APPLE MIcRosmowGY, June 1975, p. 819-825Copyright 0 1975 American Society for Microbiology

Vol. 29, No. 6Printed in U.SA.

Potential Pathogens in the Environment: Cultural Reactionsand Nucleic Acid Studies on Klebsiella pneumoniae from

Clinical and Environmental Sources'RAMON J. SEIDLER*, MARTIN D. KNITTEL, AND CAROL BROWN

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331,* and Pacific NorthwestEnvironmental Research Laboratory, U. S. Environmental Protection Agency, Corvallis, Oregon 97330

Received for publication 10 February 1975

The phenotypic and nucleic acid properties of Klebsiella pneumoniae havebeen studied on cultures obtained from six different habitats (humans, vegeta-bles, seeds, trees, rivers, and pulp mills). The 19 cultural reactions of 107 isolatesvaried significantly only in tryptophanase activity and dulcitol fermentation.The percentage of guanine plus cytosine base composition of 41 isolates variedfrom 53.9 to 59.2%. The range of percentage of guanine plus cytosine basecomposition for environmental klebsiellas was broader than that for the culturesof human origin. The range of deoxyribonucleic acid relative reassociation(homology) to the human K. pneumoniae reference strain extended from 5% to100% and the chromosome molecular weights ranged from 2,200 x 106 to 3,000 x106. The species of K. pneumoniae is thus molecularly more heterogeneous thanpreviously thought and most isolates of human, pulp mill, and river origin aregenetically indistinguishable. The presence of K. pneumoniae therefore repre-sents a deterioration of the microbiological quality of the environment andshould be considered of public health significance. At the present time the healthsignificance of the molecularly more divergent strains, primarily of vegetable andseed origin, their relationship to klebsiellas of human origin, or to other genera ofthe Enterobacteriaceae is unclear.

Klebsiella pneumoniae, members of the fam-ily Enterobacteriaceae, comprise a group ofnonmotile, nonsporeforming, lactose-ferment-ing, gram-negative rods. These bacteria areusually heavily encapsulated and are typicallyassociated with nosocomially acquired infec-tions of the genito-urinary, respiratory, andsepticemic foci in debilitated, chronically ill, orpostoperative situations (27). K. pneumoniae isalso found as the primary agent of coliformmastitis in dairy herds (3). Classically,Enterobacter (formerly Aerobacter) andKlebsiella have been difficult to distinguishbecause of many similar cultural reactions andthese two genera have not always been properlyidentified (20). As recommended by Hormaecheand Edwards (16), and subsequently demon-strated in other studies (3, 11), a few keycultural tests can be used to differentiate thetwo genera.

Several recent studies have demonstratedthat bacteria which conform to the culturaldefinition of K. pneumoniae can be isolatedfrom a variety of natural environments (1, 7, 10,18, 19, 24). These observations coincide with the

'Technical paper no. 3980, Oregon Agricultural Experi-ment Station.

increasing incidence of multiple antibiotic re-sistant nosocomially acquired K. pneumoniaeinfections in humans as well as infections indomestic and wild animals (5, 27, 28, 29).Because of the subtle phenotypic differencesbetween Enterobacter and Klebsiella and thepotential significance of high densities ofKlebsiella in the natural environment, we haveinitiated several studies on the molecular taxon-omy and ecology of the klebsiellas. In this reportthe molecular heterogeneity of the K.pneumoniae species is documented on the basisof nucleic acid studies.

MATERIALS AND METHODSBacterial cultures. K. pneumoniae cultures were

selected from among lactose-positive and oxidase-negative colonies appearing on m-endo LES agar(Difco) that had been streaked with aliquots fromsamples of industrial waste waters, river water, vege-tables and plant seeds (7). Several reference K.pneumoniae cultures isolated from human infectionswere obtained from the American Type CultureCollection, Rockville, Md., J. Matsen, University ofMinnesota School of Medicine, Minneapolis, andCenter for Disease Control, Atlanta, Ga. The culturesand their origin are listed in Table 1.

Identification of isolates and growth media.819

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SEIDLER, KNITrEL, AND BROWN

TABLE 1. Origin of strains used in nucleic acid analyses

Source or reference

Klebsiellapneumoniae1388213883804715574141143UOMS-1UOMS-5MUSC-20040120370450500840890930941021161181314064-194082-20A006051002R301R302R303R304R306R307V-10ibV-104aV-112V-233V-236V-244V-171V-151Ka-1M5A1

Enterobacter aerogenes13048

E. cloacae13047

ATCC; human serotype 64ATCC; neotype strain, human serotype 3ATCC; human serotype 1ATCC; leaf nodule endosymbiont, serotype 24J. Matsen, humanJ. Matsen, humanUniv. Oregon Medical School, humanUniv. Oregon Medical School, humanMedical University South Carolina, humanPulp mill; ammonium base sulfitePulp mill; ammonium base sulfitePulp mill; ammonium base sulfitePulp mill; ammonium base sulfitePulp mill; ammonium base sulfitePulp mill; KraftPulp mill; KraftPulp mill; KraftPulp mill; KraftPulp mill; KraftPulp mill; defiberizationPulp mill; defiberization

Living fir tree (1)Living fir tree (1)RiverRiverRiverRiver downstream from pulp mill, serotype 64 (7)River downstream from pulp mill, serotype 15 (7)River downstream from pulp millRiver downstream from pulp mill, serotype 47 (7)River downstream from pulp millRiver downstream from pulp millPotatoMushroom, serotype related to 6 and 7 (7)Potato, serotype 26 (7)Radish, serotype 11 (7)Radish, serotype 21 (7)Green onion, serotype 47 (7)Turnip seeds, serotype 23 (7)Carrot seeds, serotype 23 (7)Soil (19)2,3 butylene glycol fermentation, serotype related to 8, 11, 21, 26,and 69 (20)

ATCC; neotype strain, human

ATCC; neotype strain, human

Biochemical examination of all isolates was accom-

plished according to the recommendations of Edwardsand Ewing (11) and the Manual of ClinicalMicrobiology (3).

Cultures for deoxyribonucleic acid (DNA) extrac-

tion were propagated in 2 to 4 liters of nutrient broth(BBL) with the addition of 1% glucose or in a brothconsisting of 1% tryptone (Difco) plus 0.3% yeastextract (Difco).

DNA preparation. Saline-ethylenediaminetetraa-cetic acid washed and frozen cells were thawed andsuspended in saline-ethylenediaminetetraacetic acidand lysed by the addition of 1 to 2% sodium laurylsulfate (22). DNA purification involved a modifiedMarmur technique by using neutralized phenol de-proteinization immediately after cell lysis and ribo-nuclease treatment (22).The capsular polysaccharide was removed from the

Strain

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VOL. 29, 1975 ENVIRONMENTAL KLEB'

DNA solution after the last ethanol precipitation. TheDNA was dissolved in 0.1 strength standard salinecitrate (0.1 x SSC) and an equal volume of 2.5 Mpotassium phosphate, pH 7.5, was added. An equalvolume of 2 methoxy-ethanol was added, dropwise,until the initial precipitate could no longer be dis-solved by gentle swirling. The slurry was centrifugedat 10,000 rpm, and then the clear supernatant wasdecanted. The DNA was precipitated with N-propanol and collected on a glass rod. The above is amodification of the procedure of Bellamy and Ralph(4).

Determination of percentage guanine plus cyto-sine (% G+C) base composition. The % G+C basecomposition of purified DNA was determined by thethermal denaturation procedure as described by Mar-mur and Doty (23). The % G+C content was calcu-lated from the midpoint of the thermal melting curve(Tm) by using equation 3 of DeLey (9).

Quantitative measurements of DNAreassociation. Two procedures were used in deter-mining DNA homology between environmental andhuman K. pneumoniae strains. In one procedure, theDNA membrane filter technique was used and hy-bridization was measured by the competition assay(13, 17). About 20% of the input homologous radioac-tive DNA renatured to the membrane-bound DNA.This resulted in some 10,000 counts/min of radioactiv-ity. Approximately 400 counts/min of DNA wasretained on blank filters. The second technique uti-lized measurement of Cot..5 values of renaturing DNAmixtures (26). Membrane filter competition assays

SIELLA: NUCLEIC ACIDS 821

were performed in 2x SSC, whereas optical measure-ment of renaturing DNA mixtures were in 3x SSCplus 20% Me2SO. DNA duplex formation occurred atT. -25 C.Determination of genome size. Genome sizes were

estimated by the technique of DNA renaturationkinetics (26). Renaturation occurred in 3x SSC plus20% Me,SO at T. -25 C. Escherichia coli WP2 wasused as the molecular weight standard.

RESULTS

Over 100 presumptive K. pneumoniae strainsfrom nine different ecological habitats wereexamined for their reactions in 19 diagnosticmedia (Table 2). In general, isolates from allgroups conformed to the acceptable pattern(s)exhibited by the species, K. pneumoniae (4, 11).Perhaps the most atypical group of strainscomes from white fir trees (1). Although some ofthese isolates exhibited a weak production ofH2S and nearly two-thirds did not grow inKCN, all were confirmed as K. pneumoniaeserotype 68 by the Center for Disease Control.The single most variable cultural reaction

was the ability to produce indole. In general,strains of plant-associated K. pneumoniae hada somewhat higher incidence for indole produc-tion than would be expected from the publishedkey (11). Nearly one-third of the vegetable,

TAwx 2. Percentage of positive cultural reactions of

Test or substrate

IndoleMethyl RedVoges-ProskauerCitrateH2SUreaseKCNMotilityGelatinLysineArginineOrnithinePhenylalanineMalonateGlucose gasLactoseSucroseMannitolDulcitol

613.391980

959803.3

100100

100971009910032

K. pneumoniae from various environments

Origina

Vege- 1 Seeds [Trees 1 Pulp River Human CDC ATCC Othertables _______jmill clinical CC AC te

20.012.092.0881280NDc

00

10000

ND9684100ND10032

255

100100080

00

8500

ND88100100ND10070

10038100100631003800

100000

100100100ND100ND

00

1001000

95100

00

100000

10010010010010047

00

100100

0100ND

0ND100

00

ND100100100ND10067

170

100100

092ND

0

10000

ND100100100ND10017

00

100100

0100100

00

100000

10010010010010043

330

100100

0100100

00

100000

1001001006710033

500

1001000

100100

00

100000

10010010010010050

a Number of cultures included in each group: vegetables, 25; seeds, 20; trees, 8; pulp mills, 19; river, 9; humanclinical, 12; CDC, 7; ATCC, 3; other, 4.

bKey of expected cultural reactions taken from Edwards and Ewing (11).cND, Not done.

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SEIDLER, KNITTEL, AND BROWN

seed, and tree strains were positive for this trait(18/53).The % G+C of 41 strains of K. pneumoniae

from various habitats is summarized in Table 3.The % G+C content for all groups ranged from53.9% to 59.2%. There was no significant differ-ence in the average G+C contents among any ofthe groups. However, the groups represented bypulp mill and environmental klebsiellas con-tained isolates with more divergent base compo-sition than the human group (54.7 to 59.2%G+C, 53.9 to 58.2% G+C and 56.2 to 57.8%G+C, respectively).One of the main goals of the present study

was to determine the degree of genetic related-ness of the environmental K. pneumoniaestrains with the human reference strain. TheDNA polynucleotide diversity was ascertainedby DNA hybridization experiments (Table 4).The strains of K. pneumoniae were divided intofour groups which reflect their origin in nature.A broad range of relative reassociation (homol-ogy) values were observed for these phenotypi-cally indistinguishable K. pneumoniae strains.Most of the human isolates were homogeneousillustrating some 75% or higher relatedness tothe neotype reference strain DNA, 13883. Com-parable high values were also obtained with fiveout of seven pulp mill isolates and all fiveisolates from rivers. However, only the leafnodule symbiont (8) exhibited a high homologywith the human reference DNA in the plant-vegetable-seed group of strains. The averagehomology value for the remaining eight out ofnine vegetable isolates is 35%. This low averagevalue is comparable to that observed for onehuman strain UOMS-1 and for two strains frompulp mills, 094 and 012.The more conventional DNA membrane filter

hybridization technique was used in early stud-ies. Experiments involving hybridization trialswith 141 as the reference included strains 131,116, and 094. Comparable relative reassociationvalues were obtained by both optical and mem-brane filter hybridization procedures (agree-ment within 10%).The question now arises as to the significance

of the K. pneumoniae strains which exhibit lessthan 50% homology with the human neotypereference strain, 13883. It therefore was relevantto examine further DNA reassociation levelsamong these low homology strains in an at-tempt to clarify their taxonomic status andpossible health significance. Additional experi-ments were studied by using neotype strains ofEnterobacter spp. as reference DNA (Table 5).E. aerogenes and E. cloacae share similarity inpolynucleotide sequences over one-third of theirgenome, whereas E. aerogenes and K.

TABLE 3. Midpoint of thermal melting curve and %G + C mean base composition ofDNA from K.

pneumoniae from various habitats

Pulp mills004012037045050084089093094102116118Average

% G + C baseOrigin T7 compo-

sitiona, b

Environmental isolatesM5A1UW-1V-l0lbV-104aV-112V-151V-171V-233V-236V-2444069-194082-20A006002051R-302R-304R-306R-307Ka-1ATCC 15574Average

Human isolatesUOMS-1UOMS-5MUSC-113882141143ATCC 13883ATCC 8047Average

93.5 0.593.894.1 ± 0.493.5 + 0.493.093.592.6 + 0.393.6 0.392.9 + 0.293.293.292.2 + 0.193.3

92.693.692.092.193.793.493.793.691.992.592.493.293.293.592.993.293.693.693.793.593.593.1

93.193.592.893.392.9 40.592.9 0.293.593.193.1

57.858.459.257.856.657.755.658.056.357.057.054.757.2

55.658.054.254.458.257.458.258.053.955.455.2c57.Oc57.057.856.357.158.058.058.257.857.856.8

56.857.856.257.257.757.757.856.956.9

a % G + C base composition was calculated usingequation 3 of DeLey (9).

'Results of two or more determinations.c Reported in reference 2.

pneumoniae 13883 exhibit a slightly greaterextent of relative reassociation. These data arein agreement with other published studies (6).

L

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ENVIRONMENTAL KLEBSIELLA: NUCLEIC ACIDS

TAz 4. Relative reassociation and % G + C ofhuman and environmental K. pneumoniae DNA

% Rel- %G+COrigin Strain ative basereasso- compo-

ciation sition

Human ATCC 13883a 100 57.8Human 13882° 100 57.2Human MUSC-2 74 56.2Human 141" 91 56.3Human UOMS-5 98 57.7Human UOMS-1 40 56.8

River 006 91 57.0River 002 81 57.8River 051 85 56.3River R-302 97 57.1River R-307 92 58.2

Mushroom V-104a 23 54.4Radish V-236 13 53.9Green onion V-244 5 55.4Potato V-112 70 58.2Radish leaves V-233 55 55.2Carrot seeds V-151 26 57.4Turnip seeds V-171 48 58.2

Psychotria leaf nod- ATCC 15574 93 57.8ule symbiont

Pulp mill:Raw mill effluent 116" 91 57.0Primary pond 102 90 57.0Primary pond 045 90 57.8Aeration basin 037 87 59.2Secondary pond 084 73 57.7Primary pond 094" 41 56.3Primary pond 012 32 58.4

aReference DNA from ATCC 13883 K. pneumoniaeneotype strain.

b Percentage of relative DNA reassociation alsodetermined by the membrane filter competition pro-cedure (17).

K. pneumoniae R-307, which shows high relat-edness to the reference strain 13883, also ex-hibits the expected 50% reassociation with E.aerogenes. That is, R-307 is just as differentfrom E. aerogenes as is 13883. It is moresignificant that two lower homology vegetableisolates of K. pneumoniae (V-104a and V-233)are also less related to E. aerogenes and E.cloacae than to the 13883 neotype strain. It isclear that such genetically divergent strains arenot Enterobacter spp. which are masqueradingas K. pneumoniae.The low relative reassociation exhibited by

the human strain UOMS-1 prompted experi-ments with environmental isolates also showinglow relatedness values to 13883. Three strains ofenvironmental K. pneumoniae exhibiting high,intermediate, and low levels of relative reas-

sociation with 13883 were chosen for such astudy. Strain V-233 was found to be less relatedto UOMS-1 than to the neotype K. pneumoniaereference strain. Strain R-302 which has 97%relative reassociation with 13883, exhibited theexpected 40% value range with UOMS-1. Thus,R-302 and 13883 share a common 40% of thegenome with human strain UOMS-1. On theother hand, mushroom isolate V-104a sharedmore than twice the level of polynucleotidesimilarity with UOMS-1 than with 13883.Genome sizes of selected strains were esti-

mated from the renaturation kinetics of DNA(Table 6). The genome sizes of all strainsexamined were in the range of 2,200 x 106 to3,000 x 106 daltons. This range is typical forspecies of the Enterobacteriaceae (2, 12, 14). Ingeneral, K. pneumoniae strains with genomesizes similar to 13883, showed the highest rela-tive reassociation values to this human strain.Thus, six out of seven K. pneumoniae isolateswith 2,200 x 106 to 2,600 x 106 daltons genomesize exhibited 55% or greater relative reassocia-tion values with 13883. With one exception,strains with larger genomes (2,700 x 106 to 3,000x 106) and the Enterobacter species showed lessthan 50% relatedness.

DISCUSSIONThe biochemical testing of cultural reactions

on over 100 K. pneumoniae strains illustratedsome phenotypic variability existed among theisolates. A variety of indole, methyl red, Voges-Proskauer, and citrate patterns was observedand this variability appears comparable to otherstudies on K. pneumoniae from the environ-

TABLE 5. Relative reassociation values for additionalreference strains ofDNA

% RelativeDNA species Strain reasso-

ciation

Enterobacter aerogenes ATCC 13048a 100E. cloacae ATCC 13047 33Klebsiellapneumoniae ATCC 13883 49K. pneumoniae river R-307 52K. pneumoniae V-233 18K. pneumoniae V-104a 11

E. cloacae ATCC 13047a 100K. pneumoniae V-104a 33K. pneumoniae V-233 45

K. pneumoniae UOMS-la 100K. pneumoniae V-233 29K. pneumoniae R-302 43K. pneumoniae V-104a 57

a Reference DNA.

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TABLE 6. Genome sizes calculated from renaturationrates

CalculatedSource ofDNA Cot0., (mol a per goenom

daltons)

Escherichia coli 1.08 ± 0.06 (4) 2,200bKlebsiella pneumoniae:13883 1.11 0.06 (7) 2,30015574 1.08 2,200R-301 1.12 2,300MUSC-2 1.18 2,400UOMS-5 1.19 2,400UOMS-1 1.23 2,500V-233 1.28 2,600R-302 1.29 2,600R-303 1.31 2,700V- 104a 1.32 2,700V-10ib 1.36 2,800V-112 1.39 2,800V-171 1.47 3,000E. cloacae13047 1.25 2,600E. aerogenes13048 1.28 2,600

a Corrected for effect of G +C content on renatura-tion rate constant (26).

°Value based on two literature reports (12, 26).

ment (1, 7, 10, 24). A few strains representingmost ecogroups produced tryptophanase, a traitnot classically recognized as being associatedwith K. pneumoniae but acknowledged to bepresent in some 6% of the more recent clinicalisolates (11). As is typical, a variable number ofstrains fermented dulcitol. Careful analyses ofthe cultural reactions tested indicated thatvariable properties in tryptophanase, dulcitol,and indole, methyl red, Voges-Proskauer, cit-rate patterns were not correlated with habitat oforigin nor with DNA reassociation levels. Theauthors of a recent study on 339 strains of K.pneumoniae of clinical origin also recognizedcultural variability and subdivided their iso-lates into eight biotypes on patterns of dulcitol,sorbitol, and tartrate fermentation (25). Themost common patterns of dulcitol, sorbose, andd-tartrate fermentations were - - - (109strains), - - + (70 strains) and - + - (52strains). Unfortunately, the phenotypic bi-otypes did not correlate with capsule serotypingand no nucleic acid studies were performed.The DNA thermal melting curves indicated

that the average G+C base composition for allK. pneumoniae isolates was 57%. This overallaverage is indistinguishable from the averageobtained for each of the four ecogroups. Thesevalues are similar to most literature reports

which indicate a G+C range of about 53 to 57%(9, 21, 23). The present overall range of 54 to59% may be more broad simply because moreisolates were examined from diverse origins.The 5 to 6% spread is much broader than theusual 2% range typical of other bacterial species(15). In fact, the G+C base composition spreadfor each genus of the enteric bacteria is typicallyabout 2% (21). This larger variation in basecomposition is indicative of a higher degree ofmolecular heterogeneity among strains of the K.pneumoniae species.The 5 to 100% range observed in the relative

DNA reassociation levels among Klebsiellaclearly confirms the molecular heterogeneity ofthis group. However, it is most significant thatthe strains of K. pneumoniae examined fromrivers, most pulp mill isolates and strain V-112from potato are only as different from thehuman reference DNA as are most of the humancultures themselves.We might speculate that strains having simi-

lar genetic composition presumably have thesame potential to induce infections as do strainsof known human origin. However, we recognizethe limitations in the DNA hybridization tech-nique and do not claim the procedure is specifi-cally capable of detecting differences in levels ofvirulence. We are stating that, in terms of thetests we do comprehend, the cultural reactions,serotyping, and genetic analyses, most environ-mental isolates are indistinguishable fromstrains of human origin.Although the number of strains are limited,

the evidence indicates a good correlation existsbetween the % G+C base composition, DNAhomology, and genome size. Cultures with ge-nome sizes and G+C contents most divergentfrom the reference DNA, also have the lowesthomology. This is typified by isolates V-l0iband V-104a. Isolates with a base compositionsimilar to the reference strain, but having largergenome sizes (V-112, V-171) also exhibit de-pressed homology levels. Additional studies arenecessary before this apparent anomaly of phe-notypic uniformity and genetic heterogeneitycan be fully understood. This apparent anomalyis theoretically explainable since the 19 culturalreactions examined represent only 2% or less ofthe total genes present in a bacterium. Inaddition, we do not know whether there isamino acid sequence homology in the enzymaticreactions the isolates have in common.The inference here is that one must consider

the possibility it is merely fortuitous that thehuman and vegetable klebsiellas have manyphenotypic properties in common. Enterobacterand Klebsiella are specific examples of bacteria

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ENVIRONMENTAL KLEBSIELLA: NUCLEIC ACIDS

which share numerous phenotypic propertiesbut nevertheless, differ in some 60% of thepolynucleotide sequences in the genome (6).Unfortunately, many relevant questions stillremain concerning the evolutionary relation-ships of the low homology level klebsiellas. Arethey ancestors ofhuman Kiebsiella? Are the lowhomology strains closely related to each other orto other already described genera/species of theenteric group? Finally, what are the publichealth implications of bacteria which are ofenvironmental origin, conform to the culturaldefinition of K. pneumoniae, but are geneticallydistantly related to the human pathogenic eco-types? The answers to these and other impor-tant questions are currently under study.

ACKNOWLEDGMENTSThis work was supported in part by a grant to R.J.S. from

the Oregon State University Research Council. We appreciatereceiving unpublished material from E. J. Ordal pertaining tothe molecular taxonomy of K. pneumoniae. The editorialcomments offered by Larry Moore and Charles Hagedorn aregratefully acknowledged.

LITERATURE CITED1. Aho, P. E., R J. Seidler, H. J. Evans, and P. N. Raju.

1974. Distribution, enumeration, and identification ofnitrogen-fixing bacteria associated with decay in livingwhite fir trees. Phytopathology 64:1413-1420.

2. Bak, A. L., C. Christiansen, and A. Stenderup. 1970.Bacterial genome size determined by DNA renatura-tion studies. J. Gen. Microbiol. 64:377-380.

3. Balir, J. E., E. H. Lennette, and J. P. Truant (ed.). 1970.Manual of clinical microbiology. The Williams andWilkins Co., Baltimore.

4. Bellamy, A. R., and R. K. Ralph. 1968. Recovery andpurification of nucleic acids by means of cetyltrime-thylammonium bromide, p. 156-160. In L. Grossmanand K. Moldare (ed.), Methods in enzymology, vol. 12.Academic Press Inc., New York.

5. Braman, S. K., R. J. Eberhart, M. A. Asbury, and G. J.Hermann. 1973. Capsular types of Klebsiellapneumoniae associated with bovine mastitis. J. Am.Vet. Met. Assoc. 162:109-111.

6. Brenner, D. J., A. G. Steigerwalt, and G. R. Fanning.1972. Differentiation of Enterobacter aerogenes fromKlebsiellae by deoxyribonucleic acid reassociation. Int.J. Syst. Bacteriol. 22:193-200.

7. Brown, C., and R. J. Seidler. 1973. Potential pathogens inthe environment: Klebsiella pneumoniae, a taxonomicand ecological engima. Appl. Microbiol. 25:900-904.

8. Centifanto, Y. M., and W. S. Silver. 1964. Leaf-nodulesymbiosis. I. Endophyte of Psychotria bacteriophila. J.Bacteriol. 88:776-781.

9. DeLey, J. 1970. Reexamination of the association be-tween melting point, buoyant density, and chemicalbase composition of deoxyribonucleic acid. J. Bacte-riol. 101:738-754.

10. Duncan, D. W., and W. E. Razzell. 1972. Klebsiellabiotypes among coliforms isolated from forest environ-

ments and farm produce. Appl. Microbiol. 24:933-938.11. Edwards, P. R., and W. H. Ewing. 1972. Identification of

Enterobacteriaceae, 3rd. ed. Burgess Publishing Co.,Minneapolis.

12. Eigner, J. 1968. Molecular weight and conformation ofDNA, p. 386-429. In L. Grossman and K. Moldave(ed.), Methods in enzymology. Vol. 12. Academic PressInc., New York.

13. Gillespie, D., and S. Speigelman. 1965. A quantitativeassay for DNA-RNA hybrids with DNA immobilized ona membrane. J. Mol. Biol. 12:829-842.

14. Gillis, M, J. DeLey, and M. DeCleene. 1970. Thedetermination of molecular weight of bacterial genomeDNA from renaturation rates. Eur. J. Biochem.12:143-153.

15. Hill, L. R. 1966. An index to deoxyribonucleic acid basecompositions of bacterial species. J. Gen. Microbiol.44:419-437.

16. Hormaeche, E., and P. R. Edwards. 1960. A proposedgenus Enterobacter. Int. Bull. Bacteriol. Nomenc.Taxon. 10:71-74.

17. Johnson, J. L., and E. J. Ordal. 1968. Deoxyribonucleicacid homology in bacterial taxonomy: effect of incuba-tion temperature on reaction specificity. J. Bacteriol.96:893-900.

18. Knowles, R., R. Neufeld, and S. Simpson. 1974. Acety-lene reduction (nitrogen fixation) by pulp and papermill effluents and by Klebsiella isolated from effluentsand environmental situations. Appl. Microbiol.28:608-613.

19. Line, M. A., and M. W. Louitit. 1971. Non-symbioticnitrogen-fixing organisms from some New Zealandtussock-grassland soils. J. Gen. Microbiol. 66:309-318.

20. Mahl, M. C., P. W. Wilson, M. A. Fife, and W. H. Ewing.1965. Nitrogen fixation by members of the tribeKlebsiella. J. Bacteriol. 89:1482-1487.

21. Mandel, M., and R. Rownd. 1963. DNA base compositionin the Enterobacteriaceae: an evolutionary sequence?In C. A. Leone (ed.), Taxonomic biochemistry andserology. Ronald Press, New York.

22. Marmur, J. 1961. A procedure for the isolation of deoxyri-bonucleic acid from microorganisms. J. Mol. Biol.3:208-218.

23. Marmur, J., and P. Doty. 1962. Determination of the basecomposition of deoxyribonucleic acid from its thermaldenaturation temperature. J. Mol. Biol. 5:109-118.

24. Nunez, W., and A. R. Colmer. 1968. Differentiation ofAerobacter-Kiebsiella isolated from sugarcane. Appl.Microbiol. 16:1875-1878.

25. Richard, C. 1973. Etude antigenique et biochimique de500 souches de Klebsiella. Ann. Biol. Clin. 31:295-303.

26. Seidler, R. J., and M. Mandel. 1971. Quantitative aspectsof deoxyribonucleic acid renaturation: base composi-tion, state of chromosome replication, and polynucleo-tide homologies. J. Bacteriol. 106:608-614.

27. Selden, R., S. Lee, W. L. Wang, J. V. Bennett, and T. C.Eickhoff. 1971. Nosocomial Klebsiella infections: Intes-tinal colonization as a reservoir. Ann. Intern. Med.74:657-664.

28. Steinhauer, B. W., T. C. Eickhoff, J. W. Kislak, and M.Finland. 1966. The KlebsielLa-Enterobacter-SerratiaDivision. Clinical and epidemiologic characteristics.Ann. Intern. Med. 65:1180-1194.

29. Wyand, D. S., and D. W. Hayden. 1973. Klebsiellainfections in muskrats. J. Am. Vet. Med. Assoc.163:589-591.

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