JOURNAL OF CLINICAL MICROBIOLOGY, June 1981, p. 1099-11040095-1137/81/061099-06$02.00/0

Vol. 13, No. 6

Antibiotic Resistance in Providencia stuartii Isolated InHospitals

PATRICK J. McHALE,' CONOR T. KEANE,' AND GORDON DOUGAN2*Department of Clinical Microbiology, Dublin University, Adelaide Hospital, Dublin 8,' and Department of

Microbiology, Moyne Institute, Trinity College, Dublin 2,2 IrelandReceived 12 January 1981/Accepted 13 March 1981

A total of 238 isolates of Providencia stuartii obtained from infected patientsin six Dublin hospitals were grouped by using serological and bacteriocin typingmethods and tested for sensitivity to a number of antimicrobial agents. Mostisolates were resistant to several of these agents. Resistance to tetracycline,resistance to penicillin, resistance to polymyxin, and probably resistance tonitrofurantoin were intrinsic. Plasmid screening coupled with resistance transferstudies showed that both chromosome-encoded and plasmid-encoded resistancemechanisms were clinically important. Ampicillin resistance was both chromo-somally and plasmid encoded, whereas resistance to kanamycin and resistance tocarbenicillin were exclusively plasmid encoded. Gentamicin resistance was morecommon than kanamycin resistance, and although gentamicin-resistant strainscontained aminoglycoside acetyltransferase activity, no association could be dem-onstrated with plasmid deoxyribonucleic acid in the strains tested. Unlike minimalinhibitory concentrations for kanamycin, minimal inhibitory concentrations forgentamicin varied over a wide range. P. stuartii isolates obtained from severaldifferent countries were tested for comparison. As a group, these strains were lessresistant, but they did exhibit similar resistance properties.

The genus Providencia includes the speciesProvidencia stuartii, Providencia alcalifaciens,and Providencia rettgeri (formerly Proteus rett-geri) (2). P. stuartii is one of the most antibiotic-resistant gram-negative bacilli and has been im-plicated in nosocomial infections, particularlythose affecting the urinary tract (13, 14). Respi-ratory tract and burn infections have also beenreported, but septicemia is uncommon and gen-erally restricted to elderly, severely debilitatedpatients (8, 9). The reasons for the emergence ofthis organism as a pathogen are not known.However, the high incidence of resistance tomany of the commonly used antibiotics and tocertain disinfectants may have played a part.The presence ofplasmids in Providencia specieshas been demonstrated previously, and the prop-erties of transmissable plasmids from strainsisolated in many countries have been described(6). A number of antibiotic resistance determi-nants have been located on these plasmids, in-cluding determinants for ampicillin resistance(Apr), chloramphenicol resistance, and tetracy-cline resistance. However, because P. stuartiiexhibits such an unusually wide range of resist-ance properties, chromosomal determinantscould also be responsible for clinically significantresistance.

P. stuartii strains were isolated from infectedpatients in a group of Dublin hospitals over an8-year period. Since these isolates were resistantto many antibiotics, including gentamicin, weundertook a detailed study of the genetic basisof the resistance. P. stuartii strains isolated inseveral other countries were collected and char-acterized in a similar manner for comparison.

MATERIALS AND METHODSBacterial strains and culture conditions. All

Dublin strains of P. stuartii studied were primaryisolates from patients in the Federated Dublin Vol-untary Hospitals and were isolated between January1972 and December 1979. The Federated Dublin Vol-untary Hospital association consists of seven hospitals.No P. stuartii strains were isolated in one of these, ahospital for children. The other six hospitals contain950 beds and cover most disciplines, including burnsand genitourinary units. The P. stuartii isolates wereidentified by their biochemical profiles or by the API20E system (Analytab Products, Plainview, N.Y.). Asingle colony of each P. stuartii isolate was inoculatedonto two nutrient agar slants and a dorset egg slantand stored at room temperature. The organisms wereinoculated onto fresh slants at intervals. The strainsused as recipients in resistance transfer experimentswere DUF186, a spontaneous rifampin-resistant (Rf)P. stuartii strain which contained no detectable plas-mid deoxyribonucleic acid (DNA) and was ampicillin

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1100 McHALE, KEANE, AND DOUGAN

sensitive (Ap'), gentamicin sensitive (Gm'), and kan-amycin sensitive (Km'), and Escherichia coli J5-3pro- met- Rf' (4). Strains were cultured in nutrientbroth no. 2 (Oxoid Ltd.) or on nutrient agar (Oxoid)plates.

P. stuartii strains isolated in other countries were

obtained from E. Dowsett (Essex, England), A. Em-merson (London, England), A. McAllister (Glasgow,Scotland), D. J. Stickler (Cardiff, Wales), M. Nishida(Osaka, Japan), R. N. Jones (Oregon), G. Miller (NewJersey), J. A. Retsema (Connecticut), C. L. Heifetz(Michigan), R. P. Wenzel (Virginia), and J. L. Penner(Toronto, Canada).Typing of isolates. Antisera prepared against the

O-somatic antigens of five selected Dublin P. stuartiistrains were used to designate five serotypes (FOl toF05), as described previously (10). The strains were

also typed on the basis of sensitivity to 12 bacteriocins(McHale, manuscript in preparation).Antibiotic sensitivity tests. The disk diffusion

method was used to determine the sensitivity of eachisolate to kanamycin (30 ig), gentamicin (10 ig), am-picilHin (10 or 25 ,ig), carbenicillin (100 ,ug), sulfafura-zole (100 kg), trimethoprim (1.25 fig), tetracycline (10Mg), nitrofurantoin (200,ug), penicillin G (2 U), poly-myxin (2 gg), amikacin (10 Mg), and cefuroxime (30 Mg).Minimum inhibitory concentration (MIC) tests useddoubling dilutions of antibiotic in diagnostic sensitivitytest agar (Oxoid).

Antibiotic resistance transfer. Transfer experi-ments were performed Jby the method of Datta et al.(4). Equal (2-ml) volumes of donor (Rf) and recipient(Rf) late logarithmic cultures were mixed and incu-bated at 37°C for 1 h or overnight. Transconjugantswere purified on selective media and tested for sero-

type in the case of strain DUF186 (serotype F02) or

for the abiity to ferment lactose in the case of strainJ5-3.Plasmid screening. A modification of the proce-

dure of Hansen and Olsen was used (5). Fresh over-

night cultures of P. stuartii were diluted 1:100 into 100ml of nutrient broth and grown for 3 h at 37°C withshaking. The cultures were chilled on ice, and the cellswere harvested by centrifugation at 6,000 x g and 4°Cfor 10 min. The cells were washed once in TE buffer[50 mM tris(hydroxymethyl)aminomethane, 10 mMethylenediaminetetraacetic acid, pH 8.0], resuspendedin 0.6 ml of the same buffer, and transferred to clean50-ml polycarbonate centrifuge tubes. Lysozyme [0.2ml of a 5.0-mg/ml solution in 0.25 M tris(hydroxy-methyl)aminomethane hydrochloride, pH 8.0] was

added, and after 10 min of incubation at 37°C, 9 ml oflysing buffer (TE buffer containing 4% [wt/vol] so-

dium dodecyl sulfate, pH 12.35) was added and mixedwell but gently. Then incubation was continued at37°C for an additional 20 min to achieve completelysis of the cells, 0.6 ml of 2.0 M tris(hydroxymethyl)-aminomethane hydrochloride (pH 7.0) was added, fol-lowed immediately by 0.24 ml of 5.0 M NaCl, and thetubes were left on ice for 2 h. Chromosomal DNA was

removed by centrifugation at 20,000 x g and 4 OC for30 min. Each supernatant was decanted into a cleancentrifuge tube, and 5.5 ml of ice-cold isopropanol wasadded to precipitate the plasmid DNA. The tubes

were incubated overnight at -20°C, and the precipi-tates were pelleted by centrifugation at 6,000 x g and4°C for 10 min. The supernatants were discarded, andthe pellets were dried under a vacuum; each pellet wassuspended in 100 jul of TE buffer and stored at -20°C.Plasmid DNA was detected by electrophoresis of 10-to 50-Ml samples on 0.7% (wt/vol) agarose gels by themethod of Meyers et al. (11).

Aminoglycoside-inactivating enzyme assays.The cellulose phosphate paper binding system of Ben-veniste and Davies (1) was used to assay selectedstrains for the presence of aminoglycoside phospho-rylating, adenylating, and acetylating enzyme activi-ties.

RESULTS

P. stuartii strains were isolated from urine(142 strains), wound (93 strains), and sputum (3strains) specimens of patients in the FederatedDublin Voluntary Hospitals between 1972 and1979. All strains isolated were urease negative.Serological typing indicated that the majority(56%) of these isolates were of one serotype, FOI(Table 1). Bacteriocin typing was consistent withthe serotyping results, indicating that strainswithin serological groups were related, whereasstrains that could not be serotyped were heter-ogeneous. Isolates of serotypes FO1 and F02were cultured from both urine and wound spec-imens and from patients in all six hospitals.Antibiotic resistance properties of Dub-

lin isolates and isolates from other coun-tries. All P. stuartii isolates were tested forsensitivity to the antibiotics listed above. Mostwere multiply resistant, and as a group the Dub-lin strains were more resistant than the isolatesfrom other countries (Table 2). All isolates fromboth groups were resistant to tetracycline, pen-icillin G, and polymyxin, and almost all wereresistant to nitrofurantoin. Together with evi-dence obtained from plasmid screening and re-sistance transfer studies, these results suggestthat these resistance properties are intrinsic.

In both groups of P. stuartii, Apr was morecommon than carbenicillin resistance (Cbr).Many Apr isolates were carbenicillin sensitive

TABLE 1. Distribution of serotypes among P.stuartii strains isolated in Dublin hospitals from

1972 to 1979

Serotype No. of isolates % of total

FO1 133 55.9F02 36 15.1F03 27 11.3F04 5 2.1F05 3 1.3Polyagglutinable 3 1.3Not typablea 31 13.0

" Not serotypable with the five antisera used.

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P. STUARTII ANTIBIOTIC RESISTANCE 1101

TABLE 2. Antibiotic resistance properties of P. stuartii isolates% of strains resistant to:o

Source ~str Tetra- Penicil- Ampi- beni e_

Sulfaf- ftro d- Cefu- Kana- Genta- Amika-cycline lin G cillin lin prim urazole toin acid roxime mycin micim cin

Dublin 238 100 100 92.4 83.1 81.5 80.6 100 71 3.8 36.9 83.6 0

Overseas 160 100 100 52.5 16.2 26.2 60 88.6 20 3.8 17.5 51.8 5

a The values represent the percentages of isolates resistant to each antibiotic.

TABLE 3. Cb' strains among Apr Dublin isolates ofdifferent serotypes

Serotype No. ofAp' No. of Ap' % Ap' Cb'Cb

FO1 3 132 2.3F02 10 25 40.0F03 7 26 26.9F04 0 3F05 0 3Polyagglutinable 0 2Not typablea 3 29 10.3

a Not serotypable with the five antisera used.

A B C D E.... ..... ..

26-50 51-100 :>100

z100P

mm<nz

25 <n

FIG. 1. MICs of gentamicin (open bars) and kan-

amycin (cross-hatched bars) against P. stuartii iso-lates from Dublin hospitals.

(Cb8), whereas all CW organisms were also Apr.Only 10.5% of the Apr Dublin isolates were Cb8,compared with 65.3% of the Ap' isolates fromother countries. Within the Dublin group, theApr Cb8 phenotype was more common in sero-

types F02 and F03 than in serotype FO1 (Table3).Although gentamicin resistance (Gmr) was

common in both the Dublin isolates (83.6%) andthe isolates from other countries (51.8%), kana-mycin resistance (Kmr) was less frequently en-

countered (36.9 and 17.5%, respectively). Wedetermined the MICs for gentamicin and kana-mycin for the Dublin strains and detected a

difference in the patterns of sensitivity (Fig. 1).Strains were either very sensitive or very resist-ant to kanamycin, whereas they exhibited a widerange of MICs for gentamicin (between 1.0 and

FIG. 2. Plasmid screening of P. stuartii strains ona 0.7% agarose gel. Track A, Molecular weightmarkers R27 (112 Md) and LT2 (60 Md); track B,United States isolate; track C, FO1 Dublin isolate;track D, F03 Dublin isolate; track E, F02 Dublinisolate; track F, United States isolate; track G, F05Dublin isolate; track H, molecular weight markersTpll6 (143 Md), RP4 (35 Md), pPS002 (10.4 Md), andpAC184 (2.8 Md). See reference 5 for molecularweights.

100 ,ug/ml). All KmT strains were neomycin re-

sistant, and all Gmr strains were also tobramycinresistant. The MICs for neomycin and tobra-mycin were lower than the MICs for kanamycinand gentamicin, respectively. A high proportionof the Dublin strains were resistant to nalidixicacid, trimethoprim, and sulfafurazole, whereasnearly all of the strains in both groups were

sensitive to amikacin and cefuroxime.Plasmid screening of selected strains. We

screened more than 100 Dublin isolates and 30isolates from other countries for the presence ofplasmid DNA. The Dublin strains included iso-lates of the five serotypes and isolates from allsix hospitals. Examples from each of the othersources were also included. Dublin isolates fromwithin a particular serological group usuallygave similar plasmid profiles, although excep-tions did occur. Most serotype FO1 isolatestested harbored a single plasmid of approxi-mately 50 megadaltons (Md) (Fig. 2, track C).

F G H

1002

75-

50-

25-

H[nh- _. ._ . - h i -.__C1 1-5 6-25

M IC pg)

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FIG. 3. Plasmid screening of transconjugant strains on 0.7% agarose gels. Tracks A and I, Molecularweight markers R27 (112 Md) and LT2 (60 Md); track B, strain DUF186; track C, DUF186 recipient ofApr Cbrfrom an F05 strain; track D, DUF186 recipient ofApr Cb' Kmr from F05; track E, F04 Dublin Apr CbY Kmrdonor; track F, DUF186 recipient of Kmr from F04; track G, F01 Dublin Apr Cbr donor; track H, DUF186recipient ofAp' Cb' from F01; track J, DUF179; track K, DUF186 recipient of Apr CbY Gm` from DUF1 79;track L, DUF186 recipient ofApr Cbr Gmr from DUF179. CHROM., Chromosome. Arrow 1, 112 Md; arrow 2,60 Md.

Isolates of serotypes F02, F03, and F04 allharbored single plasmids of between approxi-mately 40 and 60 Md (Fig. 2, tracks D and E).Isolates of serotype F05 and some isolates fromthe United States harbored two plasmids (Fig.2, tracks F and G). Many of the isolates fromother countries harbored single plasmids similarin size to the plasmids in the Dublin isolates.However, some of these strains contained plas-mids of less than 15 Md, and strains whichharbored no detectable plasmids were encoun-tered more frequently (Fig. 2, track B).Transfer of antibiotic resistance markers

to P. stuartii and E. coli We tested 45 Dublinisolates and 15 isolates from other countries forthe ability to transfer resistance markers to P.stuartii DUF186 and E. coli J5-3. All Apr Cbrstrains tested transferred these markers tostrains DUF186 and J5-3 at high frequencies(>i0-3), indicating that these determinants wereencoded on transmissible plasmids. In all casesa single plasmid was detected in the transcon-jugants (Fig. 3, tracks G and H). All Apr CbWstrains failed to transfer the Apr determinant ata detectable frequency. We isolated spontaneousApr Cb' derivatives of DUF186 but not of J5-3.Thus, the Apr Cb' phenotype was almost cer-tainly due to chromosomal mutations in theclinical isolates. Similar findings were reportedby Hedges (6). Different selective pressures mayhave been responsible for the emergence ofthesetwo types of ampicillin Apr resistance.

Ail Km' P. stuartii strains tested transferredthis resistance to both DUF186 and J5-3, and weshowed that the Kmr transconjugants acquireda plasmid in all cases. Neomycin resistance andKmr were always transferred together. Moststrains transferred Km' together with Ap' and

Cbr, and plasmid screening showed that a singleplasmid was responsible (data not shown). AI-though most FO1 isolates harbored plasmids ofsimilar sizes, these plasmids were not alwaysidentical since some encoded Apr and CbW andothers encoded Apr, Cb', and Km'. One serotypeF04 strain was Apr Cb' Kmr. Only the Km'marker could be transferred from this strain,and this marker was located on a 60-Md plasmid(Fig. 3, tracks E and F). Dublin serotype F05isolates transferred Km' 100-fold less efficientlythan Ap' and Cbr. Transconjugants receivingonly Apr and Cb' harbored a single 50-Md plas-mid (Fig. 3, track C), whereas Apr CbW Kmrtransconjugants harbored this 50-Md plasmidtogether with a plasmid of 90 Md (Fig. 3, trackD).

Plasmid-associated transfer of Gmr was notdetected for any of the strains tested. As shownabove, all detectable plasmids could be trans-ferred from many isolates to strain DUF186 byselecting for either Apr or Km', but Gmr wasnever transferred together with these markers.Direct selection for Gmr transconjugants on nu-trient agar containing rifampin and gentamicinproduced a small number of colonies which wereGmT, KICr, and amikacin resistant (Akr), con-tained no plasmid DNA, grew slowly in brothand on solid media, and were more sensitive tocarbenicillin than strain DUF186. These colo-nies were serotype F02, suggesting that the Gmrwas due to a spontaneous chromosomal muta-tion in strain DUF186. This was confirmed byplating strain DUF186 onto nutrient agar con-taining gentamicin. The mutants describedabove were isolated. Since direct selection withgentamicin enriched for spontaneous mutants ofstrain DUF186, we took a different approach,

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P. STUARTII ANTIBIOTIC RESISTANCE 1103

involving indirect selection of transconjugants.One Gmr Dublin isolate, DUF179 (serotype

FO1), was studied in detail. This strain was AprCbW Gmr Km8 and harbored a single 50-Md plas-mid (Fig. 3, track J). A mating mixture of strainsDUF179 and DUF186 was diluted and spreadonto nutrient agar containing rifampin and am-picillin to select for Apr transconjugants. Platescontaining a suitable number of Rf ' Apr colonieswere replica-plated onto nutrient agar contain-ing rifampin and gentamicin. About 1 in 300colonies grew on the gentamicin containing agar.Such colonies were Rf F Apr CbV Gmr Km8 Aiand grew normally in broth. When examined forplasmids, both Rf Apr 0C Gm8 colonies isolatedfrom the master plates and Rf' Apr CÇr Gm'colonies from the replica plates contained a sin-gle 50-Md plasmid (Fig. 3, tracks K and L).Cultures of Rf' ApT Cbh GmT colonies were matedwith a nalidixic acid-resistant (NxT) derivative ofJ5-3, and the mating mixture was selected onnutrient agar containing nalidixic acid and eitherampicillin or gentamicin. Colonies grew only onthe plates containing ampicillin, and they wereNxT ApT Cb' Gm and harbored the single 50-Mdplasmid. These results indicate that the GmTdeterminant was not linked to the 50-Md plas-mid.Aminoglycoside-inactivating enzymes.

Aminoglycoside-resistant donor strains and thecorresponding transconjugants were assayed forthe presence of aminoglycoside-inactivating en-zymes. None of the Gm8 KmA strains produceddetectable levels of these enzymes. All of theKmr donors and transconjugants contained aphosphorylase with the activity profile of ami-noglycoside 3'-phosphotransferase I. This en-zyme has been found in P. stuartii by otherworkers (15). All Gmr strains tested produced anacetyltransferase with the activity profile ofami-noglycoside 2'-N-acetyltransferase 2. The Gmrtransconjugants isolated by the replica-platingmethod all produced this enzyme, whereas GmTKm' Akr mutants did not. No adenyltransferaseactivity was detected in any strains tested. Thus,our results suggested that aminoglycoside-inac-tivating enzyme activity is responsible at least inpart for clinically significant Gmr and KmT.

DISCUSSIONOur results show that both plasmid-encoded

resistance and chromosome-encoded resistanceto antimicrobial agents are common in clinicalisolates of P. stuartii. A number of observationssuggest that these organisms may possess un-usual cell envelope properties which contributeto the resistance properties. Unlike most gram-negative bacilli, which lyse spontaneously in the

lysing buffer used in the plasmid screening tech-nique, most P. stuartii isolates only lysed afterpretreatment with lysozyme. While attemptingcuring experiments, we found that P. stuartiistrains were resistant to high levels of ethidiumbromide (>500 ,ug/ml) and acridine orange(>750 ,tg/ml) (McHale, unpublished data).Strains were also intrinsically resistant to poly-myxin, which acts at the cell walls of sensitivebacteria. All of these unusual properties couldbe mediated in the cell envelope, although fur-ther studies wiil be needed to confirm this sug-gestion conclusively.Most of the Dublin isolates were resistant to

the antimicrobial drugs commonly used to treaturinary tract infections, including nitrofuran-toin, trimethoprim, and sulfafurazole. Most werealso resistant to gentamicin, a drug in wide-spread use as a broad-spectrum antibioticagainst gram-negative infections. These resist-ance properties probably explain the emergenceof P. stuartii in hospital-acquired urinary tractand wound infections. Drugs such as amikacinand cefuroxime may be more useful in treatingP. stuartii infections since nearly all strainstested were sensitive to these antibiotics.Both plasmid and chromosomal genes have to

be considered in studies on antibiotic resistance.ApT was commonly encoded on a plasmid, par-ticularly in Dublin isolates, but chromosomalresistance was also clinically important in strainswhich were obtained from an outbreak of cross-infection (3). Ap' among Providencia strainsfrom many countries has also been reported byHedges (6).The results of the aminoglycoside resistance

determinations were complex. Spontaneous KmTGmT AkT mutants were isolated during thisstudy, but they did not appear to be clinicallysignificant since they grew poorly and were verysensitive to carbenicillin. The mechanism of re-sistance in these spontaneous mutants is notknown. KmT and neomycin resistance were ex-clusively plasmid encoded in the clinical isolatesand were due to the production of aminoglyco-side 3'-phosphotransferase I. GmT was morecommon among P. stuartii isolates than Km'.Ail Gm' strains were also tobramycin resistant.Although the MICs for these two antibioticsvaried over a wide range, all GmT isolates testedcontained the enzyme aminoglycoside 2'-N-ace-tyltransferase 2. No association could be estab-lished between plasmid DNA and GmT in any ofthe strains. GmT and aminoglycoside 2'-N-ace-tyltransferase 2 activity were transferred to-gether to strain DUF186 but were not linked onthe single 50-Md ApT CbT plasmid transferred,since this plasmid could be retransferred inde-

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pendently of Gmr to strain J5-3. Thus, the Gmrdeterminant had not transposed onto this plas-mid. Gmr has been shown to be encoded on atransposon in other gram-negative bacteria (12,16).Gmr must have been transferred to strain

DUF186 either on an undetected plasmid or, ifit was chromosomally encoded in strainDUF179, by some other mechanism, possiblyinvolving a bacteriophage or low-level Hfr for-mation in the donor cells. Other reports (7) onchromosomally determined Gmr implicate im-paired uptake of the antibiotic as the mode ofresistance rather than enzymatic inactivation.However, all of our Gmr strains produced anaminoglycoside acetyltransferase. The level ofresistance was variable. This variability mayhave been due to secondary mutations withinthe bacterial chromosome which varied theamount of enzyme being produced or its release.Selective pressures, such as the use of gentami-cin, probably led to these variations. Certainly,gentamicin unlike kanamycin, is used widely inthe Dublin hospitals, and Gmr is found in thePseudomonas aeruginosa and Staphylococcusaureus strains isolated in these hospitals. Thissuggests that there is a strong selection for Gmrin the organisms found in these hospitals.

ACKNOWLEDGMENTS

We thank all people who sent us isolates of P. stuartii, G.Dowd for assisting in our experiments, M. Kehoe for readingthe manuscript, and others in the Moyne Institute (especiallyGillian Johnson).

This work was supported by a grant to P.M. by the Labo-ratory Medicine Development Fund and by a grant to G.D.from the Irish Medical Research Council.

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2. Brenner, D. J., J. J. Farmer, F. W. Hickman, M. A.Asbury, and A. G. Steigerwalt. 1977. Taxonomic and

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nomenclature changes in Enterobacteriaceae. U.S. De-partment of Health, Education and Welfare Publication(CDC)78-8356. Government Printing Office, Washing-ton, D.C.

3. British Medical Journal. 1977. Unusual infection in anintensive care unit. Br. Med. J. 1:111.

4. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, andM. Richmond. 1971. Properties of an R factor fromPseudomonas aeruginosa. J. Bacteriol. 108:1244-1249.

5. Hansen, J. B., and R. H. Olsen. 1978. Isolation of largebacterial plasmids and characterization of the P2 incom-patibility group plasmids pMG1 and pMG5. J. Bacteriol.135:227-238.

6. Hedges, R. W. 1974. R factors from Providence. J. Gen.Microbiol. 81:171-181.

7. John, J. F., C. E. Rubens, and W. E. Farrar. 1980.Characterization of gentamicin resistance in nosocomialinfections. Am. J. Med. Sci. 271:25-30.

8. Keane, C. T., L. F. English, and R. Wise. 1975. Provi-dencia stuartii infections. Lancet ii:1045.

9. Kocka, F. E., S. Srinivasan, M. Mowjood, and H. S.Kantor. 1980. Nosocomial multiply resistant Providen-cia stuartii: a long-term outbreak with multiple bio-types and serotypes at one hospital. J. Clin. Microbiol.11:167-169.

10. McHale, P., L. English, A. Speekenbrink, C. Keane,and R. Wise. 1978. Kanamycin resistance in Providen-cia stuartii. J. Antimicrob. Chemother. 4:273-278.

11. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Fal-kow. 1976. Simple agarose gel electrophoretic methodfor the identification and characterization of plasmiddeoxyribonucleic acid. J. Bacteriol. 127:1529-1537.

12. Nugent, M. E., D. H. Bone, and N. Datta. 1979. Atransposon, Tn732, encoding gentamicin and tobramy-cin resistance. Nature (London) 282:422-423.

13. Overturf, G. D., J. Wilkins, and R. Ressler. 1974.Emergence of resistance of Providencia stuartii to mul-tiple antibiotics: speciation and biochemical character-ization of Providencia. J. Infect. Dis. 129:353-357.

14. Penner, J. L., N. A. Hinton, I. B. R. Duncan, J. N.Hennessy, and G. R. Whiteley. 1979. O-serotyping ofProvidencia stuartii isolates collected from twelve hos-pitals. J. Clin. Microbiol. 9:11-14.

15. Price, K. E., T. A. Pursiano, M. D. DeFuria, and G. E.Wright. 1974. Activity of BB-K8 (amikacin) againstclinical isolates resistant to one or more aminoglycosideantibiotics. Antimicrob. Agents Chemother. 5:143-152.

16. Rubens, C. E., W. F. McNeil, and W. E. Farrar. 1979.Transposable plasmid deoxyribonucleic acid sequencein Pseudomonas aeruginosa which mediates resistanceto gentamicin and four other antimicrobial agents. J.Bacteriol. 139:877-882.

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