelikelihood of contamination of shellfish with fecal matter andhence with human pathogens (1, 11, 15, 30, 34, 35). Failureto detect evidence of Salmonella and Shigella species isgenerally regarded as an indication of the safety of theshellfish with regard to pathogenic bacteria.Although potential contamination of shellfish with fecal

matter is known to be a health hazard, sewage discharge intomarine ecosystems frequently is allowed. Amendments tothe United States Clean Water Act permit communities toapply for waivers of secondary sewage treatment, and manycommunities of southeastern Alaska have applied for suchwaivers; in some cases the requests also seek permission toavoid chlorination of the discharge because of the potentialimpact of residual chlorine on fish and shellfish in thereceiving waters (J. Hastings, U.S. Environmental Protec-tion Agency Region 10, personal communication). In thesecases the ability of human pathogens to survive in coldmarine waters and of shellfish to bioconcentrate such patho-gens does not appear to be viewed by the local municipalitiesas a threat to human health; part of the lack of concernundoubtedly relates to the inadequacy of epidemiologicaldata on the outbreaks of foodborne disease in these regions,the failure to isolate the classical fecal indicator organism,Escherichia coli, from shellfish collected in these regions,and the fact that shellfish generally are adequately cooked tokill most potential human pathogens.

Besides the concern about fecal contamination of human

* Corresponding author.

foods from marine ecosystems, starting in the late 1960s itwas recognized that various bacteria indigenous to estuarineand marine waters also are potential human pathogens andthat these pathogens can be concentrated in shellfish, pre-senting a human health risk (2, 3, 6, 9, 16, 23, 26, 31, 32, 38,40, 43). Most concern centers around several species ofVibrio, such as Vibrio parahaemolyticus. Recent studieshave also identified shellfish as sources of Vibrio cholerae,Vibrio vulnificus, and other Vibrio species in cases of humaninfections (21). Some of these human pathogens can surviveand some can grow at the low temperatures that characterizemarine ecosystems. In this study, we examined the associa-tion of potential human pathogens with edible crabs collect-ed in cold marine ecosystems.

MATERIALS AND METHODS

Crabs examined. Several species of edible crabs, consid-ered potential carriers of human pathogens, were used inthese studies (Table 1). The crabs were collected in northernwaters, primarily in Alaskan continental shelf regions; somecrabs collected off the coast of Maine also were used. Theprimary criteria for the selection of crabs for use in this studywere that they be edible, large, from cold waters, andavailable for examination in an appropriate physiologicalstate; the availability of suitable samples for study was acritical factor in determining the selection of the crabs usedin different aspects of the study.Enumeration of bacteria associated with crab tissues. Tis-

sue samples from muscle, gill, and gut were macerated inone-half strength sterile Rila salts solution in water (pH 8.4),one part tissue to one part diluent (wt/vol). Hemolymph wascollected with a sterile syringe with a 23-gauge needle bypenetrating the intersegmental membrane between the pos-terior of the carapace and the abdomen after the site wasdisinfected with alcohol. Serial dilutions of samples were

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BACTERIA ASSOCIATED WITH CRABS FROM COLD WATERS 1055

TABLE 1. Sources of crabs used in this study

cclletionBottomSpecies Site of cellection collection water

Tanner crab Southern Bering Oct. 1975 2.0(Chionoecetes opilio) Sea

Near Kodiak Oct. 1975 3.5Island

Dungeness crab Near Kodiak Oct. 1975 3.5(Cancer magaster) Island

Near mouth of Jan. 1981 6.5ColumbiaRiver(Oregon andWashington)

King crab (Paralithodes Southern Bering Oct. 1979 3.0camtschatica) Sea

Near Kodiak Oct. 1979 4.5Island

Rock crab (Cancer Off the coast of 1980 6-12irroratus) Maine

surface-spread in triplicate onto the following: marine agar

2216 for enumeration of total heterotrophs, MacConkey agar

for enumeration of gram-negative enteric bacteria, andTCBS ager for enumeration of Vibrio species. Replicate setsof plates were incubated at 35°C aerobically for 3 days toenumerate mesophilic bacteria and at 5°C for 10 days toenumerate psychrotrophic and psychrophilic bacterial popu-

lations; the counts from the incubation temperature givingthe highest numbers were recorded as the viable CFUs.After incubation, colonies were counted, and the concentra-tions of bacteria in the original samples were calculated as

number per gram (wet weight). Because it was necessary to

freeze some of the crabs for up to 2 weeks before enumera-

tion procedures could be conducted, bacterial counts per-

formed immediately after collection were compared withbacterial counts from crabs collected at the same time andfrozen. No significant differences between the counts were

found.Scanning electron microscopic examination of crab tissues.

Shell, gill, and muscle tissues from rock crabs were exam-ined by scanning electron microscopy to directly view theassociated microorganisms. Small pieces of tissues were

exposed to primary fixation (phosphate-buffered glutaralde-hyde fixative, 0.1 M, pH 7.2) for 24 h. The fixative waschanged four to five times during this period. Tissues wereexposed to the secondary fixative, osmium tetroxide, for 1 hduring which the fixative was changed four to five times. Thetissues were rinsed several times with distilled water andincubated in 100 ml of distilled water on a rotary shaker for12 h to eliminate excess fixative. The water was thenremoved from the tissues by successive treatment with 20,30, 50, and 99.5% acetone, and finally the samples wereplaced in a desiccator under an atmosphere of 100% acetonefor 12 h. The acetone was replaced with liquid carbondioxide, and the specimen was subjected to critical pointdrying by using a Polaron critical point dryer. Specimens weremounted on aluminum stubs and surface coated with gold byusing a Polaron sputter coater. The specimens were thenviewed for surface structure and associated microorganismswith an ISI-40 scanning electron microscope.

Bioaccumulation of indicator bacteria. Rock crabs wereexposed experimentally to selected bacteria in microcosmsto determine whether waterborne bacteria become concen-

trated in crab tissues and whether human pathogens canbecome associated with crab tissues. Microcosm studieswere conducted in 20-gallon (ca. 75.7-liter) tanks. Layers ofdolomite sands (10 cm) were added to the tanks as artificialsediments. The tanks were filled with Rila marine saltssolution and kept at 5°C in a cold room. The tanks werecontinuously aerated, and the water was recirculatedthrough a glass wool and activated charcoal filter. During theexperiments, oxygen concentration, pH, and temperaturewere monitored daily. The ranges of the above parameterswere: oxygen, 8.5 to 9.0 ppm; pH, 7.5 to 8.0; and tempera-ture, 5.0 to 6.0°C.Two crabs were placed into each tank. Bacterial cultures

grown in Trypticase soy broth at 35°C for 24 h were added tothe tanks to yield a final concentration of 200 to 250 CFU/ml.Replicate tanks were each inoculated with one of the follow-ing bacterial species: Klebsiella pneumoniae ATCC 13883,Pseudomonas fluorescens (P. V. Liu, University of Louis-ville), V. cholerae ATCC 11623, V. parahaemolyticus ATCC17802, Beneckea harveyi 392 (J. W. Hastings, HarvardUniversity), and E. coli ATCC 25922. The water was sam-pled every 24 h to determine viable bacterial counts. Whenthe indicator bacteria could not be detected in the watercolumn, the tanks were reinoculated to preclude artificialdie-off of the bacteria or removal by the filtration system.At 48-h intervals, 1-ml samples of hemolymph were col-

lected from the crabs with sterile syringes, and the hemo-lymph was examined for the presence of the indicatorbacteria. Water samples also were collected at these timesfor analysis of indicator bacterial populations. At 2, 4, 7, and14 days, one replicate crab from each of the treatmentgroups was sacrificed, and the shell, gill, gut, and musclewere examined for the presence of the indicator bacteria.To detect the indicator organisms, samples were plated

onto Trypticase soy agar, eosin-methylene blue agar (EMB),MacConkey agar, and thiosulfate-citrate-bile salts-sucroseagar (TCBS). P. fluorescens was detected on Trypticase soyagar by its characteristic fluorescence when examined undera UV lamp. B. harveyi was also detected on Trypticase soyagar by examination of the plates in the dark for characteris-tic bioluminescent colonies. E. coli and K. pneumoniaewere detected on EMB and MacConkey agar by the charac-teristic coloration of the colonies. V. parahaemolyticus andV. cholerae were detected on TCBS agar and by usingspecific fluorescent antibodies followed by observation forstained cells with a fluorescent microscope. Results wererecorded as the presence or absence of the indicator bacte-ria. Additionally, a quantitative study was performed byusing V. cholerae added to the microcosms to give aconcentration of 50 per ml of water.Postmortem changes of bacterial populations associated

with crab tissues. In addition to these studies with rockcrabs, studies were conducted in microcosm tanks withDungeness crabs to trace the fate of bacteria after the deathof crabs. For these studies, a shipment of Dungeness crabs,collected at the mouth of the Columbia River in January 1981,was placed into microcosm tanks. Crabs that were alive atthe time of receipt but that died within 12 h of placement intothe microcosm tanks were selected. These crabs were al-lowed to remain in the microcosm tanks. Determination ofdeath was based upon lack of movement and lack of irritabil-ity (failure to grasp a glass rod with claws). At 0, 4, 8, 24, 48,72, 96, and 120 h after the start of the study, gill, muscle, andhemolymph samples were obtained from replicate crabs.The tissues were prepared as described earlier for enumera-tion of viable bacteria. Marine agar, TCBS agar, and EMB

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1056 FAGHRI ET AL.

agar were used to enumerate bacteria from different tissuesto determine the postmortem distribution of bacteria withinthe crab.

Identification of microorganisms associated with crab tis-sues. Samples of muscle and gill tissues from Dungenesscrabs collected near Kodiak Island and tanner crabs collect-ed in the southern Bering Sea were macerated and immedi-ately added to tubes of enrichment media. Four differentenrichment media were used to maximize the probability ofisolating bacteria potentially pathogenic to humans. Theinitial enrichment media used were Trypticase soy brothwith and without 3% marine salts added and brilliant greenbile broth with and without 3% marine salts added. Initialreplicate enrichment samples were incubated for 24 h aerobi-cally at 5 and 35°C. The enrichment cultures were thenstreaked onto isolation media. The isolation media usedwere Trypticase soy agar, marine agar 2216, EMB agar,

EMB agar with 3% marine salts, SS agar, SS agar with 3%marine salts, and TCBS agar.

All distinguishable colonies were isolated for identifica-tion. A total of 763 isolates were selected for further charac-terization. Cultures were maintained on either Trypticasesoy agar or marine agar 2216, and isolates from these mediawere used for further taxonomic studies. Gram-negativerods were identified by the tests of the API 20E identificationsystem, supplemented with temperature growth range (5 to43°C), salt tolerance (0 to 15%), and several physiologicaland nutritional utilization tests and serological typing forvibrios. Gram-positive cocci were identified by morphology,mannitol salt reaction, acid from glycerol, catalase produc-tion, coagulase production, reaction on blood agar, aerobicand fermentative carbohydrate utilization, and optochin andbacitracin sensitivity tests. Gram-positive rods were exam-ined for the presence of endospores and oxygen require-ments. Serological testing of gram-positive anaerobic endo-spore formers was also performed for the identification ofClostridium botulinum.

Additionally, 318 isolates from Dungeness crabs collectedat the mouth of the Columbia River and rock crabs collectedoff the coast of Maine were selected at random from marineagar enumeration plates for taxonomic studies; these isolateswere from gill, hemolymph, and muscle tissues. The APIsystem tests supplemented with the tests described abovewere used to characterize and identify the bacterial isolates.

Pathogenicity studies. The pathogenicity of selected cul-tures isolated from the crabs collected in Alaskan coastalwaters and identified as potential human pathogens was

tested in mice. A total of 25 isolated strains were tested. Thebacterial cells were washed free of medium by centrifugationand resuspended in sterile physiological saline. The bacterial

concentration was adjusted to ca. 106 per ml. Test animals(adult CFW male mice, 6 to 8 weeks old) were inoculated byintravenous, intramuscular, and oral administration routes.All tests were run in triplicate for each isolate and route ofadministration. For intravenous and intramuscular routes ofinoculation, 0.25 ml of cell suspension was used. For oraladministration, the cell suspension was added to the drinkingbottles of animals; healthy mice normally consume 2 to 3 mlof water per day. The animals were observed for behavioralsymptoms for 1 week after exposure to the bacterial isolatesor until death occurred. Animals that survived 1 week were

killed by etherization and an autopsy was performed; ani-mals that died during the week were examined immediately.The lung, liver, mesentery, intestine, and spleen were exam-ined for gross pathology, and macerated tissues were inocu-lated onto appropriate media to determine whether thebacterial strain being tested could be recovered from thesetissues.

RESULTS

Enumeration of bacteria associated with crab tissues. Thecounts of viable microorganisms enumerated from crabtissues by using different media are shown in Table 2. Thegreatest concentrations of bacteria occurred in the gills.Muscle tissues had relatively few bacteria. There were

significant differences between the counts from crabs col-lected from different regions. The lowest counts were for thecrabs collected away from areas of human population, e.g.,crabs from the Bering Sea. The highest counts were from thecrabs collected near areas of human habitation, e.g., crabscollected near Kodiak, Alaska, and those from off the coastof Maine.

Scanning electron microscopic examination of crab tissues.Scanning electron microscopic examination of the shells ofrock crabs revealed many cracks of differing sizes andshapes. Bacteria and fungi were associated with many ofthese fissures (Fig. 1A). Many coccoid-shaped bacteria ap-peared to be growing through the shell. Based upon theshapes of some of the breaks in the shell surface and theclose association of bacteria, it appears that chitinase-producing microorganisms, many of which resemble strepto-cocci, may contribute to the breaks in the shell surface. Theopenings through the shell provide one route through whichmicroorganisms can reach underlying muscle tissues. Nu-merous diverse bacteria of different morphologies occurredin localized regions of crab gill tissue (Fig. 1B). Unlike theshell and gill tissues, the surfaces of muscle tissues of activecrabs showed a lack of microorganisms (Fig. 1C).

Bioaccumulation of indicator bacteria. The results from the

TABLE 2. Concentration of viable bacteria associated with tissues of crabs

Organisms per g of tissue (wet wt)Type of

Area and time of collection Marine agar TCBS MacConkeycrab _______________Muscle Gill Muscle Gill Muscle Gill

Dungeness Chiniak Bay, Kodiak Island, 1975 4 x 102 2 x 105 <50 1 X 102 <50 <101Columbia River, 1981 1 x 10l 2 x 10' <50 2 x 101 <50 <10'

Tanner Chiniak Bay, Kodiak Island, 1975 2 x 102 3 x 104 <50 5 X 101 <50 <10lUgak Bay, Kodiak Island, 1975 2 x 101 1 x 104 <50 1 x 10l <50 <10'Bering Sea, 1975 1 x 10l 1 x 103 <50 1 x 10l <50 <101

King Chiniak Bay, Kodiak Island, 1981 3 x 102 1 x- 106 <50 1 x 103 <50 1 X 101Bering Sea, 1981 5 x 101 1 x 103 <50 2 x 102 <50 <lo1

Rock Atlantic Ocean, Maine, 1981 2 x 102 1 x 107 <50 1 x l0 <50 I x l0'

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FIG. 1. Scanning electron micrographs of tissues of a rock crab(Cancer irroratus) and the associated microorganisms. (A) Shell of arock crab showing the cracks in the surface and bacteria growing onthe surface. The appearance of bacteria associated with the crackssuggests that the bacteria have chitinolytic activities which areresponsible for forming these cracks. Other chitinoclastic microor-ganisms also may be associated with the shell, particularly in theareas of cracks. (B) Gill filament surface showing morphologicallydiverse bacteria. Localized aggregation of bacteria on the gill mayreflect areas of mucus secretion, areas of high nutrient concentra-tion, or a characteristic clumping mechanisms of the host defensesystem. (C) Muscle tissue showing lack of bacteria on the surface.

microcosm studies showed differential survival of the indica-tor microorganisms added to the tanks and selective bioac-cumulation within the tissues of the crab (Table 3). E. colicould not be recovered from water or sediment 24 h after

inoculation of the microcosms and never was detected in anytissues of the crabs. K. pneumoniae and P. fluorescens wereisolated from water and gill tissue but not from other tissues.B. harveyi was readily isolated from the water column, butnot from crab tissue until the crabs had been exposed to thisbacterium for more than 1 week; after 1 week of exposure,B. harveyi was found in gill tissue. V. cholerae and V.parahaemolyticus could readily be isolated from the shell,gut, and gill tissues. The hemolymph and muscle tissueswere not sterile, but none of the indicator organisms weredetected in the hemolymph or muscle tissues of active crabs.The Vibrio species accumulated in the gill tissues of thecrabs at concentrations that were higher than those in thesurrounding water. In a quantitative study, V. choleraereached a concentration of 5 x 103 per g in gill tissuecompared to a maximal concentration of 5 x 100 per ml inthe water.Postmortem changes of bacterial populations associated

with crab tissues. Although the concentrations of bacteria inhemolymph and muscle tissues were low in active Dunge-ness crabs, the bacterial concentrations associated withthese tissues increased significantly after the crabs died(Table 4). Bacteria, which had a mean concentration of lessthan 102 per ml in the hemolymph of active crabs and whichwere not even detectable in the hemolymph of some activecrabs, reached a concentration of over 108 bacteria per ml inthe hemolymph within 120 h of the death of the crab. Theconcentrations of bacteria in muscle tissues of dead crabsexceeded 103 bacteria per g, a rise of over two orders ofmagnitude from the healthy state. The counts on TCBS andEMB media indicated that Vibrio species, but not entericbacteria, increased in numbers in the gill, hemolymph, andmuscle tissues of the crabs after death.The microcosm studies with rock crabs exposed to indica-

tor bacteria also indicated that hemolymph in many cases isfree of bacteria. In active crabs the hemolymph remainedfree from indicator bacteria during the 2 weeks of exposure,although there were high numbers of indicator bacteria in theexternal environment, i.e., the tank water, shell surface, andgill tissue. In those cases, however, in which animals wereinjured in the tank due to fighting between crabs or stressedby a failure in the aeration system, the animals usually weresluggish, and bacteria appeared in hemolymph. High num-bers of gram-negative bacteria in hemolymph, >102 bacteriaper ml, usually coincided with a reduction in the numbers ofhemocytes, indicating that the spread of bacteria through thetissues was accompanied by a loss of host defense cells.Taxonomy of bacteria associated with crab tissues. The

diversity of bacterial species associated with the tissues ofthe crabs was greatest for the Dungeness crabs collectednear Kodiak Island and rock crabs collected off the coast ofMaine and lowest for the tanner crabs collected in the

TABLE 3. Indicator bacteria detected in microcosm water andcrab tissues

Detected in:Bacterium

Water Gill Shell Gut Hemolymph Muscle

B. harveyi + + - - - -E. coli - -K. pneumoniae + +P. fluorescens + +V. cholerae + + + +V. parahaemolyticus + + + +

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TABLE 4. Postmortem distribution of bacteria in tissues of Dungeness crabs"

Bacteria per g of tissue (wet wt)Time after Marine agar TCBSdeath (h)

Muscle Gill Hemolymph Muscle Gill Hemolymph

0 1 x lo, 2 x 103 5 x 10' <1 x 10 x lo, 1 x 104 1 x 10' 2 x 104 3 x 102 <1 x101 1 x 102 x lo,8 8 x 101 2 x 105 9 x 103 <1 x lo, 2 x 102 2 x 103

24 1X102 4x105 1x105 <1xlo, 1X102 6x10348 4 x 102 4 x 106 2 x 107 <1 X lO, 1 X 102 5 x 10672 8 x 102 5 x 107 9 x 108 1 x 102 5 x 103 7 x 10695 1 x 103 4 x 107 7 x 108 1 x 102 5 x 103 2 x 106120 2x103 6x106 6x108 5x10' 1x103 1x106

a In EMB, <10 bacteria per g of tissue (wet weight) were recovered from all tissues examined up to 120 h after death.

Bering Sea (Tables 5 and 6). More bacterial species wereisolated from gill than from muscle tissue. In addition togram-negative rods and coccobacilli, gram-positive cocciwere isolated with a high frequency. In the crabs from nearKodiak Island and off the coast of Maine, several gram-negative bacterial species were found in both gill and muscletissues that are not frequently found in surrounding watersand sediments.Although E. coli, the widely used indicator of fecal con-

tamination, was not isolated in any case, the other gram-negative enteric bacteria isolated from the Dungeness crabscollected near Kodiak Island can be considered as possibleindicators of human pollution. The frequency of occurrenceof the bacterial isolates in the muscle of Dungeness crabscollected near Kodiak was, by genus, Staphylococcus >Sarcina > Acinetobacter > all others. In the gill tissue ofDungeness crabs from near Kodiak, the isolation frequencywas Streptococcus > Pseudomonas > Alcaligenes > Mor-axella = Acinetobacter = Aeromonas = Citrobacter =Enterobacter > Sarcina = Yersinia = Staphylococcus =Klebsiella. In the Dungeness crabs collected off the mouth ofthe Columbia River, Acinetobacter, Moraxella, Vibrio, andAchromobacter species were the most abundant gram-nega-tive rods and Pseudomonas species were numerically lessimportant. In comparison, several Pseudomonas specieswere among the numerically dominant isolates from the rockcrabs collected off Maine. Additionally, Acinetobacter, Mor-axella, Klebsiella, Beneckea, Vibrio, Achromobacter, andCitrobacter species were numerically important isolatesfrom rock crab tissues. C. botulinum, Salmonella spp., andShigella spp. were not detected in crab tissues.

Serological testing with polyvalent antiserum failed todetect V. cholerae type 01 associated with any of the crabs,but vibrios belonging to the nonagglutinable group werefound. Numerical taxonomic analyses indicated that V.parahaemolyticus and V. alginolyticus were associated withcrab tissues. V. parahamolyticus and V. vulnificus werefound in association with crab gill tissue from Dungenesscrabs collected off the mouth of the Columbia River but notfrom crabs collected in the Bering Sea or near Kodiak. V.alginolyticus, however, was detected in crabs collected at alllocations.

Staphylococcus and Micrococcus species also were abun-dant in the muscle and gill tissues of all crabs. Coloniesrandomly chosen from marine agar cultures of gill, hemo-lymph, and muscle showed a significantly higher number ofgram-positive cocci (25%) compared to environmental sea-water and sediment samples (<1%) at the sites of crabcollection. All Staphylococcus isolates were coagulase nega-

tive. According to the identification scheme of Kloos andSchleifer (29), the Staphylococcus isolates from the crabsincluded S. epidermidis and S. hominis.

Pathogenicity of selected bacteria associated with crabs.Studies with mice demonstrated the pathogenicity of severalof the bacterial isolates. Mice developed behavioral symp-toms, lethargy, and disinterest in food 24 h after intravenousinjection with strains of coagulase-negative Staphylococcusspecies isolated from crabs from both the Bering Sea andnear Kodiak Island. The symptoms lasted for 3 days, afterwhich the animals recovered. Autopsy failed to show anypathological abnormalities. All 27 mice exposed to Y. entero-colitica isolated from the Dungeness crabs collected nearKodiak Island showed behavioral symptoms 24 h afterinoculation, regardless of the route of administration. Symp-toms included staggering, lethargy, and lack of responsive-ness. One mouse died 5 days after intravenous injection andY. enterocolitica was recovered after autopsy. All miceinoculated with a Klebsiella isolate from crabs collected nearKodiak Island showed overt symptoms of disease, and 33%(20 individuals) of the mice died. Death occurred withintraperitoneal and intravenous injection but no deaths oc-curred after oral administration. K. pneumoniae of the samebiotype as the culture used for inoculation was recoveredfrom the blood, liver, spleen, and kidneys of the dead mice.

DISCUSSION

Many studies have shown the inadequacy of standardcoliform counts as an indicator of fecal contamination ofmarine ecosystems and the safety of shellfish collected fromareas impacted by sewage effluents; E. coli is rapidly elimi-nated from seawater, whereas other bacteria in sewageeffluents, including human pathogens, may survive for long-er periods of time (8, 13, 14, 33). The microcosm studiesconfirmed the rapid disappearance of E. coli from seawaterat low temperatures and indicated that E. coli is not protect-ed by association with crabs. Other bacteria, such as Klebsi-ella species, however, showed prolonged survival in associa-tion with crab tissues. The microcosm studies indicated thepotential for accumulation of human pathogens in tissues ofcrabs collected in regions contaminated with sewage. Thetaxonomic analyses of bacteria associated with crabs collect-ed near Kodiak Island, Alaska, indicate possible contamina-tion with sewage; even though E. coli was not isolated fromthese crabs, Klebsiella species and other enteric bacteriawere isolated from the tissues of these crabs. Enteric bacte-ria occur in noncontaminated environmental samples, buttheir coexistence in a marine environment indicates a proba-

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TABLE 5. Microorganisms identified from tissues of crabs collected near and away from Kodiak Island, Alaska

Bacteria identified in:Crabs Gill Muscle Hemolymph

Dungeness, Kodiak Island Acinetobacter spp. Acinetobacter spp. ND'1Aeromonas hydroxvlaAlcaligenes spp.Citrobacter freundiiEnterobacter agglomeransKlebsiella pneumoniaeMoraxella spp.Pasteurella spp.Pseudomonas maltophiliaPseudomonas fluorescensSarcina spp.Staphylococcus spp.,

coagulase negativeStreptococcus group DYersinia enterolitica

Alcaligenes spp.Micrococcus spp.Moraxella spp.Pasteurella spp.Pseudomonas spp.Sarcina spp.Staphylococcuis spp..

coagulase negativeStreptococcus group D

Dungeness, ColumbiaRiver

Acinetobacter spp.Achromobacter spp.Aeromonas spp.Alcaligenes spp.Micrococcus spp.Moraxella spp.Pseudomonas spp.Staphylococcus spp.,

coagulase negativeVibrio spp.

Acinetobacter spp.Achromobacter spp.Alcaligenes spp.Micrococcus spp.Moraxella spp.Pseudomonas spp.Staphylococcuis spp.,

coagulase negative

Acinetobacter spp.Vibrio spp.

Acinetobacter spp.Achromobacter spp.Aeromonas spp.Alcaligenes spp.Beneckea spp.Citrobacter spp.Klebsiella spp.Micrococcus spp.Moraxella spp.Pseudomonas spp.Pseudomonas fluorescensStaphylococcus spp.,


Acinetobacter spp.Achromobacter spp.Micrococcus spp.Moraxella spp.Staphylococcus spp.,

coagulase negative

Acinetobacter spp.Achromobacter spp.Alcaligenes spp.Beneckea spp.Klebsiella spp.Micrococcus spp.Pseudomonas spp.Staphylococcus spp.,


Tanner, Bering Sea Acinetobacter calcoaceticusAlcaligenes spp.Micrococcus spp.Moraxella spp.Staphylococcus spp.,

coagulase negative

Micrococcus spp.Staphylococcus spp.,

coagulase negative

a ND, Not determined.

ble terrestrial source. It is the general profile of bacterialspecies, which included Klebsiella, Citrobacter, and Yer-sinia species, that is suggestive of sewage contaminationrather than the finding of any one indicator species. Thebacterial species associated with crabs collected in populat-ed areas were quite different from those from crabs collectedaway from areas of human habitation, as evidenced by thecomparison of isolates from the crabs collected near KodiakIsland and those collected in the pristine Bering Sea.Of significant concern was the isolation of Y. enterocoli-

tica, a human pathogen that can grow at 5°C as well as 37°C,from crabs collected near Kodiak Island; the pathogenicityof the isolated strain of Y. enterocolitica was demonstratedin vivo. The pathogenicity of the Y. enterocolitica isolatessuggests that they may be of human origin. Strains of this

bacterium from human sources have been reported to dem-onstrate typical pathogenicity for laboratory animals, where-as environmental isolates from water and food reservoirslack such pathogenicity (7). At the time of this study, KodiakIsland did not have a sewage treatment facility, and rawsewage effluent was directly released into coastal waters.The crabs were collected north of the sewage outfall, andthese crab populations are known to regularly migrate pastthe sewage outfall and into areas of commercial collection(Alaska Department of Fish and Game, personal communi-cation). This raises the possibility that crabs migrating pastthe sewage outfall were contaminated and bioaccumulatedbacteria from the contaminated water column in their gilltissues. Extensive taxonomic surveys in this region did notreveal widespread distribution of such bacterial populations

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TABLE 6. Number of bacterial genera observed from tissues ofcrabs collected in different regions

No. of genera found in:Site of collection

Gill Muscle Hemolymph

Kodiak 13 9 0Columbia River 9 7 2Bering Sea 5 2 0Maine 12 5 9

in water and sediment (19, 20), which may indicate that thesebacteria are able to survive in much higher numbers inassociation with crab tissues than in surrounding environ-ments.

Besides the potential for association of bacteria fromsewage with crabs, the possible accumulation of indigenousmarine bacteria that may be human pathogens has beeninvestigated. Detailed taxonomic studies of Vibrio speciesfrom marine ecosystems have led to a reevaluation of thetaxonomy of this group and the definition of several new

species that are potential human pathogens (4, 39, 41).Several investigators have found Vibrio species associatedwith tissues of blue crabs collected in temperate waters (10,12, 37, 42). Recent studies have shown that V. choleraenaturally occurs in temperate estuaries and that cases ofcholera in the Gulf Coast region of the United States haveresulted from the ingestion of contaminated shellfish, includ-ing inadequately cooked crab meat (5, 26, 44). It has beensuggested that V. cholerae is a ubiquitous inhabitant ofestuarine ecosystems, but this organism was not found inassociation with the crabs examined in this study. Severalfactors influence the survival of V. cholerae in estuarineecosystems, including salinity and nutrient concentration,and special enumeration procedures are necessary to ensure

recovery of this organism from natural habitats (26, 36);environmental isolates of V. cholerae do not grow at 5°C,and cold temperatures appear to restrict the natural distribu-tion of this bacterial species.

V. vulnificus, which has been reported in association witheels and shellfish (32, 39, 41), was found in association with

gill tissues of crabs collected off the Oregon and Washingtoncoasts but not in tissues of crabs collected in Alaskancontinential shelf regions. Similarly, V. parahaemolyticuswas isolated from Dungeness crabs collected off the mouthof the Columbia River but not from Alaskan crabs. V.

parahaemolyticus has been shown to undergo an annual

cycle in temperate estuaries during which it becomes associ-ated with chitin exoskeletons of invertebrates (22, 24). V.

parahaemolyticus apparently is restricted to temperate wa-

ters that reach temperatures high enough for it to completeits annual cycle (25). Previous surveys have failed to detectV. parahaemolyticus in cold Alaskan waters, although the

closely related species V. alginolyticus has been isolatedfrom these ecosystems (43). Our results support the restrict-

ed ecological distribution of V. parahaemolyticus. AlthoughVibrio species are among the major bacterial species occur-

ring in Alaskan waters, those of concern with respect to

human health appear to be absent in cold marine waters.The frequent isolation of Staphylococcus and Micrococ-

cus species from crab tissues at first seemed surprising;these gram-positive cocci are much more abundant in associ-

ation with crab tissues than in surrounding waters and

sediments (19, 20). Recent reports, however, have indicatedthat gram-positive cocci are normally found in marine habi-

tats (17, 18, 27). All of the Staphylococcus isolates from thecrabs examined in this study were coagulase negative (S.epidermidis and S. hominis). These species have been isolat-ed from other marine ecosystems (17). Some coagulase-negative Staphylococcus species are human pathogens (28),and the in vivo pathogenicity tests showed that the crabisolates were pathogenic for mice.An important question is whether the bacteria associated

with crabs contaminate the muscle tissues, the portion of thecrab that is eaten. The direct scanning electron microscopicobservations and the viable enumeration procedures indicat-ed that most bacteria are associated with the surface tissuesof the gills and shell. Hemolymph and muscle, although notsterile, normally had low populations of bacteria, and bacte-ria added to microcosms generally were not found in hemo-lymph or muscle tissues. However, after the death of a crab,high numbers of bacteria were found in hemolymph andmuscle tissues. V. parahaemolyticus has previously beenfound in muscle tissues of blue crabs (16). Separate studiesto be published later indicate that rock crabs have anextensive cellular defense system that limits the bacterialcontamination of muscle tissue; the impairment of the crab'sdefenses renders the muscle susceptible to contamination.The muscle tissues of crabs that are stressed by depletion ofoxygen or are injured and die within holding tanks can

rapidly become contaminated with bacteria, including hu-man pathogens.These results raise questions concerning the safety of

crabs collected in regions receiving untreated sewage efflu-ent and about consuming crabs that have died or that havebeen severely stressed before processing. The finding ofpotential human pathogens in tissues of crabs collected near

Kodiak Island underscores the need to carefully evaluaterequests for waivers for primary and secondary sewagetreatment in this region. The inability to isolate E. coli fromcrab tissues suggests the need for using alternative indicatorsof fecal contamination. The occurrence of gram-positivecocci in crab tissues that are potential human pathogensbroadens the scope of concern. With respect to indigenouspathogenic Vibrio species, it appears that crabs from coldwaters are far less likely to act as vectors of these pathogensthan shellfish from temperate waters. Continued studics are

needed to assure the safety of crabs and to establish ade-quate test procedures for assessing the probabilty of con-

tamination with potential human pathogens.

ACKNOWLEDGMENTSThis study was supported by the Bureau of Land Management

through interagency agreement with the National Oceanic andAtmospheric Administration under which a multiyear program,responding to the needs of petroleum development of the Alaskancontinental shelf, is managed by the Outer Continental Shelf Envi-ronmental Assessment Program (OCSEAP) Office.We thank the Alaska Department of Fish and Game for help in

obtaining crabs, E. J. Krichevsky and M. I. Krichevsky for assist-ance with data management, J. W. Hastings and P. V. Liu forsupplying cultures, K. Jabarizadeh and C. Short for laboratoryassistance, and R. Apkarian for the scanning electron microscopy.

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bacteria associated with crabs from cold waters with emphasis on the occurrence of potential

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