MFR PAPER 1144

A Comparative Study of theBacterial Flora of the Hemolymphof Callinectes sapidus

R. R. Colwell is with the Depart­ment of Microbiology, Universityof Maryland, College Park, MD20742. T. C. Wicks is with the De­partment of Biology, GeorgetownUniversity, Washington, DC 20016.H. S. Tubiash is with the BiologicalLaboratory, National MarineFisheries Service, NOAA, Oxford,MD 21654.

R. R. COLWELL, T. C. WICKS, and H. S. TUBIASH

ABSTRACT-The bacterial flora of blue crabs (Callinectes sapid us) fromChesapeake Bay was examined. Hemolymph of normal, healthy blue crabs wasfound non-sterile, with total bacterial counts ranging from <100 to >3.0 x 10 5

on a seawater-based medium and 0 to >10 5 on Standard Methods Agar.Counts of 0-10 3 were observed on TCBS agar. Vibrio spp., including Vibrioparahaemolyticus, were the major taxonomic groups found in the crabhemolymph. A comparison of crab data with that of Chesapeake Bay oystersrevealed a qualitative difference in that the crab hemolymph flora was almostentirely Vibrio spp., whereas the oyster flora included species of a variety ofother genera.

INTRODUCTION

The normal bacterial flora of bluecrabs (Cal/inectes sapidus) fromChesapeake Bay has not been exten­sively studied. Interest in the micro­biology of shellfish, traditionally, hasfocused on human pathogens found incommercially important species, as inthe case of the blue crab. Many of thepapers published on the microflora ofthe blue crab have concerned pathogensrather than the normal microbial florapresent in the healthy animal (Couch,1967; Fishbein et a!., 1970; Krantz eta!., 1969; Rosen, 1967; Sawyer, 1969;Williams-Walls, 1968). In addition, in­formation has been gathered concerningthe bacteriology of processing, han­dling, packaging, or storage of crab­meat (Loaharanu and Lopez, 1970;Ulmer, 1961; Ward and Tatro, 1970).

This study was conducted to deter­mine the total viable, aerobic, hetero­trophic, bacterial flora of blue crabhemolymph in freshly caught,aquarium-held, and market crabs duringthe summer months and to identify andclassify the bacterial s trains isolated,using the methods of numerical tax­onomy. Numerical taxonomy as amethod for the analysis of taxonomicdata for bacteria was first proposed bySneath (1957) and has been used, withmodifications of the original methods,

by various investigators (Society forGeneral Microbiology, 1962; Beers eta!., 1962; Silvestri eta!., 1962;SokalandSneath, 1963). Colwell and Liston(1961), in one of the earliest numericaltaxonomy studies, examined marinebacteria. Subsequently, several otherinvestigators examined the taxonomy ofmarine bacteria (Floodgate and Hayes,1963; Hansen et a!., 1965; Liston et aI.,1963; Pfister and Burkholder, 1965;Qualding and Colwell, 1964). Results ofthese studies proved helpful in identify­ing and classifying the bacterial florafrom blue crab hemolymph.

MATERIALS AND METHODS

Healthy adult blue crabs (Callinectessapidus) weighing 150-230 g were ob­tained from seafood markets or col­lected from several areas in ChesapeakeBay and Chincoteague Bay during thesummer of 1970. Male and female crabspossessing hard carapaces ("soft-shell"crabs undergoing ecdysis were not in­cluded) were sampled to determine totalnumbers and types of aerobic, hetero­trophic bacteria comprising their nor­mal microflora during the summermonths when the water temperatureranged between 23°-32°C. Sources ofcrabs, bacterial isolates, and dates ofsampling are given in Table I.

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The isolates from the holding tankswere obtained from animals maintainedin the laboratory for more than I week.Crabs obtained from the fish marketwere held in a tank for I to 2 days beforesampling. Crabs from the Rhode Riverin Chesapeake Bay were sampled im­mediately after capture. Crabs fromMarumsco Bar were also sampled in thefield without delay after capture. Thelargest sample of animals examined wasa catch made off Franklin City, Va. Thehemolymph was sampled 12-16 h aftercapture.

Total viable counts of aerobic,heterotrophic bacteria in blue crabhemolymph were carried out as follows.After removing the fluid with a sterile25-gauge needle and syringe wettedwith a sterile solution of 5 percent sodi­um polyanethol sulfonate (G RO­BAX)I,Z, an anticoagulant reportedlyeffective for isolating bacteria fromblood (Morello and Ellner, 1969), I mlof the hemolymph was withdrawn fromeach crab, either through the posteriorhinge of the carapace or from the base ofthe coxa of the flippers. A dilution serieswas made, using a sterile diluent com­posed offour salts and distilled water inthe concentration: NaCI, 24.0 g; KCI,0.78 g; MgClz, 5.3 g; MgS04 • 7HzO, 7.0g; and distilled water, I liter. One-tenthml of each dilution was inoculated ontoagar plates, in triplicate, and spreadevenly with a sterile, bent glass rod. Thethree media used were: MSYE (Pro­teose peptone, I g; yeast extract, 1 g;agar, 15 g; four salts solution, as above;pH adjusted to 7.4-7.6 with 0.1 NNaOH), SMA, the Standard MethodsAgar (Casein digest, 5.0 g; yeast ex­tract, 2.5 glucose, 1.0 g; agar, 15 g; dis­tilled water, I liter; pH adjusted to 7.0with NaOH) and TCBS, the Thio­sulfa te-citrate-bile salts-sucrose agar3

I Roche Diagnostics, HotTman-LaRoche, Nutley,N.J.'Reference to trade names does not imply en­dorsement by lhe National Marine Fisheries Ser­vice, NOAA.

Table I.-Source and date of Isolation of bacterialstrains Included In the numerics' taxonomy analysis.

3Baltimore Biological Laboratories, Baltimore,Md.'Difco Laboratories, Detroit, Mich.

'HT ~ Holding tank. Georgetown University: FM ~ Fishmarket. District of Columbia; MB = Marumsco Bar. Md.(Chesapeake Bay): RR = Rhode River, Md. (ChesapeakeBay): FC = Franklin City, Va. (Chincoteague Bay).

(yeast extract, 5.0 g; polypeptone pep­tone, 10 g; sodium citrate, 10.0 g;sodium thiosulfate, 10.0 g; oxgall, 5.0 g;sodium cholate, 3.0 g; sucrose, 20.0 g;NaCI, 10.0 g; iron citrate, 1.0 g; thymolblue, 0.04 g; bromthymol blue, 0.04 g;agar, 14.0 g; distilled water, I liter; pH8.6). The inoculated media were incu­bated at 25°C for 48 h, after whichcounts were made and the media againincubated for an additional 5 days for arepeat count.

Forty-nine cultures were isolated,purified, and subjected to the testingprocedure used for numerical tax­onomy. Colonies were selected ran­domly and were streaked three times toensure purity of the cultures. Afterpurification, all cultures were tested forability to grow on a medium (MSYE)containing the major ionic constituentsof seawater. All cultures isolated fromthe SMA and TCBS media also grew onMSYE and were, therefore, maintainedon MSYE.

Taxonomic tests were carried out oneach pure culture for morphology andmotility, using wet-mount preparationsof 24-h broth cultures under phase con­trast; gram stain; growth characteristicson MSYE agar after 48 h incubation at25°C; growth at 4, 15,25,37, and 45°C;growth at NaCI concentrations of 0.0,3.0,7.0, and 10.0 percent; growth at pH4.0,5.0,6.0,7.0,8.0, and 9.0; hemolysisof human blood; utilization of citrate;aerobic and anaerobic utilization of glu­cose and sucrose, employing MOFmedium4 (Leifson, 1963); utilization ofgalactose, mannitol, ribose, lactose,mannose; production of catalase, ox­idase (Kovacs, 1956), indole, acetyl

SMA TCBSMYSENo.

Source sampled

Counts on MSYE were comparableamong sampling locations. Greatervariations were noted for counts onSMA, with the lowest counts on SMAnoted for Rhode River animals. Crabsfrom Marumsco Bar gave highestcounts on SMA. The salinity of thewater at Rhode River was lower than atMarumsco Bar and both the RhodeRiver and Marumsco Bar crabs weresampled immediately after capture.Hence, no correlation was noted be­tween time of sampling after capture

DISCUSSION

colony type appearing on TCBS agarwas yellow, 2 mm in diameter, and simi­lar to Vibrio alginolyticus. Results oftotal viable counts are given in Table 2.No change in counts was observed be­tween 48 hand 7 days.

The strain clusters obtained from thecomputer analysis of the taxonomicdata are shown in Figure 1. All strainsgrouped atS ;;3 57 percent and the majorphenons detected are shown in FigureI, A through 1. Strains clustered in Phe­non A were not found to be closely re­lated, indicated by the low similarityvalues (62-64 percent). Phenon B con­sisted of strains more closely related toeach other (5 = 72-74 percent). PhenonC comprised the largest group, consist­ing of strains 41 through II (Figure I)clustering atS ;;3 75 percent. Three sub­groups (I, II, III) were observed inPhenon C (Figure 1).

Phenon D, strain 9-24 (Figure I)formed at 5 values ;;3 71-75 percent, in­dicating relatively high relationshipamong the strains. Phenon E formed at72 percent 5 and was considered dis­tinct from Phenon D. Phenons F and Gformed at relatively low 5 values, 65-67percent 5 and 68 percent 5, respec­tively. Phenon H (intra 5 values of62-63 percent) and Phenon I completedthe groupings obtained in the analysis.

Laboratoryholding tank 3 1.8 x lOS 3 X 10' 10

Fish market 2 6 x 1()4 4 x 103 1Rhode River 6 2.1 x lOS 60 0Marumsco Bar 4 1.3 x lOS 2 X 105 100Franklin

Cily. Va. 33 3.1 x 10' 2 X 10' 33Total range 48 -:100--·3.0 x 10' (}_>105 0-10'

Table 2.-Bacterial counts of the hemolymph of crabscollected at several locations on the media employedin the study. Counts given are per milliliter hemolymphof Individual crabs. Figures given for each sample setare mean values, except where otherwise indicated.

A wide range in bacterial counts forthe blue crab hemolymph was ob­served. On the MSYE medium, countsranged from < 100/ml (five animals) to>3.0 x 105/ml(fiveanimals). Counts onSMA were lower, O/ml (II animals) to> 105 mJ (two animals), indicating thatmany of the bacteria found in thehemolymph require the major saltsfound in seawater for growth. OnTCBS, the counts were from 0 (14 ani­mals) to 103 (3 animals). The dominant

RESULTS

methyl carbinol, and urease; productionof ammonia from peptone, NOz, andN0 3 ; hydrolytic activity on starch,gelatin and casein; methyl red test; andutilization of alanine, proline, glutamicacid or methionine as sole carbon andnitrogen sources.

Drug sensitivity of each pure culturewas determined using penicillin, 10units; Chloromycetin, 30 J.1g; tetracy­cline, 30 J.1g; dihydrostreptomycin, 10J.1g; and colimycin, 10 )J.g5. Sensitivitywas recorded without quantification ofthe diameter ofthe inhibition zone. Sen­sitivity to the pteridine compound(0/129) was also recorded (Shewan eta!., 1954).

All media, with the exception of theblood agar, were prepared with the saltsolution, described above, as diluent. Inmost cases, dehydrated medium wasmixed directly with the salt solution asdiluent, but some media required spe­cial preparation. Media for the caseinhydrolysis, methyl red, Voges­Proskauer, and carbohydrate utilizationtests were prepared double strength indistilled water, and a double strength,sterile salt solution was added (I: IvolJvol) after sterilization.

A total of 126 features used in thecomputer analysis were scored, withthe code: 0 (negative), I (positive), or 3(not tested or not applicable). Thecoded data were entered for analysis inan IBM 360/40 system equipped withdiscs and magnetic tape drives.Georgetown University TaxonomyPrograms (GTP-I, 2, 4, and 5) wereused in the analysis (Colwell, 1964;Colwell and Liston, 1961; Moffett andColwell, (968). The programs havebeen documented for the IBM Compu­ter Users Library.

'Baltimore Biological Laboratory, Bioquest Divi­sion of Becton-Dickinson. Inc., Sensi-Disks.

HTFMRRMBFCFCFCFCFCFCFCFCFCFC

Source'

7/15f7071191707/21/708/221708/27/708/271708/271708/27/708/27/708/271708/271708/271708/27/708/27170

Date of isolation

1-1011-2728-3334-39

40414243444546474849

Strain number

30

100

95

90

85

rf-

a::: 80<l-l

:2(/)

75

~0

70

65

60

55

A

36 48 51 17 37 47 34 42 30 16 29 4 32 43 5 10 14 9 3 25 26 27 22 13 20 4512 8 18 41 49 50 36 28 46 2 6 31 33 44 7 I II 39 40 24 23 15 21 19 35

Flgure 1.-Sorted outputlrom the taxonomic analy.l. by computer. Groups were formed by highest linkage sorting.

and the salinity and the number of bac­teria in crab hemolymph detected onSMA, a medium employed for detectingnonmarine bacteria. Also, no correla­tion between sex and total viable counton SMA was detected. However, it wasclear that the proportion of nonmarinebacteria in crab hemolymph was higherfor fish market and Marumsco Barcrabs. The holding tank and FranklinCity samples exhibited approximatelythe same proportion of nonmarine bac­teria (bacteria capable of growth onSMA).

The genera of bacteria found in crabhemolymph were obtained from thetaxonomic data analyses, and each ofthe eight phenons were classified.Strains 38 and 12 (Phenon A) weregram-positive, motile rods, 1.3-3.0 Mmlong; capable of utilizing glucose andsucrose aerobically and anaerobically;growing at temperatures of 4°_37°C andpH 7.0-9.0; sensitive to penicillin,Chloromycetin, and colimycin; resis-

tant to dihydrostreptomycin; beingoxidase positive, indole negative, andcitrate positive; utilizing alanine andproline as sole carbon and nitrogensources; growing in 7-10 percent NaCl;and were classified as Bacillus sp.

Phenon B included five strains, one ofwhich was the reference strain of Vibrioparahaemolyticus (strain Sak-3).

Phenon C was the largest group, with27 strains comprising the cluster. All ofthe strains of Phenon C were roundended, motile, gram-negative rods,1.3-3.0 Mm long x 0.5-1.3 Mm wide andoccurring singly, not as pairs or chains.These strains grew at pH 4.0-9.0 and attemperatures of 15°-45°C, with half ofthe isolates capable ofgrowth at 4°C andin 3-10 percent NaCI. However, 92 per­cent of the isolates were capable ofgrowth without added NaCI. Thestrains formed convex, entire, translu­cent colonies yellow to white in color onMYSE agar. All fermented glucoseanaerogenically and, in general, pro-

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duced acid in sucrose, galactose, man­nose, mannitol, and ribose and were lac­tose negative. All strains hydrolyzedstarch, produced NH 3 from peptone,reduced nitrates, produced catalase,hydrolyzed casein, utilized citrate as asole carbon source, were oxidase posi­tive and methyl red positive. None ofthe strains of Phenon C producedurease or acetylmethylcarbinol, buthalf produced indole. All were sensitiveto Chloromycetin; 64 percent were sen­sitive to dihydrostreptomycin; 36 per­cent to colimycin, and 16 percent topenicillin. Phenon C was classified asVibrio spp.

Phenon 0 strains were straight,gram-negative rods, 1.3-3.0 Mm long,occurring singly and in short chains(less than five cells). Colonies 2-5 mm indiameter and a translucent yellow orwhite were formed on agar. Glucoseand sucrose were utilized aerobicallyand anaerobically, without gas. Acidwas produced from mannose; nitrate

was reduced and NH 3 produced frompeptone. All of the strains of Phenon 0grew in 3-10 percent NaCI and moststrains utilized citrate and were indoleand methyl red negative. Phenon 0strains were catalase positive and hy­drolyzed casein. This phenon was alsoidentified as comprising members of thegenus Vibrio.

Phenon E consisted of two cultures,strains 23 and 26, clustering at 72 per­cent 5. These strains were gram­positive, motile rods, occurring singly,1.3-3.0 11m long x 0.7-1.5 11m wide.Both strains grew at 15°_45°C and fer­mented glucose and sucrose and pro­duced acid from mannose. They did nothydrolyze casein and were oxidase andcatalase negative. They were sensitiveto penicillin, chloromycetin, tetracy­cline, and colimycin, but not to dihy­drostreptomycin. The strains did notproduce indole, were methyl red nega­tive and citrate positive, did not pro­duce urease, and grew in 7-10 percentNaC!. Phenon E was identified as aBacillus sp.

The two strains, 15 and 27 (Phenon F)were identified as A cinetobacter sp.based on their inability to utilize thecarbohydrates tested, their oxidasenegativity, and catalase positivity.

The distinguishing features of Phe­non G (strains 21 and 22) which clus­tered at 68 percent S were variable gramstain, rod shape and yellow colonies.The strains fermented glucose and su­crose, but did not reduce nitrates )lorhydrolyze casein. They were oxidasepositive, sensitive to dihydrostrep­tomycin, indole and methyl red nega­tive, citrate positive, urease negativeand grew in the presence of 0-10 per­cent NaCI. Phenon G was identified asa Flavobacterium sp.

Phenons H and I were not identified.They were motile, gram-negative rodswhich produced yellow to white col­onies on MSYE agar at 15°_37°C and atpH 7.0-9.0. Strains in Phenons H and Ifermented glucose and sucrose. Theother characteristics tested variedamong the strains of the two groups.

In summary, the isolates from thehemolymph of the blue crab belonged tothe genera Vibrio, A cinetobacter,Flavobacterium, and Bacillus. Thelargest number of isolates, 38 of the 49isolates examined, were identified asVibrio spp.

A taxonomic distribution by source

of the animals proved to be interesting.The holding tank isolates were allVibrio spp., including phenons B, C(subgroups I, II and III), and O. Thefish market isolates were Vibrio spp.,including the B, C, and 0 phenons.However, representatives of the othergenera were also isolated from the fishmarket samples. Rhode River isolateswere all identified as Vibrio spp. (Phe­non C, subgroups I and II). TheMarumsco Bar isolates included phe­nons A, C, 0, and H (Vibrio, Bacillus,and unidentified strains). The FranklinCity isolates were predominantly ofPhenon C (subgroups I and II) withsome Band 0 phenons also observed tocomprise the flora. Thus, the commonfeature of the crab hemolymph was thepreponderance of Vibrio spp., particu­larly Phenon C vibrios which were pres­ent in crab hemolymph from all loca­tions. A significant difference observedbetween the Marumsco Bar and fishmarket crabs and crabs from other loca­tions in Chesapeake Bay was the pres­ence of Bacillus, Acinetobacter,Flavobacterium, and other unidentifiedbacteria in the hemolymph.

The analysis was carried further inthat the data for the 49 strains of thisstudy were combined with data ob­tained from 181 other isolates fromChesapeake Bay blue crabs (unpub­lished data). The data for the 230 strainswere subjected to computer analysisand the results showed 26 clusters at5;361 percent with the largest clustercontaining 68 strains. The 49 strainsfrom the study reported here showed 22strains clustered in the large, 68 strainphenon, identified as the genus Vibrio.

Thus, the results of the extended tax­onomic analysis substantiates the ob­servation that the predominant bacteriain the hemolymph of blue crabs areVibrio spp.

An additional comparison was madeof the data for the 49 crab isolates withdata for 161 strains isolated from theEastern oyster, Crassostrea virginica,which is found in Chesapeake Bay (un­published data). The entire set of datafor the 210 strains was analyzed bycomputer and it was found that the crabisolates, for the most part, did not clus­ter with and were distinct from the oys­ter strain clusters. Seven of the crabstrains did cluster with oyster strains at5 ;361 percent. These seven strainswere largely of one phenon, Phenon 0,

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members of which did not predominatecrab hemolymph. The results suggestthat the Phenon 0 Vibrio is more com­mon in oysters than in crabs, with thephenon C Vibrio being more common incrabs. Further support for this observa­tion can be drawn from the observationsof Lovelace et al. (1968) who did a bac­teriological analysis ofoysters collectedfrom the Marumsco Bar and EasternBay areas ofChesapeake Bay and founda predominance of Vibrio, Pseudomon­as, Achromobacter spp., in that order,with a smaller percentage of Coryne­bacterium, Cytophaga-Flavobac­terium, Micrococcus-Bacillus, andenteric species. Pseudomonas, A chro­mobacter and Corynebacterium spp.were not isolated from crab hemolymphin this study and only a few Flavobac­terium, Bacillus, and other unidenti­fied species were noted. However, thenumber of strains included in this studywas relatively small. Nevertheless, theindication is that the composition of themicrobial flora of the blue crab does notreflect geographical differences sostrongly nor is so varied as that of theoyster (Lovelace et aI., 1968).

In summary, the total number of via­ble, aerobic, heterotrophic bacteriafound in hemolymph of healthy bluecrabs is large and variable. Analysis oftaxonomic data by computer revealedthe predominant genus present in thehemolymph to be Vibrio, with membersof Bacillus, Acinetobacter andFlavobacterium present in much small­er numbers. Clearly the hemolymph ofmost healthy blue crabs is not sterile.

ACKNOWLEDGMENTS

This study was supported by SeaGrant Project #04-3-158-7, NationalOceanic and Atmospheric Administra­tion, and Grant #GB-35261 X from theNational Science Foundation. The au­thors wish to acknowledge the helpfuladvice and kind cooperation of AaronRosenfield, National Marine FisheriesService, NOAA, Oxford, Md.

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Couch, J. A. 1967. A new species ofLagenophrys (Ciliata: Peritrichida Lageno­phryidae) from a marine crab, Cal/inectessapidus. Trans. Am. Microsc. Soc. 86:204-211.

Fishbein, M., I. J. Mehlman, and J. Pitcher.1970. Isolation of Vibrio porahaemolyticusfrom the processed meat ofChesapeake Bay bluecrabs. Appl. Microbiol. 20: 176-178.

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Kovacs, N. 1956. Identification of Pseudomonaspyocyanea by the oxidase reaction. Nature(Lond.) 178:703.

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Loaharanu, P., and A. Lopez. 1970. Bacte­riological and shelf-life characteristics of canned,pasteurized crab cake mix. Appl. Microbiol.19:734-741.

Lovelace, T. E., H. TUbiash, and R. R. Colwell.1968. Quantitative and qualitative commensalbacterial "ora of Crassostrea virginica inChesapeake Bay. Proc. Natl. Shellfish. Assoc.58:82-87.

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Pfister, R. M., and P. R. Burkholder.1%5. Numerical taxonomy of some bacteriaisolated from antarctic and tropical seawaters. J.Bacteriol. 90:863-872.

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Sawyer, T. K. 1%9. Preliminary study on theepizootiology and host-parasite relationship ofParamoeba sp. in the blue crab, Callinectessapidus. Proc. Natl. Shellfish. Assoc. 59:6Q..64.

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Silvestri, L, M. Turri, L R. Hill, and E. Gilardi.1962. A quantitative approach to the systemat­ics ofactinomycetes based on overall similarity.In Microbial Classification. Twelfth Symp. Soc.Gen. Microbiol. 12:333-360.

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MFR Paper 1144. From Marine Fisherie,> Review, Vol. 37, Nos. 5~,

May.,}une 1975. Copies of this paper, in limited numbers, are availablefrom 083, Technical Information Division, Environmental Science Infor­mation Center, NOAA, Washington, DC 20235. Copies of Marine FisheriesReview are available from the Superintendent of Documents, U.S. Gov­ernment Printing Office, WaShington, DC 20402 for $1.10 each.

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