Antibiotic Resistance in Providencia stuartii Isolated In

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1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1981, p /81/ $02.00/0 Vol. 13, No. 6 Antibiotic Resistance in Providencia stuartii Isolated In Hospitals 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 Ireland Received 12 January 1981/Accepted 13 March 1981 A total of 238 isolates of Providencia stuartii obtained from infected patients in six Dublin hospitals were grouped by using serological and bacteriocin typing methods and tested for sensitivity to a number of antimicrobial agents. Most isolates were resistant to several of these agents. Resistance to tetracycline, resistance to penicillin, resistance to polymyxin, and probably resistance to nitrofurantoin were intrinsic. Plasmid screening coupled with resistance transfer studies showed that both chromosome-encoded and plasmid-encoded resistance mechanisms were clinically important. Ampicillin resistance was both chromosomally and plasmid encoded, whereas resistance to kanamycin and resistance to carbenicillin were exclusively plasmid encoded. Gentamicin resistance was more common than kanamycin resistance, and although gentamicin-resistant strains contained aminoglycoside acetyltransferase activity, no association could be demonstrated with plasmid deoxyribonucleic acid in the strains tested. Unlike minimal inhibitory concentrations for kanamycin, minimal inhibitory concentrations for gentamicin varied over a wide range. P. stuartii isolates obtained from several different countries were tested for comparison. As a group, these strains were less resistant, but they did exhibit similar resistance properties. The genus Providencia includes the species Providencia stuartii, Providencia alcalifaciens, and Providencia rettgeri (formerly Proteus rettgeri) (2). P. stuartii is one of the most antibioticresistant gram-negative bacilli and has been implicated in nosocomial infections, particularly those affecting the urinary tract (13, 14). Respiratory tract and burn infections have also been reported, but septicemia is uncommon and generally restricted to elderly, severely debilitated patients (8, 9). The reasons for the emergence of this organism as a pathogen are not known. However, the high incidence of resistance to many of the commonly used antibiotics and to certain disinfectants may have played a part. The presence of plasmids in Providencia species has been demonstrated previously, and the properties of transmissable plasmids from strains isolated in many countries have been described (6). A number of antibiotic resistance determinants have been located on these plasmids, including determinants for ampicillin resistance (Apr), chloramphenicol resistance, and tetracycline resistance. However, because P. stuartii exhibits such an unusually wide range of resistance properties, chromosomal determinants could also be responsible for clinically significant resistance. P. stuartii strains were isolated from infected patients in a group of Dublin hospitals over an 8-year period. Since these isolates were resistant to many antibiotics, including gentamicin, we undertook a detailed study of the genetic basis of the resistance. P. stuartii strains isolated in several other countries were collected and characterized in a similar manner for comparison. MATERIALS AND METHODS Bacterial strains and culture conditions. All Dublin strains of P. stuartii studied were primary isolates from patients in the Federated Dublin Voluntary Hospitals and were isolated between January 1972 and December The Federated Dublin Voluntary Hospital association consists of seven hospitals. No P. stuartii strains were isolated in one of these, a hospital for children. The other six hospitals contain 950 beds and cover most disciplines, including burns and genitourinary units. The P. stuartii isolates were identified by their biochemical profiles or by the API 20E system (Analytab Products, Plainview, N.Y.). A single colony of each P. stuartii isolate was inoculated onto two nutrient agar slants and a dorset egg slant and stored at room temperature. The organisms were inoculated onto fresh slants at intervals. The strains used as recipients in resistance transfer experiments were DUF186, a spontaneous rifampin-resistant (Rf) P. stuartii strain which contained no detectable plasmid deoxyribonucleic acid (DNA) and was ampicillin 1099

2 1100 McHALE, KEANE, AND DOUGAN sensitive (Ap'), gentamicin sensitive (Gm'), and kanamycin sensitive (Km'), and Escherichia coli J5-3 pro- met- Rf' (4). Strains were cultured in nutrient broth 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. Emmerson (London, England), A. McAllister (Glasgow, Scotland), D. J. Stickler (Cardiff, Wales), M. Nishida (Osaka, Japan), R. N. Jones (Oregon), G. Miller (New Jersey), 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. stuartii strains were used to designate five serotypes (FOl to F05), 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 each isolate to kanamycin (30 ig), gentamicin (10 ig), ampicilhin (10 or 25,ig), carbenicillin (100,ug), sulfafurazole (100 kg), trimethoprim (1.25 fig), tetracycline (10 Mg), nitrofurantoin (200,ug), penicillin G (2 U), polymyxin (2 gg), amikacin (10 Mg), and cefuroxime (30 Mg). Minimum inhibitory concentration (MIC) tests used doubling dilutions of antibiotic in diagnostic sensitivity test agar (Oxoid). Antibiotic resistance transfer. Transfer experiments 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 incubated at 37 C for 1 h or overnight. Transconjugants were purified on selective media and tested for serotype in the case of strain DUF186 (serotype F02) or for the abiity to ferment lactose in the case of strain J5-3. Plasmid screening. A modification of the procedure of Hansen and Olsen was used (5). Fresh overnight cultures of P. stuartii were diluted 1:100 into 100 ml of nutrient broth and grown for 3 h at 37 C with shaking. The cultures were chilled on ice, and the cells were harvested by centrifugation at 6,000 x g and 4 C for 10 min. The cells were washed once in TE buffer [50 mm tris(hydroxymethyl)aminomethane, 10 mm ethylenediaminetetraacetic acid, ph 8.0], resuspended in 0.6 ml of the same buffer, and transferred to clean 50-ml polycarbonate centrifuge tubes. Lysozyme [0.2 ml of a 5.0-mg/ml solution in 0.25 M tris(hydroxymethyl)aminomethane hydrochloride, ph 8.0] was added, and after 10 min of incubation at 37 C, 9 ml of lysing buffer (TE buffer containing 4% [wt/vol] sodium dodecyl sulfate, ph 12.35) was added and mixed well but gently. Then incubation was continued at 37 C for an additional 20 min to achieve complete lysis of the cells, 0.6 ml of 2.0 M tris(hydroxymethyl)- aminomethane hydrochloride (ph 7.0) was added, followed immediately by 0.24 ml of 5.0M NaCl, and the tubes were left on ice for 2 h. Chromosomal DNA was removed by centrifugation at 20,000 x g and 4 OC for 30 min. Each supernatant was decanted into a clean centrifuge tube, and 5.5 ml of ice-cold isopropanol was added to precipitate the plasmid DNA. The tubes J. CLIN. MICROBIOL. were incubated overnight at -20 C, and the precipitates were pelleted by centrifugation at 6,000 x g and 4 C for 10 min. The supernatants were discarded, and the pellets were dried under a vacuum; each pellet was suspended 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 the method of Meyers et al. (11). Aminoglycoside-inactivating enzyme assays. The cellulose phosphate paper binding system of Benveniste and Davies (1) was used to assay selected strains for the presence of aminoglycoside phosphorylating, adenylating, and acetylating enzyme activities. RESULTS P. stuartii strains were isolated from urine (142 strains), wound (93 strains), and sputum (3 strains) specimens of patients in the Federated Dublin Voluntary Hospitals between 1972 and 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 with the serotyping results, indicating that strains within serological groups were related, whereas strains that could not be serotyped were heterogeneous. Isolates of serotypes FO1 and F02 were cultured from both urine and wound specimens and from patients in all six hospitals. Antibiotic resistance properties of Dublin isolates and isolates from other countries. All P. stuartii isolates were tested for sensitivity to the antibiotics listed above. Most were multiply resistant, and as a group the Dublin strains were more resistant than the isolates from other countries (Table 2). All isolates from both groups were resistant to tetracycline, penicillin G, and polymyxin, and almost all were resistant to nitrofurantoin. Together with evidence obtained from plasmid screening and resistance transfer studies, these results suggest that these resistance properties are intrinsic. In both groups of P. stuartii, Apr was more common 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 FO F F F F Polyagglutinable Not typablea " Not serotypable with the five antisera used.

3 VOL. 13, 1981 TABLE 2. P. STUARTII ANTIBIOTIC RESISTANCE 1101 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- Amikacycline lin G cillin lin prim urazole toin acid roxime mycin micim cin Dublin Overseas a The values represent the percentages of isolates resistant to each antibiotic. TABLE 3. Serotype Cb' strains among Apr Dublin isolates of different serotypes No. ofap' No. of Ap' % Ap' Cb' Cb FO F F F F Polyagglutinable 0 2 Not typablea I a Not serotypable with the five antisera used H[nh _.._. -. h i - C :>100 M IC pg) z 100P mm<n z 25 <n FIG. 1. MICs of gentamicin (open bars) and kanamycin (cross-hatched bars) against P. stuartii isolates 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 from other countries. Within the Dublin group, the Apr Cb8 phenotype was more common in serotypes F02 and F03 than in serotype FO1 (Table 3). Although gentamicin resistance (Gmr) was common in both the Dublin isolates (83.6%) and the isolates from other countries (51.8%), kanamycin resistance (Kmr) was less frequently encountered (36.9 and 17.5%, respectively). We determined the MICs for gentamicin and kanamycin for the Dublin strains and detected a difference in the patterns of sensitivity (Fig. 1). Strains were either very sensitive or very resistant to kanamycin, whereas they exhibited a wide range of MICs for gentamicin (between 1.0 and A B C D E FIG. 2. Plasmid screening of P. stuartii strains on a 0. 7% agarose gel. Track A, Molecular weight markers 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 Dublin isolate; track F, United States isolate; track G, F05 Dublin isolate; track H, molecular weight markers Tpll6 (143 Md), RP4 (35 Md), pps002 (10.4 Md), and pac184 (2.8 Md). See reference 5 for molecular weights. 100,ug/ml). All KmT strains were neomycin resistant, and all Gmr strains were also tobramycin resistant. The MICs for neomycin and tobramycin were lower than the MICs for kanamycin and gentamicin, respectively. A high proportion of the Dublin strains were resistant to nalidixic acid, trimethoprim, and sulfafurazole, whereas nearly 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 30 isolates from other countries for the presence of plasmid DNA. The Dublin strains included isolates of the five serotypes and isolates from all six hospitals. Examples from each of the other sources were also included. Dublin isolates from within a particular serological group usually gave similar plasmid profiles, although exceptions did occur. Most serotype FO1 isolates tested harbored a single plasmid of approximately 50 megadaltons (Md) (Fig. 2, track C). F G H

4 1102 McHALE, KEANE, AND DOUGAN J. CLIN. MICROBIOL FIG. 3. Plasmid screening of transconjugant strains on 0.7% agarose gels. Tracks A weight and markers R27 (112 Md) and LT2 (60 I, Md); track B, strain Molecular DUF186; track from C, an F05 strain; DUF186 track D, DUF186 recipient recipient ofapr Cb' Kmr ofapr from Cbr F05; track E, F04 donor; track Dublin F, DUF186 Apr CbY recipient of Kmr from F04; track Kmr G, F01 Dublin Apr Cbr recipient of Ap' donor; track Cb' H, from F01; track J, DUF179; track K, DUF186 DUF186 track L, DUF186 recipient of Apr CbY Gm` recipient ofapr Cbr Gmr from from DUF1 DUF ; CHROM., Chromosome. 60 Md. Arrow 1, 112 Md; arrow 2, Isolates of serotypes F02, F03, and F04 all Cbr, and plasmid screening showed that a single harbored single plasmids of between approximately 40 and 60 Md (Fig. 2, tracks D and E). Isolates of serotype F05 and some isolates from the United States harbored two plasmids (Fig. 2, tracks F and G). Many of the isolates from other countries harbored single plasmids similar in size to the plasmids in the Dublin isolates. However, some of these strains contained plasmids of less than 15 Md, and strains which harbored no detectable plasmids were encountered more frequently (Fig. 2, track B). Transfer of antibiotic resistance markers to P. stuartii and E. coli We tested 45 Dublin isolates and 15 isolates from other countries for the ability to transfer resistance markers to P. stuartii DUF186 and E. coli J5-3. All Apr Cbr strains tested transferred these markers to strains DUF186 and J5-3 at high frequencies (>i0-3), indicating that these determinants were encoded on transmissible plasmids. In all cases a single plasmid was detected in the transconjugants (Fig. 3, tracks G and H). All Apr CbW strains failed to transfer the Apr determinant at a detectable frequency. We isolated spontaneous Apr Cb' derivatives of DUF186 but not of J5-3. Thus, the Apr Cb' phenotype was almost certainly due to chromosomal mutations in the clinical isolates. Similar findings were reported by Hedges (6). Different selective pressures may have been responsible for the emergence of these two types of ampicillin Apr resistance. Ail Km' P. stuartii strains tested transferred this resistance to both DUF186 and J5-3, and we showed that the Kmr transconjugants acquired a plasmid in all cases. Neomycin resistance and Kmr were always transferred together. Most strains transferred Km' together with Ap' and plasmid was responsible (data not shown). though AI- most FO1 isolates harbored plasmids of similar sizes, these plasmids were not identical always since some encoded Apr and CbW and others encoded Apr, Cb', and Km'. One serotype F04 strain was Apr Cb' Kmr. Only the Km' marker could be transferred from this and strain, this marker was located on a 60-Md plasmid (Fig. 3, tracks E and F). Dublin isolates serotype transferred F05 Km' 100-fold less than efficiently Ap' and Cbr. Transconjugants only Apr receiving and Cb' harbored a single 50-Md mid plas- (Fig. 3, track C), whereas Apr CbW transconjugants Kmr harbored this 50-Md plasmid together with a plasmid of 90 Md (Fig. 3, track D) Ṗlasmid-associated transfer of Gmr was not detected for any of the strains tested. As shown above, all detectable plasmids could be transferred from many isolates to strain DUF186 selecting for by either Apr or Km', but Gmr was never transferred together with these markers. Direct selection for Gmr transconjugants on nutrient agar containing rifampin and produced gentamicin a small number of colonies which were GmT, KICr, and amikacin resistant (Akr), contained no plasmid DNA, grew slowly in broth and on solid media, and were more sensitive to carbenicillin than strain DUF186. These colonies were serotype F02, suggesting that the Gmr was due to a spontaneous chromosomal mutation in strain DUF186. This was confirmed by plating strain DUF186 onto nutrient agar containing gentamicin. The mutants described above were isolated. Since direct selection with gentamicin enriched for spontaneous mutants of strain DUF186, we took a different approach,

5 VOL. 13, 1981 involving indirect selection of transconjugants. One Gmr Dublin isolate, DUF179 (serotype FO1), was studied in detail. This strain was Apr CbW Gmr Km8 and harbored a single 50-Md plasmid (Fig. 3, track J). A mating mixture of strains DUF179 and DUF186 was diluted and spread onto nutrient agar containing rifampin and ampicillin to select for Apr transconjugants. Plates containing a suitable number of Rf ' Apr colonies were replica-plated onto nutrient agar containing rifampin and gentamicin. About 1 in 300 colonies grew on the gentamicin containing agar. Such colonies were Rf F Apr CbV Gmr Km8 Ai and grew normally in broth. When examined for plasmids, both Rf Apr 0C Gm8 colonies isolated from the master plates and Rf' Apr CÇr Gm' colonies from the replica plates contained a single 50-Md plasmid (Fig. 3, tracks K and L). Cultures of Rf' ApT Cbh GmT colonies were mated with a nalidixic acid-resistant (NxT) derivative of J5-3, and the mating mixture was selected on nutrient agar containing nalidixic acid and either ampicillin or gentamicin. Colonies grew only on the plates containing ampicillin, and they were NxT ApT Cb' Gm and harbored the single 50-Md plasmid. These results indicate that the GmT determinant was not linked to the 50-Md plasmid. Aminoglycoside-inactivating enzymes. Aminoglycoside-resistant donor strains and the corresponding transconjugants were assayed for the presence of aminoglycoside-inactivating enzymes. None of the Gm8 KmA strains produced detectable levels of these enzymes. All of the Kmr donors and transconjugants contained a phosphorylase with the activity profile of aminoglycoside 3'-phosphotransferase I. This enzyme has been found in P. stuartii by other workers (15). All Gmr strains tested produced an acetyltransferase with the activity profile of aminoglycoside 2'-N-acetyltransferase 2. The Gmr transconjugants isolated by the replica-plating method all produced this enzyme, whereas GmT Km' Akr mutants did not. No adenyltransferase activity was detected in any strains tested. Thus, our results suggested that aminoglycoside-inactivating enzyme activity is responsible at least in part for clinically significant Gmr and KmT. DISCUSSION Our results show that both plasmid-encoded resistance and chromosome-encoded resistance to antimicrobial agents are common in clinical isolates of P. stuartii. A number of observations suggest that these organisms may possess unusual cell envelope properties which contribute to the resistance properties. Unlike most gramnegative bacilli, which lyse spontaneously in the P. STUARTII ANTIBIOTIC RESISTANCE 1103 lysing buffer used in the plasmid screening technique, most P. stuartii isolates only lysed after pretreatment with lysozyme. While attempting curing experiments, we found that P. stuartii strains were resistant to high levels of ethidium bromide (>500,ug/ml) and acridine orange (>750,tg/ml) (McHale, unpublished data). Strains were also intrinsically resistant to polymyxin, which acts at the cell walls of sensitive bacteria. All of these unusual properties could be mediated in the cell envelope, although further studies wiil be needed to confirm this suggestion conclusively. Most of the Dublin isolates were resistant to the antimicrobial drugs commonly used to treat urinary tract infections, including nitrofurantoin, trimethoprim, and sulfafurazole. Most were also resistant to gentamicin, a drug in widespread use as a broad-spectrum antibiotic against gram-negative infections. These resistance properties probably explain the emergence of P. stuartii in hospital-acquired urinary tract and wound infections. Drugs such as amikacin and cefuroxime may be more useful in treating P. stuartii infections since nearly all strains tested 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, particularly in Dublin isolates, but chromosomal resistance was also clinically important in strains which were obtained from an outbreak of crossinfection (3). Ap' among Providencia strains from many countries has also been reported by Hedges (6). The results of the aminoglycoside resistance determinations were complex. Spontaneous KmT GmT AkT mutants were isolated during this study, but they did not appear to be clinically significant since they grew poorly and were very sensitive to carbenicillin. The mechanism of resistance in these spontaneous mutants is not known. KmT and neomycin resistance were exclusively plasmid encoded in the clinical isolates and were due to the production of aminoglycoside 3'-phosphotransferase I. GmT was more common among P. stuartii isolates than Km'. Ail Gm' strains were also tobramycin resistant. Although the MICs for these two antibiotics varied over a wide range, all GmT isolates tested contained the enzyme aminoglycoside 2'-N-acetyltransferase 2. No association could be established between plasmid DNA and GmT in any of the strains. GmT and aminoglycoside 2'-N-acetyltransferase 2 activity were transferred together to strain DUF186 but were not linked on the single 50-Md ApT CbT plasmid transferred, since this plasmid could be retransferred inde-

6 1104 McHALE, KEANE, AND DOUGAN pendently of Gmr to strain J5-3. Thus, the Gmr determinant had not transposed onto this plasmid. Gmr has been shown to be encoded on a transposon in other gram-negative bacteria (12, 16) Ġmr must have been transferred to strain DUF186 either on an undetected plasmid or, if it was chromosomally encoded in strain DUF179, by some other mechanism, possibly involving a bacteriophage or low-level Hfr formation in the donor cells. Other reports (7) on chromosomally determined Gmr implicate impaired uptake of the antibiotic as the mode of resistance rather than enzymatic inactivation. However, all of our Gmr strains produced an aminoglycoside acetyltransferase. The level of resistance was variable. This variability may have been due to secondary mutations within the bacterial chromosome which varied the amount of enzyme being produced or its release. Selective pressures, such as the use of gentamicin, probably led to these variations. Certainly, gentamicin unlike kanamycin, is used widely in the Dublin hospitals, and Gmr is found in the Pseudomonas aeruginosa and Staphylococcus aureus strains isolated in these hospitals. This suggests that there is a strong selection for Gmr in 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 reading the manuscript, and others in the Moyne Institute (especially Gillian Johnson). This work was supported by a grant to P.M. by the Laboratory Medicine Development Fund and by a grant to G.D. from the Irish Medical Research Council. LITERATURE CITED 1. Benveniste, R., and J. Davies Mechanisms of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42: Brenner, D. J., J. J. Farmer, F. W. Hickman, M. A. Asbury, and A. G. Steigerwalt Taxonomic and J. CLIN. MICROBIOL. nomenclature changes in Enterobacteriaceae. U.S. Department of Health, Education and Welfare Publication (CDC) Government Printing Office, Washington, D.C. 3. British Medical Journal Unusual infection in an intensive care unit. Br. Med. J. 1: Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. Richmond Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108: Hansen, J. B., and R. H. Olsen Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pmg1 and pmg5. J. Bacteriol. 135: Hedges, R. W R factors from Providence. J. Gen. Microbiol. 81: John, J. F., C. E. Rubens, and W. E. Farrar Characterization of gentamicin resistance in nosocomial infections. Am. J. Med. Sci. 271: Keane, C. T., L. F. English, and R. Wise Providencia stuartii infections. Lancet ii: Kocka, F. E., S. Srinivasan, M. Mowjood, and H. S. Kantor Nosocomial multiply resistant Providencia stuartii: a long-term outbreak with multiple biotypes and serotypes at one hospital. J. Clin. Microbiol. 11: McHale, P., L. English, A. Speekenbrink, C. Keane, and R. Wise Kanamycin resistance in Providencia stuartii. J. Antimicrob. Chemother. 4: Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Falkow Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127: Nugent, M. E., D. H. Bone, and N. Datta A transposon, Tn732, encoding gentamicin and tobramycin resistance. Nature (London) 282: Overturf, G. D., J. Wilkins, and R. Ressler Emergence of resistance of Providencia stuartii to multiple antibiotics: speciation and biochemical characterization of Providencia. J. Infect. Dis. 129: Penner, J. L., N. A. Hinton, I. B. R. Duncan, J. N. Hennessy, and G. R. Whiteley O-serotyping of Providencia stuartii isolates collected from twelve hospitals. J. Clin. Microbiol. 9: Price, K. E., T. A. Pursiano, M. D. DeFuria, and G. E. Wright Activity of BB-K8 (amikacin) against clinical isolates resistant to one or more aminoglycoside antibiotics. Antimicrob. Agents Chemother. 5: Rubens, C. E., W. F. McNeil, and W. E. Farrar Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents. J. Bacteriol. 139: