Vol. 39, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 97-1040099-2240/80/01-0097/08$02.00/0

Antibiotic Resistance and Its Transfer Among Clinical andNonclinical Klebsiella Strains in Botanical EnvironmentstHENRY W. TALBOT, JR., DEBORAH K. YAMAMOTO, MARTIN W. SMITH, AND RAMON J.

SEIDLER*Department ofMicrobiology, Oregon State University, Corvallis, Oregon 97331

A total of 183 isolates of Klebsiella from drinking water, market vegetables,wood, sawdust, industrial effluents, and human and animal origin were examinedfor susceptibility to 10 antibacterial agents. Incidence of resistance to two or moreantibiotics tested was: 65% of the human clinical isolates, 59% among bovinemastitis, and 24% among the nonclinical isolates. The five different multipleresistance patterns among nonclinically derived Klebsiella were also found amongthe human and bovine mastitis isolates. Statistical analyses revealed that patternsof resistance among Klebsiella isolates from drinking water, market vegetables,and industrial effluents were highly correlated with each other and with resistancepatterns of human clinical isolates. Antibiotic resistance was transferred betweenKlebsiella growing in two habitat-simulated environments (growing radish plantsand aqueous sawdust suspensions). Transconjugants were detected in 5 of 21 and6 of 21 mating pairs, respectively. Average transconjugants/donor ranged from10-3 to 10-6 in Penassay broth, from 10-6 to 10'- on radish plants, and from 10-5to 108 in sawdust suspensions. Although antibiotic resistance transfer undersimulated environmental conditions can occur, regrowth of clinical strains isprobably the major cause for the widespread occurrence of antibiotic-resistantKlebsiella in the nonclinical environment.

Antibiotic-resistant strains of the coliformbacterium Klebsiella have become an increas-ingly serious complication in the treatment ofhuman and animal diseases caused by this or-ganism (1, 3, 15, 29, 32). The transfer of antibioticresistance from clinical isolates of Klebsiella hasbeen demonstrated on many occasions, usuallywith Escherichia coli recipients (3, 15, 17, 23,32, 40); this transferable resistance is mediatedby R-plasmids, which have a wide distributionthroughout the Enterobacteriaceae (3, 15, 17,32).

Klebsiella is an opportunistic pathogen and isa causative agent of several kinds of infectionsin humans (10, 16, 35, 37, 38) and animals (8, 14,20, 26). In addition to its clinical role, Klebsiellahas been found to occur at high cell densities inmany botanical environments. These include liv-ing trees (2, 6), wood products (12, 34), sugarcanewastes (30), pulp and textile mill effluents (11,18, 21), fresh vegetables (9, 12, 42), and finisheddrinking water stored in redwood tanks (34).Some of the Klebsiella isolated from these en-vironments have been found to be indistinguish-able from pathogenic Klebsiella isolates whenbased on biochemical, serological, and mousevirulence tests (5, 25, 33). Many of these non-clinical isolates are fecal coliform positive (4),

t Technical paper no. 4616, Oregon Agricultural Experi-ment Station, Corvallis, OR 97331.

which classifies them as Klebsiella pneumoniaesensu stricto (28, 41).

In this investigation, isolates of Klebsiella ofnonclinical as well as clinical origins were ex-amined for susceptibility to 10 antibacterialagents. Antibiotic resistance patterns of Klebsi-ella from the different sources were then com-pared to determine how the patterns of thesegroups were correlated and to provide informa-tion concerning the origin of the antibiotic-re-sistant nonclinical isolates. In addition, potentialdonors and recipients were chosen in an attemptto demonstrate, by conventional methods, con-jugal transfer of antibiotic resistance betweenKlebsiella of diverse origins. Experiments werethen conducted in two habitat-simulated envi-ronments to test for the prevalence of in situtransfer of antibiotic resistance between Kleb-siella cultures growing on radish plants or inaqueous redwood sawdust suspensions. Matingsin Penassay broth with the same donor andrecipient combinations were performed for com-parison of transfer frequencies in the simulatedhabitats.

MATERIALS AND METHODSBacterial cultures. Klebsiella cultures received or

isolated for this study were identified as to species bythe procedures of Edwards and Ewing (13) and Nae-mura et al. (28). Origins of most cultures have beenpreviously compiled (4, 9, 33, 34). Cultures used in

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successful antibiotic resistance transfer experimentsare listed in Tables 3 and 4.Those cultures listed under bovine area (classified

as nonclinical in Tables 1 and 2) were isolated from avariety of areas occupied by cows, as well as frombovine teat ends. It is possible that isolates frombedding and stall water may be recent contaminantsfrom animals that are frequently fed antibiotics bothfor prophylaxis and treatment.

Antibiotic sensitivity testing. Sensitivities weredetermined by the high concentration disk diffusiontechnique as described by Bauer et al. (7). The follow-ing antibiotic disks were used: streptomycin (10 ,ug,Sm), chloramphenicol (30 jig, Cm), nitrofurantoin (300jig, Nf), kanamycin (30 ,ug, Km), tetracycline (30 ug,Tc), neomycin (30 ,ug, Nm), ampicillin (10 ug, Ap),gentamicin (10 yg, Gm), furaltadone (30 ,ug, [Valsyn],Fu), and nitrofurazone (100,g, Ni). Fu disks wereobtained from BBL Microbiology Systems, N diskswere from Pfizer, and the remainder were purchasedfrom Difco. Mueller-Hinton (Difco) plates were inoc-ulated, and after 18 to 20 h of incubation at 37°C, zonediameters were measured. Isolates were assigned asresistant or not resistant by the standards of Lennetteet al. (24).

Correlation of antibiotic resistance patterns.Correlation values were calculated as described byKelch and Lee (19).

Bacterial cultures for antibiotic resistancetransfer experiments. Potential donors and recipi-ents isolated from various habitats were chosen basedon the results of the antibiotic sensitivity experiments;recipient cultures used were either resistant to noantibiotics or to Ap only. Control experiments dem-onstrated that the Ap resistance in these Klebsiellawas not transferable. Recipients and donors werestreaked onto MacConkey agar (Difco) containingknown amounts of antibiotics to determine the con-centrations of antibiotics to use in the conjugal mat-ings. E. coli strain W3110 (nalidixic acid resistant[Nalr], F-) was obtained from the laboratory of LyleR. Brown (Oregon State University).

Selection of spontaneous nalidixic acid-resist-ant Klebsiella mutants for use as recipients. Cul-tures were grown for 12 h in Penassay broth (Difco) at35°C. Nalidixic acid (Sigma) was then added to give afinal concentration of 40 ug/ml. The cultures wereincubated 6 to 12 h more, centrifuged, resuspended in1 ml of sterile 0.01 M tris(hydroxymethyl)amino-methane (Tris) buffer (pH 7.5), and plated ontoMacConkey agar containing 40 ,ug of nalidixic acid perml. Single resistant colonies were picked, streakedthree times for purity, and tested biochemically toconfirm identity with the parent culture (13, 28). Test-ing of cultures for independence on drug-free mediumwas also done by streaking onto glucose basal saltsagar plates.Test tube matings. A slight modification of pre-

viously described procedures (3, 17) was used. Twofresh isolated colonies of the recipient from a Mac-Conkey agar plate with nalidixic acid were inoculatedinto 1 ml of Penassay broth, and two fresh isolatedcolonies of the donor from a MacConkey agar platewere inoculated into 4 ml of Penassay broth. After thetwo cultures were incubated for 6 h at 35°C, 0.2 ml of

the recipient suspension was added to the donor cul-ture, and this was incubated statically for 18 h at 35°C(3, 17).

After agitation to separate the mating pairs, appro-priate dilutions were plated onto MacConkey agar fordonor and recipient enumeration, and onto Mac-Conkey agar with nalidixic acid for the recipient count.Transconjugants were selected for by plating ontoMacConkey agar with nalidixic acid and various com-binations of antibiotics to which the donor was resist-ant. None of the donors was resistant to this concen-tration (40 Lg/ml) of nalidixic acid.

Antibiotic resistance transfer on radishes.Confirmed Nalr recipients were shaken in Penassaybroth plus nalidixic acid (40 ,ug/ml) for 22 to 24 h at35°C. Cultures were diluted in sterile 0.01 M Trisbuffer (pH 7.5) to yield 103 to 104 colony-forming units(CFU)/ml of diluent. One gram ofradish seeds (varietySparkler) was soaked in 5 ml of the diluted culture for30 min. Some 102 to 103 viable Klebsiella were ab-sorbed by 1 g of seeds by this inoculation regimen.

After the diluent was aseptically drained, the seedswere placed in boxes (14 by 20 by 6 inches [ca. 35.6 by50.8 by 15.2 cm] filled with nonsterile sandy-loam,using sterile gloves. Donor cultures grown in Penassaybroth were diluted to yield 106 to 107 CFU/ml ofdiluent. A 12-ml amount was pipetted over four rowscontaining 1 g of exposed radish seeds. Recovery stud-ies showed that this concentration of Klebsiella pipet-ted over presoaked seeds resulted in some 104 to 105viable donors per g of seeds. After the addition of thedonor, the seeds were covered with soil. Repeatedattempts to recover antibiotic-resistant Klebsiellafrom uninoculated soil and seeds failed. Thus, anti-biotic-resistant Klebsiella enumerated in these exper-iments could only arise from growth of the inoculumplaced onto seeds.

Radish plants were removed at weekly intervals,the small amount of adhering sandy loam was shakenoff the roots, and the plants were placed into sterile,preweighed beakers. After weighing to determine theamount of plant material being sampled, 70 ml ofsterile 0.01 M Tris buffer was added, and the sampleswere blended in a Waring model 700A blender for 2 to3 min at room temperature. Total recipient countswere enumerated by the spread plate method onMacConkey agar with nalidixic acid (40 yg/ml). Trans-conjugants were enumerated as described in the testtube matings.

Radishes were grown in the Oregon State Univer-sity greenhouse with a daytime temperature of 240C(750F) and a night temperature of 180C (650F) under18 h of white fluorescent lighting. Plants were wateredwhen needed and grown as if for human consumption.

Matings in sawdust suspensions. Aqueous sus-pensions of redwood sawdust were prepared by adding2.0 g of sterile sawdust to 1,200 ml of sterile, double-distilled water. This suspension was allowed to standat room temperature for 2 days while nutrients wereleached from the sawdust; at this time sterile 0.1%sodium hydroxide was added to neutralize the extract.Recipients and donors were grown as described inPennassay broth (recipients with 40 Mg of nalidixicacid per ml) and then diluted 102 in sterile Tris buffer(pH 7.5). The suspension was inoculated with a recip-

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ient culture to initial density of 103 to 104 CFU/ml.Donors were added a few minutes later at a similarconcentration. After 3 days of static incubation atroom temperature, donors and recipients were enu-

merated together on MacConkey agar, and recipientswere enumerated on MacConkey agar containing 40,ug of nalidixic acid per ml. To recover transconjugants,the suspension was filtered through 0.45-um mem-brane filters and immediately placed onto MacConkeyagar with nalidixic acid and the other appropriateantibiotics. All plates were incubated at 35°C.Confirmation ofantibiotic resistance and iden-

tity of transconjugants. All presumptive transcon-jugants were picked from and streaked onto mediacontaining antibiotics to verify antibiotic resistance.Transconjugants were then inoculated into diagnosticmedia for identification and comparison with reactionsobtained with nonresistant recipient cultures (13, 28).This was done to confirm that presumptive transcon-jugants were not actually nalidixic acid-resistant do-nors. Frequencies of antibiotic resistance transfer werereported as transconjugants/donor (calculated per

gram with radishes).

RESULTS

A total of 183 isolates of Klebsiella of diverseorigins were examined for susceptibility to 10antibacterial agents used to treat infectionscaused by gram-negative bacteria. The percent-age of antibiotic-resistant isolates obtained fromvarious habitats is presented in Table 1.As anticipated, the incidence of antibiotic re-

sistance was greater among the clinical isolatesas compared with those from nonclinical sources.

The fecal coliform-positive K. pneumoniaesensu stricto comprised >70% of the clinicalisolates and an even larger percentage of theresistant strains. K. oxytoca made up 12% of theclinical isolates and about 20% of the total anti-biotic-resistant strains. Within the clinical

group, Klebsiella from human infections had alower incidence of resistance to Sm than did themastitis isolates (37.9 versus 17.5%), but higherpercentages of resistance to Nf, Km, Nm, andAp. The incidence of Fu (Valsyn) resistance wassimilar among Klebsiella from human and bo-vine infections and among isolates from the bo-vine area. This was unexpected since Valsyn useis restricted to the treatment of veterinary infec-tions (Norwich Pharmacal Co., personal com-

munication). Valsyn resistance was also foundamong some Klebsiella cultures obtained fromredwood.Among the nonclinical isolates, bovine area

Klebsiella were primarily resistant to Sm(47.8%) and Tc (30.4%). Two of the 19 Klebsiellaisolated from fresh market produce were resist-ant to Ap and Nf, and two cultures from potabledrinking water were resistant to Sm and Km,whereas three isolates from industrial effluentswere resistant to combinations of Sm, Tc, andNf. A large percentage of Klebsiella from allenvironments were resistant to Ap. Antibiotic-resistant strains from the nonclinical sourceswere composed of the species K. pneumoniaesensu stricto and K. oxytoca (28, 41).

Isolates possessing resistance to two or moreantibacterial agents were considered multiplyresistant in this investigation. It was found thatisolates with resistance to three or more drugswere, in all but two instances, of clinical origin.These two isolates of nonclinical origin werefrom textile mill effluents. Among the humanclinical strains, a total of 20 different patterns ofmultiple antibiotic resistance were observed. Anadditional six different patterns were foundamong the mastitis isolates. Of the five differentresistance patterns detected among nonclinical

TABLE 1. Incidence of antibiotic resistance among 183 clinical and nonclinical Klebsiella isolates% of isolates resistant to: % of isolates

resistant toOrigin No. tested more than

Sm Cm Nf Km Tc Nm Ap Gm Fu one anti-biotic

Mastitis 29 37.9 6.7 6.7 3.5 17.2 3.5 72.4 0 24.1 59Human clinical 57 17.5 1.8 40.5 8.8 24.6 10.5 98.3 1.8 19.3 65Total clinical 86 24.4 3.5 30.2 7.0 20.9 8.1 90.7 1.2 23.3 63

Bovine area 23 47.8 4.4 0 0 30.4 0 52.2 0 17.4 49Drinking water 27 7.4 0 0 7.4 0 0 66.7 0 0 15Market vegetables 19 0 0 10.5 0 0 0 78.9 0 0 11IndustrialaEffluents 19 10.5 0 5.3 0 10.5 0 89.4 0 0 16Otherb 9 0 0 0 0 0 0 77.7 0 33.3 33

Total nonclinical 97 15.3 1.0 3.1 2.1 9.3 0 71.1 0 7.2 24Grand total 183 19.7 2.2 16.1 4.4 16.1 3.9 80.3 0.56 15.0 42

a Pulp Mill, textile mill, and potato processing effluents.b Five from redwood chips or redwood tank (34), three from decay in white fir (2), and one from soil (4). Fu

resistance was in the redwood isolates. None of the isolates was resistant to nitrofurazone.

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strains (ApSm, ApFu, ApNf, ApTc, ApKm), all

were also present among the human and mastitiscultures. Overall, 65% of the human clinical iso-lates, 59% of the bovine mastitis strains, and 24%ofthe nonclinical isolates were multiply resistant(Table 1).A correlation matrix of antibiotic resistance

for the Klebsiella isolated from various habitatswas prepared (Table 2) based on the informationcontained in Table 1. Larger correlation valuesindicate patterns of antibiotic resistance for thehabitats being compared vary in a similar way;

smaller values reflect less similarity between thesources in resistance patterns. The matrix re-

vealed that Klebsiella isolated from drinkingwater, market vegetables, and industrial ef-fluents had correlations between the groups of0.98; furthermore, each of these groups had cor-

relations of 0.90 or greater with human clinicalKlebsiella. The nonclinical Klebsiella, with theexception of bovine area isolates, had highercorrelations with human clinical isolates thanwith mastitis isolates. Bovine area isolates hada high correlation with mastitis isolates (0.91),but lower correlations with the other groups.Table 3 compiles representative results of an-

tibiotic resistance transfer experiments carriedout in Penassay broth. Klebsiella isolated fromthe bovine environment (tap water in the stall,and sawdust bedding) transferred resistance toSm, Tc, and Fu into the E. coli recipient. OtherKlebsiella isolates which also supported meas-

urable transfer of antibiotic resistance into E.coli were chosen as potential donors for variousKlebsiella of nonclinical and clinical origins.Klebsiella from clinical infections in dairy cattleand humans transferred various markers intoKlebsiella from vegetables, from drinking water,and from the bovine area. Also, two Klebsiellafrom drinking water stored in redwood reservoirs(34) transferred resistance to Sm into Klebsiellaalso isolated from botanical environments.Transfer frequencies for these experimentsranged from 10-3 to 10-6 transconjugants perdonor.

In light of these successful antibiotic resist-ance transfer experiments and the known highcell densities of Klebsiella in botanical milieu(6, 9, 11, 18, 34, 42), two habitat-simulated en-vironments (aqueous sawdust suspensions andradish plants) were used to measure possible insitu resistance transfer between various Klebsi-

TABLE 2. Correlation matrix of antibiotic resistance among Klebsiella isolates from various habitatsa

MasMastitis Bovinearea Human clini- Drinking wa- Market vege- Industrial ef-MastitisBovine cal ter tables fluents

Mastitis

Bovine area 0.91

Human clinical 0.85 0.64

Drinking water 0.87 0.65 0.90

Market vegetables 0.84 0.58 0.98 0.98

Industrial effluents °0.90 0.71 0.9498 0.98 0.98a Correlation values were calculated as described by Kelch and Lee (19) to analyze the antibiotic resistance

patterns of the Klebsiella from each habitat. Larger values indicate high correlation; smaller values reflect lesssimilarity between the sources in resistance patterns.

TABLE 3. Antibiotic resistance transfer detected with clinical and nonclinical Klebsiella after growing inPenassay broth

Donor (origin) Recipient (origin) Markers transferred

UG20 (bovine faucet water) E. coli W3110 Nalr Sm, Tc, FuUG6 (unused sawdust bedding) E. coli W3110 Nalr Sm, TcUG24 (unused sawdust bedding) E. coli W3110 Nalr Sm, TcUG35 (used sawdust bedding) E. coli W3110 Nalr SmUG14 (bovine mastitis) V104 (market vegetable) SmUG28 (bovine mastitis) UG10 (bovine stall water) CmUT465 (human blood) MH15 (hospital salad) NfUT465 (human blood) JH42 (potable drinking water) TcSL5 (human sputum) CM 1 (potable drinking water) Nm13 (potable drinking water) V236 (market vegetable) Sm5 (potable drinking water) E44.5-1 (redwood chips) Sm

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ella isolates. Table 4 contains data for thosemating pairs that transferred resistance in thesimulated environments.

Antibiotic resistance transfer in Penassaybroth was detected with 12 of 21 donor-recipientcombinations, 5 of 21 on radishes, and with 6 of21 in aqueous sawdust suspensions. Transfer ofresistance was detected between strains of K.pneumoniae sensu stricto and between thesestrains and K. oxytoca. Resistance to Sm, Cm,Km, and Nm was transferred, and in most in-stances transfer of at least two antibioticmarkers was detected. No transconjugants weredetected on radishes after week 1 of sampling.In the sawdust experiments, only multiply anti-biotic resistance transfer frequencies were re-ported since spontaneous mutations resulting inresistance to one antibiotic could be suspectedwhen dealing with frequencies in the 10' range.

However, control experiments never revealed

spontaneous antibiotic-resistant Klebsiella. Fur-thermore, transconjugants were checked for bio-chemical reactions to confirm that they were notactually nalidixic acid-resistant donors.

Transfer frequencies (expressed as transcon-jugants/donor) ranged in magnitude from 10'to 106 in Penassay broth, with most frequenciesbetween lo-4 and -5. On radishes, transfer fre-quencies at the first week of sampling werebetween 10-6 and 10-7 (10-4 to 10-5 transconju-gants/recipient) and, in redwood sawdust sus-pensions, between 10-5 and 10-'. As expected,cell densities of Klebsiella in the simulated hab-itats (105 to 106 CFU/g on radishes, 104 to 105CFU/ml in sawdust suspensions) were consid-erably less than those in Penassay broth (10'CFU/ml). The relative differences in the fre-quencies of transconjugants appeared to be a

function of cell densities in the various experi-ments.

TABLE 4. Antibiotic resistance transfer. Penassay broth and habitat-simulated environmentsTransfer frequencya

Donor (origin) Recipient (origin) MarkersSawdustssulected Penassay RadSegbSadutsusbroth pensions

SL5 (human PS401LR (bovine Km 3.4 x 10-5 NSc NSsputum) mastitis) Nm 2.4 x 10-5 NS NS

KmNm 2.9 x 10-5 NS 4.8 X 10-7KmNmSm NS NS 4.8 X 10-7

UG13 (bovine masti- Km 1.0 x 10-5 NDd NStis) Nm 1.3 x 10-5 1.9 x 10-7 NS

KmNm 6.7 x 10-6 1.9 x 10-7 6.1 x 10-"KmNmSm NS NS 3.7 x 10-8

PC2 (potable dfinking Km 3.5 x 10-6 ND NSwater) Nm 3.0 x 10-6 1.9 x 10-7 NS

KmNm 3.0 x 10-6 ND 6.1 x 10-8KmNmSm NS NS 5.0 x 10"

MH29 (human urine) Km 5.2 x 10-5 1.7 x 10-6 NSNm 6.2 x 10-5 1.9 X 10-6 NSKmNm 4.6 x 10-5 7.6 x 10-7 1.2 x 10-5KmNmSm NS NS 3.3 x 10-6

U010994 (human Km 4.2 x 10-3 ND NSurine) Nm 5.0 x 10-3 1.9 X 10-7 NS

KmNm 4.3 x 10-3 ND ND

UG28 (bovine DS21 (potable drink- Sm 5.3 x 10-3 1.7 x 10-7 NSmastitis) ing water) Cm 2.1 x 10-5 ND NS

SmCm 1.3 x 10-5 ND NDCM,1 (potable drink- Sm 1.0 x 10-3 ND NS

ing water) Cm 9.2 x 10-4 ND NSSmCm 8.3 x 10-4 ND 1.7 x 10-7

Vlll (market vegeta- Sm 5.0 x 10-6 NS NSble) Cm 5.2 x 10-7 NS NS

SmCm 4.3 x 10-7 NS 1.4 x 10-6'Expressed as transconjugants/donor (calculated per gram with radishes). Results of independent experi-

ments with the same donor-recipient combinations revealed that in some cases transfer frequencies could varyby 10-fold.

b Frequency based on approximate donor count at inoculation.'NS, Not sampled.d ND, Transconjugants not detected.

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102 TALBOT ET AL.

DISCUSSION

Multiply antibiotic-resistant bacteria compli-cate the clinical treatment of infections by re-ducing the number of potentially useful chemo-therapeutic agents. This is a special problem incompromised hosts with limited abilities to com-bat infections (38, 39). It has been documentedthat a large percentage of human clinical Kleb-siella possess multiple antibiotic resistance andup to 80% of these resistant isolates possesstransmissible R-plasmids (3, 40). In the presentreport, 65% of the human clinical isolates, 59%of the bovine mastitis isolates, and 24% of thenonclinically derived Klebsiella were resistantto two or more antibiotics. The five differentantibiotic resistance patterns found for the non-clinically derived Klebsiella were also repre-sented among the clinical cultures.When the antibiotic resistance patterns for

Klebsiella isolated from different habitats werecompared, it was found that with the exceptionof bovine area isolates, the nonclinical cultureswere highly correlated with each other and withhuman clinical isolates. Although the correlationdata cannot prove the common origin of anti-biotic resistance, they suggest that most anti-biotic-resistant Klebsiella isolated from environ-mental sources may first have been ofhuman oranimal origin, since it is unlikely that they wouldencounter selective pressures to develop thesesame patterns of resistance. Further support forthis suggestion is the fact that the majority ofthe multiply antibiotic-resistant Klebsiella iso-lated from nonclinical habitats in this study werefecal coliform positive. These fecal coliform-pos-itive K. pneumoniae species constituted some85% of the clinical isolates in a related investi-gation (4). It has also been shown that fecalcoliforn-positive clinical isolates of Klebsiellaare capable of rapidly proliferating and coloniz-ing a variety of botanical milieu (22). Therefore,it is reasonable to expect that regrowth of anti-biotic-resistant, fecal coliform-positive K. pneu-moniae wiLl occur in nature.

In the present study multiply antibiotic-re-sistant Klebsiella were isolated from potabledrinking water, on fresh market vegetables, inpulp and textile mill effluents, and in sawdust.Some of these Klebsiella possessed transferableantibiotic resistance, but it is unlikely that re-sistance transfer in these habitats has a majorrole in the proliferation of antibiotic resistanceamong other nonclinical Klebsiella strains. Inthis study, transfer of resistance was detectedbetween clinical and nonclinical Klebsiella byconventional methods and also in two habitat-simulated botanical environments. In the latter

experiments, transfer frequencies were lowerand transconjugants were not detected on rad-ishes after week 1 of sampling. Although thesimulated environments in one case lacked thenatural interactions between indigenous bacteriaand were performed under controlled laboratoryconditions of inoculation and incubation, theseexperiments demonstrated that the potential ex-ists for resistance transfer between Klebsiella onplant matter. There are also other botanicalhabitats where similar cell densities (104 to 106CFU per g or per ml) of Klebsiella exist thatmay enable antibiotic resistance transfer to oc-cur, such as pulp and textile mill effluents (11,18, 21). However, this probably has less impacton environmental habitats as compared with thepresence and regrowth of human clinical anti-biotic-resistant Klebsiella from sewage (40), forexample.As noted above, antibiotic resistance transfer

frequencies were lower in the habitat-simulatedenvironments as compared with Penassay broth,which presumably provided optimum conditionsfor resistance transfer. The wide variations ob-served in transfer frequencies between habitatsfor each donor-recipient pair could be partiallyattributable to the lower cell densities found inthe simulated botanical habitats. There are pos-sibly other factors affecting resistance transferefficiency in natural environments. The varia-tions in transfer frequencies that were observedbetween various donor-recipient combinationsin Penassay broth are probably due to differ-ences in donor-recipient compatibility, as well asother molecular and genetic elements.Whether due to regrowth of clinical strains or

environmental resistance transfer, antibiotic-re-sistant Klebsiella on fresh vegetables and insawdust pose a potential health problem withrespect to human nosocomial infections and bo-vine mastitis, respectively. For example, studieshave illustrated a correlation between bovineteat colonization by Klebsiella and the use ofsawdust bedding (31). Klebsiella counts in usedsawdust bedding exceeded those in unused bed-ding by 1,000-fold, indicating that multiplicationoccurred. In this study, some of the Klebsiellaisolates from the bovine environment werefound to possess transferable antibiotic resist-ance and the patterns of resistance showed ahigh correlation value (0.91) with Klebsiellastrains isolated directly from mastitic animals.

In other studies, Shooter et al. found coli-forms, including Klebsiella, in foods consumedraw in hospitals, canteens, and schools (36). Hos-pital foods, and salads in particular, were foundto occasionally contain sufficient levels of coli-forms to lead to their establishment in the bowel

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ANTIBIOTIC-RESISTANT KLEBSIELLA 103

(36). Others have incriminated the bowel as thesource of some nosocomial Klebsiella infections(27, 35). Antibiotic therapy would obviously se-lect for food-bome, multiply antibiotic-resistantKlebsiella which could subsequently colonizethe bowel and selectively favor their prolifera-tion by reducing the numbers of competitive or

antagonistic normal flora.

ACKNOWLEDGMENTS

We thank those who supplied cultures for this study andNorwich Pharmaceutical Co. for the furaltadone. This re-

search was supported by U. S. Environmental ProtectionAgency research grant R-804456 and by National ScienceFoundation grant BMS 75-13799.

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2. 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.

3. Allison, M. J., J. D. Punch, and H. P. Dalton. 1970.Frequency of transferable drug resistance in clinicalisolates of Klebsiella, Aerobacter, and Escherichia, p.

94-99. Antimicrob. Agents. Chemother. 1969.4. Bagley, S. T., and R. J. Seidler. 1977. Significance of

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5. Bagley, S. T., and R. J. Seidler. 1978. Comparativepathogenicity of environmental and clinical Klebsiella.Health Lab. Sci. 15:104-111.

6. Bagley, S. T., R. J. Seidler, H. W. Talbot, Jr., and J.E. Morrow. 1978. Isolation of Klebsielleae from withinliving wood. Appl. Environ. Microbiol. 36:178-185.

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8. Braman, S. K., R. J. Eberhart, M. A. Ashbury, andG. J. Herman. 1973. Capsular types of K. pneumoniaeassociated with bovine mastitis. J. Am. Vet. Med. Assoc.162:109-111.

9. Brown, C., and R. J. Seidler. 1973. Potential pathogensin the environment: Klebsiella pneumoniae, a taxo-nomic and ecological enigma. Appl. Microbiol. 24:900-904.

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