Vol. 37, No.4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1979, p. 704-7140099-2240/79/04-0704/11$02.00/0

Comparative Study of the Aerobic, Heterotrophic BacterialFlora of Chesapeake Bay and Tokyo Bay

B. AUSTIN,' S. GARGES,' B. CONRAD,' E. E. HARDING,' R. R. COLWELL,`* U. SIMIDU,2AND N. TAGA2

Department ofMicrobiology, University ofMaryland, College Park, Maryland 207421 and Ocean ResearchInstitute, University of Tokyo, Nakano, Tokyo 164, Japan2

Received for publication 24 January 1979

A comparative study of the bacterial flora of the water of Chesapeake Bay andTokyo Bay was undertaken to assess similarities and differences between theautochthonous flora of the two geographical sites and to test the hypothesis that,given similarities in environmental parameters, similar bacterial populations willbe found, despite extreme geographic distance between locations. A total of 195aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and TokyoBay water were examined for 115 biochemical, cultural, morphological, nutri-tional, and physiological characters. The data were analyzed by the methods ofnumerical taxonomy. From sorted similarity matrices, 77% of the isolates couldbe grouped into 30 phena and presumptively identified as Acinetobacter-Mor-axella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio andAcinetobacter species were found to be common in the estuarine waters ofChesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predomi-nated in Tokyo Bay waters, at the sites sampled in the study.

With the increased interest in the microbialecology of aquatic environments in recent years,it is important to have an understanding of thenatural, or autochthonous, microbial flora, notonly in terms of biomass and potential activity,but also community structure and species com-position. Since the bacteria are well-knownagents of mineralization and transformation oforganic and inorganic matter in bays and estu-aries, it was considered useful to determinewhether bacteria in the water column in two ofthe major bays of the world supported a similarbacterial flora.Under the auspices of a cooperative research

program between the University of Marylandand the University of Tokyo, a study was initi-ated in 1974 in which water samples were col-lected using standard methods at sites selectedfor similarity in environmental parameters, andin which the methods of bacteriological analysiswere rigidly conforming to permit minimizationof variation ascribable to other than naturalfloristic differences.The pure cultures obtained in the two parallel

studies were analyzed by previously agreed-uponprocedures in the two laboratories, with ex-change of reference cultures, as well as freshisolates, so that internal quality control could bemaintained in the analyses.Although there has been rapid improvement

in the taxonomy of aquatic bacteria since theadvent of numerical taxonomy and subsequentpioneering studies of Pfister and Burkholder (31)and Quigley and Colwell (34) and the morerecent work of Bauman and associates (3, 4),Delabr6 et al. (12), and Reichelt and Baumann(35), this information has been of limited valuebecause of the relatively few organisms exam-ined, the restricted number of tests used in iden-tification of isolates, and the limited geographi-cal distribution of the organisms studied. Pfisterand Burkholder (31) attempted to compare thebacterial microflora of tropical and antarctic sea-water, a type of broadly defined study that isessential for proper assessment of the distribu-tion of bacteria in the marine environment. Bau-mann et al. (3, 4) restricted their studies to a setof selected marine isolates, for the most partfrom culture collections of various investigators,although some fresh isolates were included intheir analyses.

In the study described here, aerobic, hetero-trophic, estuarine bacteria representative of themicroflora of Chesapeake Bay and Tokyo Baywaters were compared by numerical taxonomyand molecular genetic methods, providing newinformation concerning the species compositionof microbial communities present in the watercolumn at selected sites in two major estuariesof the world.

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DISTRIBUTION OF ESTUARINE BACTERIA 705

MATERIALS AND METHODS

Isolation and maintenance of strains. Stationswere selected from track lines routinely surveyed bythe University of Maryland in Chesapeake Bay andby the Ocean Research Institute, University of Tokyo.The stations selected were the two that were the mostsimilar in physical and chemical characteristics. Selec-tion was on the basis of information available to therespective laboratories for the stations over the past15 years. Field studies designed specifically to selectlocations with the same salinity, temperature, etc.,would have been preferable, but neither time nor fundspermitted such an exhaustive search for sites exhibit-ing precisely identical parameters. In any event, an

estuarine system is dynamic, and salinity, tempera-ture, and other parameters are not constant through-out the year and from year to year. The stationsselected, on average, were the most similar, over time,based on respective laboratory records. Thus, the lo-cations, time of year, salinity, depth, amount of pol-lution, tidal cycle, time of day, etc., were carefullyconsidered, and these factors were as closely similaras circumstances permitted.Water samples were collected on 19 June 1975 from

Cape Charles in Chesapeake Bay and on 4 July 1975from Tokyo Bay during cruises aboard research vesselsthat had been scheduled a year in advance, the datesbeing the closest in time that the respective schedulespermitted. Cape Charles is a high-salinity station inChesapeake Bay, with a surface-to-sediment depth of29 m, situated approximately 32 km north of Norfolk,Va. and 26 km northeast of the James River. Thesampling station in Tokyo Bay, Japan, with a depth of17 m, is situated 2 km west of the Obitsu River.Physical and chemical characteristics of the stationsin Chesapeake Bay and Tokyo Bay are listed in Table1. Water samples were collected aseptically, using a

Niskin Bag Sampler (General Oceanics Inc., Miami,Fla.), from approximately 2 m above the underlyingsediment at Cape Charles, since the Chesapeake Bayis a typical "salt water wedge" estuary and the mostsaline water was sought, and from 1 m below thesurface of the water in Tokyo Bay. Triplicate Niskincasts were made, each collecting 2 liters of water toensure statistical validity of the sampling procedure.

Serial dilutions to 10' of the water samples were

prepared immediately after collection, using 9-ml vol-

umes of sterile marine salts solution (0.7% [wt/vol]MgSO4.7H20, 2.38% [wt/vol] NaCl, and 0.07% [wt/vol] KCI; pH 7.0), the composition of which had beenagreed upon by the two laboratory groups. For esti-mation of total counts of Chesapeake Bay water, 0.1-ml aliquots were pipetted onto Simidu medium (41),thiosulfate citrate bile salts agar (TCBS; BioQuest,Cockeysville, Md.), marine 2216 agar (50; Difco), chitinagar, and fish protein medium (0.1% [wt/vol] XM-1fish protein hydrolysate [Zapata Haynie Corp., Balti-more, Md.], 0.1% [wt/vol] Difco yeast extract, 1.5%[wt/vol] Difco agar, and 1 liter of marine salts solu-tion). The Tokyo Bay water samples were plated on

all of the above except the fish protein medium whichwas included for comparative purposes and comprisedpart of another study carried out by the University ofMaryland laboratory. All plates were spread using a

sterile glass spreader. Three replicates of each dilutionwere plated, and inoculated plates were incubated at25°C, approximating the in situ temperature, for 14days.

Cultures were randomly selected from platings ofeach of the three water samples on the media used inthe study. Thus, from each of the water samples fromthe three separate casts made at the sampling site,approximately the same number of cultures were ran-

domly picked from plates of each of the media em-ployed and from the same dilution, containing between30 and 300 colonies. The cultures were subsequentlypurified, after incubation at 25°C, by streaking andrestreaking three times on plates of 2216 agar. Heat-fixed smears from 24-h cultures were stained usingHucker's modification of the Gram stain (16) andexamined microscopically. When the cultures were

considered to be pure, they were inoculated ontoslopes of marine 2216 agar. Stock cultures were main-tained under sterile mineral oil on 2216 agar slopes at25°C and subcultured every 6 to 8 weeks. After all ofthe isolation and purification steps had been followed,a total of 195 isolates, approximately equally repre-senting the Japanese and U.S. samples, were examinedfor the biochemical, cultural, morphological, nutri-tional, and physiological characteristics included inthe analyses.

Reference strains. In addition to the fresh isolatesfrom Chesapeake Bay and Tokyo Bay, 18 referencecultures were also included in the study (Table 2).

Characterization of the strains. Each strain was

TABLE 1. Physical, chemical, and microbiological characteristics of the sampling stations

Transpar- Total counts'Depth of Dissolved ency of

Station location Sample water Salinity oxygen water, Temp Marine Fishtype sample M%O (mg/ measured (0C) Marin Simdu FCSprtish Chitin

(m) liter) from sur- agar medium medium agarface(in)agrmeim ga

Cape Charles, Water 27 25.8 7.4 3.3 25.7 2.0 x 104 8.4 x 101 6.3 x 102 4.2 x 103 8.3 x 102ChesapeakeBay

2 km west of Ob- Water 11 30.1 7.5 2.2 21.3 9.1 X 104 3.3 x 10' 1.8 x 10' _b 6.7 x 101itsu River, To-kyo Bay

Colony-forming units, i.e., total viable, aerobic, heterotrophic bacterial counts on the respective media employed.bNot done.

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TABLE 2. Reference cultures included in the numerical taxonomy analysesLaboratory Culture

Reference culture reference collection Comment/isolated fromno. no.

Acinetobacter lwoffia R12 ATCC 15309Acinetobacter iwoffi R13 Univ. of Maryland Culture

CollectionAeromonas salmonicidaa R8 ATCC 14174 Diseased brook troutArthrobacter crystallopoietes R6 ATCC 15481 SoilArthrobacter marinus Rl ATCC 25374 Renamed Pseudomonas marina a;

seawaterCorynebacterium poinsettiae R9 ATCC 9069Erwinia herbicola R18 ATCC 12887Pseudomonas aureofaciens' R24 ATCC 13985 River clayPseudomonas cepacia R25 ATCC 17759 Renamed Pseudomonas multivor-

ansa; forest soilPseudomonas maltophiliaa R20 ATCC 13637 Oropharynx of patient with mouth

cancerPseudomonas mendocina' R26 ATCC 25411 SoilPseudomonas putida" R7 ATCC 12633 Degrades aromatic acidPseudomonas stutzeria R27 ATCC 17588 Spinal fluidStaphylococcus saprophyticus" R17 ATCC 15305 UrineVibrio anguillarum R15 ATCC 14181Vibrio anguillarum" R14 ATCC 19264 Ulcerous lesion in codVibrio parahaemolyticus' R16 ATCC 17802 Food poisoning, JapanXanthomonas begoniae R21 ATCC 11725 Semituberous Begonia spp.

a Denotes type strain, obtained from the American Type Culture Collection (Rockville, Md.).

examined for a total of 115 unit characters. Wheneverpossible, marine 2216 agar was used as the basal me-dium, and, in all cases, media were prepared usingmarine salts solution as the diluent. Unless otherwisestated, all inoculated media were incubated at 25°Cfor 14 days. To assess possible test error, 20 randomlychosen strains were examined in duplicate; otherwise,tests were repeated only in the event of inconclusiveresults. For inter-laboratory quality control, a set offresh isolates was exchanged and included along withthe reference cultures in both laboratory testing re-gimes. Most ofthe tests have been described elsewhere(9, 14, 19, 25, 40); details of the remainder are givenbelow.

Utilization of substrates as the sole source ofcarbon for energy and growth. Carbon utilizationtests were carried out using the medium devised byStevenson (47). The carbon compounds included a-DL-alanine, L-arginine hydrochloride, asparagine, L-histidine hydrochloride, L-leucine, L-lysine, L-methio-nine, DL-phenylalanine, L-proline, sodium acetate, so-dium adipate, sodium citrate, sodium formate, sodiumgluconate, sodium glutamate, sodium glutarate, so-dium lactate, sodium malonate, sodium oxalate, so-dium tartrate, and L-threonine. These were sterilizedas 20% (wt/vol) solutions by tyndallizing for 1 h on 3consecutive days at 100°C, or by filtration, and addedto the supporting medium to give a final concentrationof 0.2% (wt/vol). This medium was dispensed intodivided replidishes (44) and inoculated by means of amultipoint inoculator (23). After incubation, the me-dium was examined visually at 14 days, recording thepresence or absence of growth.

Determination of the G+C content of DNA.The guanine plus cytosine content (G+C content,moles percent) of purified deoxyribonucleic acid(DNA), obtained by the method of Marmur (26), was

determined from the thermal denaturation tempera-ture (Tm) of the DNA using a Gilford 2400-S recordingspectrophotometer at 260 nm (27). The DNA prepa-rations were dialyzed to lx SSC (0.15 M NaCl, 0.015M sodium citrate) and heated at 0.5°C min-', duringwhich the optical density was monitored at 260 nm.Molar percentage of G+C was calculated from the Tmby the equation of De Ley (13).Coding of data. The characters were coded 1 for

positive or present, 0 for negative or absent, and 9 fornoncomparable or not applicable. The final n x tmatrix contained 213 strains, including referencestrains, and 115 characters recorded for all strains.Computer analyses. The data were analyzed us-

ing SsM, the simple matching coefficient (45), whichincluded positive and negative matches, and the Jac-card coefficient, Sj, which excludes negative matches(43). Clustering was by unweighted average linkage(45), and sorted similarities and dendrograms wereconstructed. The hypothetical median organism (22)for each cluster was also calculated. Programs em-ployed included UMDTAXON3 and IGPS3 programpackages available on the University of MarylandUNIVAC 1108 computer.

RESULTSTotal viable bacterial counts. Four media

were employed in the Chesapeake Bay-TokyoBay comparative study: marine 2216 agar (50);a medium designed to enhance growth of marinevibrios (41); chitin agar; and TCBS, originallydeveloped for isolation of Vibrio cholerae andsubsequently employed for isolation of Vibrioparahaemolyticus. Counts obtained on these

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media are given in Table 1. Analysis of theresults obtained for each of the three castsshowed no statistically significant differences intotal viable aerobic, heterotrophic bacterialcounts. That is, there was no significant differ-ence in counts for the three water samples thatwere collected at the same time to test for sam-ple variation. However, it can be seen that highercounts on all media except 2216 agar were ob-tained for the Chesapeake Bay water samples.The count obtained on TCBS agar was 10-foldgreater for the Chesapeake Bay water sampleand 2-fold greater for the Simidu marine vibriomedium. This difference is important in view ofthe results (see below) showing a greater pre-ponderance of vibrios in Chesapeake Bay watersamples.Clustering of the strains. Results of analy-

ses employing the SSM and SJ coefficients in-cluded 154 of the environmental isolates, repre-senting 77% of the total set, and the six referencecultures of Acinetobacter Iwoffi, Pseudomonasmaltophilia, Pseudomonas marina, Vibrio an-guillarum (two strains), and V. parahaemolyti-cus in 30 clusters at 270% similarity (S). Results

PERCENTAGE SIMILARITY

of the analysis using the SJ coefficient are givenin Fig. 1. Four clusters (phena 1, 2, 21, and 25)comprised 65 strains, including the referenceculture of V. parahaemolyticus, with the re-maining 26 groups containing between 2 and 9strains each.Identification and description of the iso-

lates. Presumptive identification of the 30 clus-ters was accomplished either by inclusion of areference culture within the cluster or by con-sultation of diagnostic keys in Bergey's ManualofDeterminative Bacteriology (8) and specialistkeys and tables including those of Shewan et al.(38), Stanier et al. (46), Bousfield (7), Jones (17),Bianchi (M. Bianchi, these de doctorat des sci-ences naturelles, Universite d'Aix-Marseille,1976), Otto and Pickett (29), and Pagel andSeyfried (30). Six generic groups comprised themajor taxa represented, including Vibrio spp.,Acinetobacter-Moraxella spp., Pseudomonasspp., Caulobacter spp., coryneforms, and group-ings of as yet unidentified gram-negative rods(see Fig. 1). A summary of selected characteris-tics of the six generic groupings is given in Table3.

NO. OFPHENON STRAINS SOURCE IDENTITY

12 C B

2 10 C B3 6 CB4 2 CB5 2 CB6 2 CB7 3 CB8 5 CB9 9 CB10 4 C BI1 7 C B12 3 CB13 3 CB14 2 C,B15 5 C.B/TB.16 5 CB/TB17 2 TB.18 4 TB19 2 TB.20 9 TB

21 22 TB

22 2 TB23 2 TB24 3 TB

25 21 TB

26 5 TB27 2 TB28 2 TB29 4 TB.30 6 TB

Vibrio porohoerno/yt/cus

V f,scheri

V ongul//orumVibrio sp.Vibr,o spV,br,o spAclnetobocter co/coocet/cusGram negotive rodsGram negative rodsGram negative rodsP morinoP mo/ltophi/ioGrom negative rodsCoryneforms"Gram negative rodsGram negative rodsAclnetobocter - Aoroxe/lo sp.Aclnetobocter - Moroxe/lo spPseudolonos sp.Grom negative rods

Coulobocter sp.Gram negotive rodsGrom negative rodsGrom negative rods

Gram negotive rods

Grom negotive rodsP pu/bdoGram negative rodsGram negative rodsGram negotive rods

FIG. 1. Simplified dendrogram prepared with the SJ coefficient and unweighted average linkage clusteringtechnique. C.B., Chesapeake Bay; T.B., Tokyo Bay.

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708 AUSTIN ET AL. APPL. ENVIRON. MICROBIOL.

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Phena 1, 3, 7, 11, and 12 were identified as V.parahaemolyticus, V. anguillarum, Acineto-bacter calcoaceticus, P. marina, and P. malto-philia, respectively, by the grouping of the ref-erence strains with the isolates. In general, thecharacteristics of the Vibrio strains matched thespecies descriptions in Bergey's Manual of De-terminative Bacteriology (8) and those given byBianchi (these de doctorat), although it wasobserved that the strains identified as V. an-guillarum were negative for the indole reaction.Although the reference strain of A. Iwoffi wasrecovered in phenon 7, A. Iwoffi is now regardedas a synonym of A. calcoaceticus (8, 30). Thecharacteristics of A. calcoaceticus (phenon 7)were in close agreement with the descriptionprovided in Bergey's Manual of DeterminativeBacteriology (8).Phenon 2 was identified as Vibrio fischeri by

micromorphology, biochemical, degradative,and physiological reactions and overall DNAcontent of 44.8% G+C (Table 3). In contrast tothe species description, none of the strains ex-hibited luminescence (39). However, lumines-cence is a characteristic that is highly dependenton medium, temperature, and other variables."Dark" strains are very commonly encounteredamong luminescent species (8, 39).Phena 4, 5, and 6 are classified as Vibrio spp.,

viz., gram-negative, curved, fernentative, oxi-dase- and catalase-positive rods, possessing over-all DNA base compositions of 46 and 49% G+C.The strains were unable to grow at temperaturesof 37°C or above. The strains isolated in thisstudy and identified as Vibrio spp. did not de-grade casein or Tween 80 or grow at tempera-tures below 100C. In contrast to the genus de-scription (39), it should be noted that none ofthe strains demonstrated a marked sensitivity topteridine. Thus, phena 4, 5, and 6 were notidentified as any of the species described andnamed in the literature and should be considerednew species.Phena 10, 16, 20, 25, 29, and 30 possessed some

ofthe characteristics ofAcinetobacterspp. How-ever, the range of overall DNA base composi-tion, 50 to 66% G+C, is higher than the range ofvalues usually associated with this genus, i.e., 40to 47% (21), and closer to that of Pseudomonas.The oxidase-negative reaction, for the present,excludes these organisms from the genus Pseu-domonas. Therefore, these phena are regardedas unidentified until further work can be doneon the taxonomy of these organisms.Phena 17 and 18 were tentatively identified

as belonging to the Acinetobacter-Moraxellagroup. Although all strains possessed the generalcharacteristics of Acinetobacter-Moraxella,

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APPL. ENVIRON. MICROBIOL.

they showed specific properties of Moraxella,i.e., they were oxidase positive and sensitive topenicillin. It has been suggested that oxidase-negative strains should be reclassified as Acine-tobacter (5); this would exclude the strains clus-tered in these phena. However, with the excep-tion of Moraxella phenylpyruvica, Moraxellaspecies, including M. lacunata, M. bovis, M.nonliquefaciens, and M. osloensis, are normallyassociated with pathological conditions and havenot been reported to occur in the aquatic envi-ronment. More importantly, the description ofthe strains isolated in this study could not bematched with any of the described species ofMoraxella or, for that matter, of Acinetobacter.Thus, it was decided to regard the phena asgroups intermediate between Acinetobacter andMoraxella until further studies on the classifi-cation of these organisms can be completed. Theoverall DNA base composition was not deter-mined for these organisms despite several at-tempts, because of problems with harvestingcells and extracting the DNA. It is possible thatthese two phena are similar to phena 10, 16, 19,20, 22, 23, 24, 25, 26, 28, 29, and 30, which alsoexhibited characteristics of Acinetobacter-Mor-axella but could not be classified in either ofthese groups due to the high G+C content of theDNA, the latter being more representative ofPseudomonas spp. Some of the clusters of gram-negative rods that remain unidentified may alsobe Pseudomonas or Alteromonas spp. However,they are indeed new species, if not new genera,and further, more precise study of the classifi-cation, identification, and nomenclature of thesestrains is in progress.Phenon 14 was identified as coryneform bac-

teria largely on the basis of the micromorphol-ogy of the strains. Unfortunately, the organismscould not be identified further, even after con-sultation of the comprehensive descriptions ofBousfield (7) and Jones (17), and may also rep-resent a new species.Phena 8, 9, 13, and 15 were not related to

bacterial species described in the available lit-erature, i.e., in diagnostic keys and/or in Ber-gey's Manual of Determinative Bacteriology(8), and will require further analysis for preciseclassification.

Strains clustered in phenon 21 produced stalksand formed "rosettes," hence were identified asCaulobacter spp. The strains were short, oxida-tive, gram-negative rods when observed in wetmounts under phase-contrast microscopy. Theycould not be identified to the species level andrepresent, in our view, a new species of Caulo-bacter.Phenon 27 was identified as Pseudomonas

putida (8, 11). Diagnostic features included

gram-negative, oxidative, oxidase-positive rodsthat were fluorescent under ultraviolet light,produced arginine dihydrolase but not lysinedecarboxylase, and utilized L-arginiie hydro-chloride and sodium citrate as the sole source ofcarbon for energy and growth, but did not hy-drolyze casein gelatin, starch, or Tween 80. Thestrains were nonmotile when observed in wetmount under phase-contrast microscopy. DNA/DNA hybridization studies of these strains withreference strains are in progress.Test error. Twenty strains examined in du-

plicate were tested on different occasions, butthe same methods were used throughout. Themajority of tests, i.e., a total of 85, gave identicalresults; these included the Gram staining reac-tion, colonial morphology, and the majority ofbiochemical degradative and nutritional char-acters. The error was calculated to be approxi-mately 2%, most of which could be attributed toa few characters, notably the pleomorphic na-ture of many of the isolates and the H25, methylred, oxidase, Voges-Proskauer, and gelatin tests,which either gave weakly positive, equivocal re-sults or were, in the case of the biochemical testresults, contradictory on retesting. The H2S testcan be variable, depending on the presence orloss of plasmids (variability due to plasmids hasbeen noted in earlier studies [S. A. Orndorff, B.Austin, L. A. McNicol, and R. R. Colwell, sub-mitted for publication]), and, in the case of themethyl red, Voges-Proskauer, and gelatin tests,there is a strain variability in the intensity of thereactions that has been observed in our ownwork with strains freshly isolated from theaquatic environment.

Variation in test results between laboratorieswas not significant and therefore permitted com-bining the data from both laboratories for acomprehensive analysis.

Selectivity of the media employed. OnlyVibrio spp. were isolated on TCBS agar, includ-ing V. parahaemolyticus, V. fischeri, and uni-dentified Vibrio spp. The Simidu medium wasalso highly selective for Vibrio spp., with V.parahaemolyticus, V. fischeri, and unidentifiedVibrio spp. being isolated. However, two iso-lates, most probably of the Acinetobacter-Mor-axella groups, were also isolated from the Sim-idu medium. The 2216 medium yielded a varietyof organisms, selecting neither for nor againstVibrio spp. It should be pointed out that the fishprotein agar was by far the most useful mediumin providing growth for a very wide variety ofbacterial genera and species, including Vibrio,Moraxella, Acinetobacter, and Pseudomonas.

DISCUSSIONThe objective of this study was to assess sim-

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ilarities and differences between microflora oftwo environmentally similar sites at widely sep-arated geographical locations. That is, the ques-tion to be answered is whether the water ofestuaries in different parts of the world willsupport a relatively similar species compositionin the microbial community structure. In spiteof the usual restraints of time, budget, cruiseschedules, and related logistical problems, it waspossible to select two sites for comparison thatwere reasonably similar and for which significantdata had been gathered in other studies. Cruiseschedules for the research vessels available tothe cooperating laboratories fortuitously over-lapped so that samples could be collected at thesame time of year. It is recognized that a moreprecise matching of sites could be achieved, butthe matching of sites was the best possible con-sidering the logistics of the situation. Further-more, it is also recognized that biomass mea-surements should have been made, i.e., directmeasurement of the total bacterial populationusing epifluorescence, Limulus lysate assay,adenosine triphosphate, or others of the morerecently developed methods for biomass esti-mation. Tests of potential activity, using 14Cheterotrophic uptake measurements, would alsobe useful. Nevertheless, the question was that ofcommunity structure and species composition,and for this reason the comparative study isconsidered to provide useful information.

It must also be recognized that the isolatesexamined in this study represent only that por-tion of the microbial community capable ofgrowth on the media employed. For example,for oligotrophs, low-nutrient media must be em-ployed for isolation and characterization (25).Nevertheless, for the purposes of this study,namely to compare microbial populations understandardized conditions of plating, media, etc.,this point does not negate the conclusions drawnfrom the results obtained.The results show that, from the counts ob-

tained on the media employed and given inTable 1 and from the numerical taxonomy anal-yses, the predominant aerobic, heterotrophicbacterial flora present in the water column ofChesapeake Bay water, at the site included inthis study, consisted of Vibrio, Acinetobacter,Pseudomonas, and coryneforms, whereas themicroflora of the water column in Tokyo Bay,at a comparable site, was predominately Acine-tobacter-Moraxella-like species, Caulobacterspp., and groups of gram-negative, rodlike bac-teria resembling Pseudomonas spp. but suffi-ciently dissimilar to be considered new species.Variation in species composition among the in-dividual water samples was not observed. Thusthe microflora of the two bodies of water appear

to be dissimilar at the sites from which samplesexamined in this study were collected. Vibriowas the numerically dominant genus in Chesa-peake Bay, regardless of the medium employed,a finding concurring with conclusions of Love-lace et al. (24), Kaneko and Colwell (18), andCook and Goldman (10). Vibrios were not pre-dominant in the microbial populations found inthe water of Tokyo Bay at the stations includedin the study. It has been suggested previously bySimidu et al. (42) that the absence of vibrios inTokyo Bay is caused by antagonistic interactionsbetween Vibrio spp. and phytoplankton and byorganic nutrients in Tokyo Bay. The presence ofV. parahaemolyticus in Chesapeake Bay isknown to be inversely related to salinity, in thatextremes of salinity affect viability (18, 36), andwarmer water temperatures are favorable fortheir growth and distribution during the warmerseasons of the year (18). In this connection, thewater at Cape Charles demonstrated compara-tively high salinity and temperature, and rela-tively few V. parahaemolyticus were isolated.Acinetobacter spp., which accounted for the

third most predominant taxon at Cape Charles,were tentatively identified as the predominatingorganisms in Tokyo Bay. Large numbers of Aci-netobacter have been isolated from the aquaticenvironment by other investigators, notably inLake Ontario (37) and Lake Superior (6). How-ever, organisms placed in this genus no doubthave been misclassified because of the relativelyloose description available for the genus. Thus,it can be concluded that Acinetobacter isolatedfrom the aquatic environment are not suffi-ciently well studied to be readily separated fromthe other gram-negative, nonmotile, nonpig-mented bacteria, a situation also true for Fla-vobacterium (14). Many of the strains recoveredfrom Tokyo Bay possessed characteristics moreappropriate for groups intermediate betweenAcinetobacter and Moraxella and, in somecases, related to Pseudomonas.Pseudomonas spp., which have been isolated

from a wide range of habitats within the aquaticenvironment (3, 10, 12, 31, 34), were recoveredin moderately large numbers from ChesapeakeBay and also from Tokyo Bay. Several of theclusters of gram-negative, rodlike bacteria werevery similar to Pseudomonas, except for a ten-dency to pleomorphism and differences in cer-tain characteristics, such as sensitivity to peni-cillin, and may ultimately prove to be new spe-cies of Pseudomonas. It would be very useful toassess the ability of these strains to degradepetroleum and to measure their resistance toheavy metals and other pollutants, since thepseudomonads, together with the coryneforms(which were surprisingly absent from the Tokyo

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Bay water samples), are associated with petro-leum degradation (1, 2) and mobilization ofheavy metals (28). The coryneform strains re-covered in this study could not be identified tothe genus level, reflective of the complexity ofthe taxonomy of the coryneform group of bac-teria and uncertainty of the validity of the clas-sification of those coryneforms found in the ma-rine environment (7, 17, 49).

Caulobacter spp. generally occur in waterscontaining low concentrations of organic nutri-ents (32, 33). Slow-growing strains of stalkedbacteria, identified as Hyphomonas polymor-pha, have been isolated in large numbers inother, more polluted areas of Chesapeake Bay(25). The occurrence of stalked bacteria in theestuarine environment would suggest that suchorganisms may have important ecological func-tions, and further studies are in progress. Thelower Chesapeake Bay, at the station includedin this study, receives much less allochthonousinput than the upper Chesapeake Bay. It wouldappear that the differences in species composi-tion noted in this study provide an index ofpollution, since the Tokyo Bay station can beconsidered to be eutrophic and more polluted,in general, than the Chesapeake Bay.

Pfister and Burkholder (31) observed a degreeof specificity between tropical and antarctic wa-ters in the distribution of bacterial taxa in theaquatic environment. Recent work in our labo-ratory has demonstrated a geographical distri-bution among petroleum-degrading bacteria anda specificity of these bacteria for water or sedi-ment in polluted and unpolluted sites in Chesa-peake Bay (1, 2). However, the distribution wasnot as striking for those genera of bacteria ca-pable of degrading petroleum as were the differ-ences for the bacterial taxa comprising the mi-croflora of Chesapeake Bay and Tokyo Bay.An interesting observation arising from this

study is that the choice of medium will exert asignificant influence on the recovery of bacterialtaxa in the marine environment, an observationin agreement with earlier studies of Goulder (15)and Vaatiinen (48). In this study, fish proteinagar was used for comparative purposes only inthe Chesapeake Bay portion of the study. Al-though the counts on the fish protein agar wereone log less than on 2216 agar, the fish proteinagar medium permitted the initial isolation ofthe widest range of taxa, that is, a greater num-ber of genera and species than the more com-monly used marine 2216 agar of ZoBell (50).This observation merits further study. In com-parison, the TCBS medium was highly selectivefor vibrios, as was the Simidu medium, both ofwhich were designed for isolation of Vibrio spp.

After isolation and culture in the laboratory,

those strains isolated on media other than 2216agar were able, subsequently, to grow on thismedium, indicating an adaptation of the isolatesto the richer medium. In a previous study ofslow-growing oligotrophic bacteria recoveredfrom the estuarine environment (25), the com-position of the isolation medium was found toexert a significant effect upon the recovery ofbacterial taxa. In particular, it was shown thatStreptothrix and other sheathed bacteria couldbe recovered only on a medium containing avery low concentration of nutrients, consistingof Chesapeake Bay water to which agar wasadded.No variation in genera and species was noted

between casts. That is, each of the separate castsyielded the same representation of genera andspecies on each of the media, with significantdifferences noted only between media. TheTCBS and Simidu media were selective for Vi-brio spp., and the other media, i.e., chitin, 2216,and fish protein agar media, selected neither fornor against Vibrio spp. Of all the media, how-ever, the fish protein concentrate mediumyielded the widest variety of genera and speciesin the case of the Chesapeake Bay water sam-ples. Clearly, the effects of media compositionare important in the recovery of bacterial taxafrom the natural environment, and this phenom-enon should be carefully considered in futurestudies of microbial community structure.

In conclusion, intrinsic differences were ob-served in the microflora of Chesapeake Bay andTokyo Bay water in a given location and season.Space and time dependency of microbial com-munity structure, of course, is well recognized.Nevertheless, the results of this study indicatethat it cannot be assumed that estuaries willcontain microbial communities similarly com-prised of representative taxa, but, rather, itshould be recognized that geographical differ-ences may be strongly influential, with the typeand concentration of nutrients present, the in-digenous plankton populations, and the nature,source, quality, and quantity of allochthonousmaterial entering an estuary acting selectivelyon the microbial populations.

ACKNOWLEDGMEENTSThis work was supported by National Science Foundation

grants OIP 7417540 and BMS 72-02227-A03. The computertime for the project was supplied, in full, through the facilitiesof the Computer Science Center at the University of Mary-land. The Japanese cooperative research was supported bygrant no. 5R064 from the Japan Society for the Promotion ofScience.

The assistance of the captains and crews of the R/VRidgely Warfifld and of the R/V Tanseimaru is gratefullyacknowledged. The authors gratefully acknowledge the assist-ance of Jayne Carney and Gary Sayler in the initial stages ofthis project.

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LITERATURE CITED

1. Austin, B., R. R. Colwell, J. D. Walker, and J. J.Calomiris. 1977. Numerical taxonomy and ecology ofpetroleum-degrading bacteria. Appl. Environ. Micro-biol. 33:60-68.

2. Austin, B., R. R. Colweli J. D. Walker, and J. J.Calomiris. 1977. The application of numerical taxon-omy to the study of petroleum degrading bacteria iso-lated from the aquatic environment. Dev. Ind. Micro-

biol. 18:685695.3. Baumann, L, P. Baumann, M. Mandel, and R. D.

Allen. 1972. Taxonomy of aerobic marine eubacteria.J. Bacteriol. 110:402-429.

4. Baumann, P., L. Baumann, and M. Mandel. 1971.Taxonomy of marine bacteria: the genus Beneckea. J.Bacteriol. 107:268-293.

5. Baumann. P., AL Doudoroff, and R. Y. Stanier. 1968.A study of the Moraxella group. II. Oxidase-negativespecies (genus Acinetobacter). J. Bacteriol. 95:1520-1541.

6. Bennett, E. A. 1969. A study of the distribution of het-erotrophic bacteria in the Great Lakes. 1. The hetero-trophs of lake water. Bacteriology Branch, Division ofLaboratories, Ontario Water Resources Commission,Ontario, Canada.

7. Bousfield, I. J. 1972. A taxonomic study of some cory-

neform bacteria. J. Gen. Microbiol. 71:441-455.8. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-

gey's manual of determinative bacteriology, 8th ed.Williams & Wilkins Co., Baltimore.

9. Colwell, R. R., and W. J. Weibe. 1970. "Core" charac-teristics for use in classifying aerobic, heterotrophicbacteria by numerical taxonomy. Bull. Ga. Acad. Sci.18:165-185.

10. Cook, T. M., and C. K. Goldman. 1976. Bacteriology ofChesapeake Bay surface waters. Chesapeake Sci. 17:40-49.

11. Cowan, S. T. 1974. Cowan and Steel's manual for theidentification of medical bacteria. Cambridge Univer-sity Press, Cambridge.

12. Delabre, AL, A. Bianchi, and M. Veron. 1973. g#tudecritique de methodes de taxonomie numerique. Appli-cation a une classification de bacteries aquicoles. Ann.Microbiol. 124:489-506.

13. De Ley, J. 1970. Re-examination of the association be-tween melting point, buoyant density, and chemicalbase composition of deoxyribonucleic acid. J. Bacteriol.101:738-754.

14. Goodfellow, M., B. Austin, and C. H. Dickinson. 1976.Numerical taxonomy of some yellow pigmented bacte-ria isolated from plants. J. Gen. Microbiol. 97:219-233.

15. Goulder, R. 1976. Evaluation of media for counting viablebacteria in estuaries. J. Appl. Bacteriol. 41:351-355.

16. Hucker, G. J., and H. J. Conn. 1923. Methods of Gramstaining. In Technical Bulletin of the New York StateAgricultural Experiment Station, no. 93.

17. Jones, D. 1975. A numerical taxonomic study of coryne-forms and related organisms. J. Gen. Microbiol. 87:57-96.

18. Kaneko, T., and R. R. Colwell. 1974. Distribution ofVibrio parahaemolyticus and related organisms in theAtlantic Ocean off South Carolina and Georgia. Appl.Microbiol. 28:1009-1017.

19. King, E. O., M. K. Ward, and D. E. Raney. 1954. Twosimple media for the demonstration of pyocyanin andfluorescein. J. Lab. Clin. Med. 44:301-307.

20. Lautrop, H. 1974. Moraxella Lwoff 1939, 173 Nom. cons.

Opin. 41, Jud. Commn. 1971, 106, p. 433-436. In, R. E.Buchanan and N. E. Gibbons (ed.), Bergey's manual ofdeterminative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

21. Lautrop, H. 1974. Acinetobacter Brisou and Prevot 1954,

717, p. 436-438. In R. E. Buchanan and N. E. Gibbons(ed.), Bergey's manual of determinative bacteriology,8th ed. The Williams & Wilkins Co., Baltimore.

22. Liston, J., W. Weibe, and R. R. Colwell. 1963. Quan-titative approach to the study of bacterial species. J.Bacteriol. 85:1061-1070.

23. Lovelace, T. E., and R. R. Colwell. 1968. A multipointinoculator for petri dishes. Appl. Microbiol. 16:944-945.

24. Lovelace, T. E., H. Tubiash, and R. R. Colwell. 1968.Quantitative and qualitative commensal bacterial floraof Crassostrea virginica in Chesapeake Bay. Proc. Natl.Shellfish. Assoc. 58:82-87.

25. Mallory, L. M., B. Austin, and R. I. Colwell. 1977.Numerical taxonomy and ecology of oligotrophic bac-teria isolated from the estuarine environment. Can. J.Microbiol. 23:733-750.

26. Marmur, J. 1961. A procedure for the isolation of deoxy-ribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218.

27. Marmur, J., and P. Doty. 1962. Determination of thebase composition of deoxyribonucleic acid from its ther-mal denaturation temperature. J. Mol. Biol. 5:109-118.

28. Nelson, J. D., and R. R. Colwell. 1975. The ecology ofmercury resistant bacteria in Chesapeake Bay. Microb.Ecol. 1:191-218.

29. Otto, L A., and M. J. Pickett. 1976. Rapid method foridentification of gram-negative, nonfermentative bacilli.J. Clin. Microbiol. 3:566-575.

30. Pagel, J. E., and P. L. Seyfried. 1976. Numerical tax-onomy of aquatic Acinetobacter isolates. J. Gen. Micro-biol. 95:220-232.

31. Pfister, R. M., and P. R. Burkholder. 1965. Numericaltaxonomy of some bacteria isolated from antarctic andtropical seawaters. J. Bacteriol. 90:863-872.

32. Poindexter, J. S. 1964. Biological properties and classi-fication of the Caulobacter group. Bacteriol. Rev. 28:231-295.

33. Poindexter, J. S. 1974. Caulobacter Henrici and Johnson1935, 83, p. 153-155. In R. E. Buchanan and R. E.Gibbons (ed.), Bergey's manual of determinative bac-teriology, 8th ed. The Williams & Wilkins Co., Balti-more.

34. Quigley, M. M., and R. R. Colwell. 1968. Properties ofbacteria isolated from deep-sea sediments. J. Bacteriol.95:211-220.

35. Reichelt, J. L., and P. Baumann. 1973. Taxonomy ofthe marine luminous bacteria. Arch. Mikrobiol. 94:283-330.

36. Sayler, G. S., J. D. Nelson, A. Justice, and R. R.Colwell. 1976. Incidence of Salmonella spp., Clostrid-ium botulinum, and Vibrio parahaemolyticus in anestuary. Appl. Environ. Microbiol. 31:723-730.

37. Seyfried, P. L. 1973. Sampling bacteria in Lake Ontarioand the Toronto Harbour, In Proceedings of the 16thCongress of Great Lakes Research. p. 163-182. Inter-national Association for Great Lakes Research, AnnArbor, Mich.

38. Shewan, J. M., G. Hobbs, and W. Hodgkiss. 1960. Adeterminative scheme for the identification of certaingenera of Gram-negative bacteria, with special refer-ence to the Pseudomonadaceae. J. Appl. Bacteriol. 23:379-390.

39. Shewan, J. M., and M. Veron. 1974. Vibrio Pancini1854, 411, p. 340-345. In R. E. Buchanan and N. E.Gibbons (ed.), Bergey's manual of determinative bac-teriology, 8th ed. The Williams & Wilkins Co., Balti-more.

40. Sierra, G. 1957. A simple method for the detection oflipolytic activity of microorganisms and some observa-tions on the influence of the contact between cells andfatty substrates. Antonie van Leeuwenhoek J. Micro-biol. Serol. 23:15-22.

41. Simidu, U., and T. Kaneko. 1973. A numerical taxonomy

VOL. 37, 1979

on May 8, 2020 by guest

http://aem.asm

.org/D

ownloaded from

714 AUSTIN ET AL.

of Vibrio and Aeromonas from normal and diseasedmarine fish. Bull. Japanese Soc. Sci. Fisheries 39:689-703.

42. Simidu, U., E. Kaneko, and N. Taga. 1977. Microbio-logical studies of Tokyo Bay. Microb. Ecol. 3:173-191.

43. Sneath, P. H. A. 1957. The application of computers totaxonomy. J. Gen. Microbiol. 17:201-226.

44. Sneath, P. H. A., and M. Stevens. 1967. A divided petridish for use with multipoint inoculatorsa J. Appl. Bac-teriol. 30:495-497.

45. Sokal, R. R., and C. D. Michener. 1958. A statisticalmethod for evaluating systematic relationships. Univ.Kansa Sci. Bull. 38:1409-1438.

46. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff.

APPL. ENVIRON. MICROBIOL.

1966. The aerobic pseudomonads: a taxonomic study. J.Gen. Microbiol. 43:159-271.

47. Stevenson, I. L. 1967. Utilization of aromatic hydrocar-bons by Arthrobacter spp. Can. J. Microbiol. 13:205-211.

48. Vaitiinen, P. 1977. Effects of composition of substrateand inoculation technique on plate counts of bacteria inthe northern Baltic Sea. J. Appl. Bacteriol. 42:437-443.

49. Yamada, K., and K. Komagata. 1972. Taxonomic stud-ies on coryneform bacteria. V. Classification of coryne-form bacteria. J. Gen. Appl. Microbiol. 18:417431.

50. ZoBell, C. E. 1941. Studies on marine bacteria. 1. Thecultural requirements of heterotrophic aerobes. J. Mar.Res. 4:42-75.

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.org/D

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