Vol. 31, No. 1ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1987, p. 70-750066-4804/87/010070-06$02.00/0Copyright © 1987, American Society for Microbiology

Characterization of Impermeability Variants of Pseudomonasaeruginosa Isolated during Unsuccessful Therapy of

Experimental EndocarditisARNOLD S. BAYER,1,2* DEAN C. NORMAN,2'3 AND KWANG SIK KIM1' 2t

Departments of Medicine and Pediatrics, Harbor-University of California, Los Angeles, Medical Center, Torrance,California 905091*; Research and Medical Services, Wadsworth Veterans Administration Medical Center, Los Angeles,

California 900733; and School of Medicine, University of California, Los Angeles, Los Angeles, California 900242

Received 7 July 1986/Accepted 21 October 1986

We characterized five amikacin-resistant variants of Pseudomonas aeruginosa isolated from aortic valvevegetations during unsuccessful therapy of experimental endocarditis. These organisms were cross resistant toother aminoglycosides. No aminoglycoside-modifying enzymes were produced by these strains. However, allfive variants demonstrated significant defects in permeability and intracellular uptake of [3lHlamikacin whencompared with the amikacin-susceptible parental strain (0 to 26% of that of the parental strain; mean, -15%).The permeability defects were unstable in vitro, with normalization after serial passage in antibiotic-freemedia. The variants grew as nonpigmented, small-colony types, with in vitro generation times -1.5 to 2 timeslonger than that of the parental strain (30 to 40 versus 20 min, respectively). Two impermeability variants werecompared with the parental strain for ability to induce experimental endocarditis in rabbits with aorticcatheters. Both variants were virulent in vivo; however, mean bacterial densities in vegetations were -2.5 logl0CFU/g lower in animals challenged with the variants than in animals challenged with the parental strain,probably reflecting a slower in vivo growth rate.

One problem limiting the success of medical therapy inhumans with Pseudomonas aeruginosa endocarditis hasbeen the development of antibiotic resistance in vivo (19).This has been most prominent and prevalent with infectionof left-sided cardiac valves and has usually involved aP-lactam agent (e.g., piperacillin [19]). We recently reportedon the development of aminoglycoside-3-lactam resistancein vivo during amikacin-ceftazidime therapy of rabbits withexperimentally induced aortic valve endocarditis caused byP. aeruginosa (3). In that study, the emergence of multiplyresistant isolates within cardiac vegetations was associatedwith poor bacteriologic efficacy in the model. The presentinvestigation (i) examined the in vitro characteristics of theamikacin resistance manifested by the P. aeruginosa vari-ants from the latter study, and (ii) delineated the in vivovirulence of these variants in the experimental endocarditismodel.

(This investigation was presented in part at the 23rdInterscience Conference on Antimicrobial Agents and Che-motherapy, Las Vegas, Nev., 24 to 26 October 1983 [A. S.Bayer, D. Norman, D. Anderson, J. 0. Morrison, and K. S.Kim, Program Abstr. 23rd Int. Conf. Antimicrob. AgentsChemother., abstr. no. 370, 1983] and the 85th AnnualMeeting of the American Society for Microbiology, LasVegas, Nev., 3 to 7 March 1985 [A. S. Bayer, D. C. Norman,and K. S. Kim. Abstr. Annu. Meet. Am. Soc. Microbiol.1985, A42, p. 8].)

MATERIALS AND METHODS

Organisms. The P. aeruginosa strain (PA-96) used tooriginally infect animals during the induction of experimental

* Corresponding author.t Present address: Division of Pediatric Infectious Diseases,

Children's Hospital of Los Angeles, Los Angeles, CA 90027.

aortic valve endocarditis was a clinical isolate used inseveral earlier efficacy studies in this laboratory (2, 3, 11). Itsidentification, serotyping, and resistance to rabbit serumwere previously described (11). The details of the inductionof endocarditis, antimicrobial therapy, and animal sacrificeswere recently given elsewhere (3). Five amikacin-resistantvariants of PA-96 from the latter study (PA-83-627, -83-630,-20, -43, and -61) were originally isolated on amikacin (50,ug/ml)-containing Mueller-Hinton agar (MHA; BBL Micro-biology Systems, Cockeysville, Md.) pour plates duringquantitative culturing of cardiac vegetations. Three of thesefive variants (PA-83-627, -43, and -61) were recovered fromanimals at day 14 of amikacin monotherapy, and two(PA-83-630 and -20) were isolated at day 14 of amikacin-ceftazidime treatment (3). PA-83-630 and PA-20 exhibitedresistance in vitro only to amikacin (not ceftazidime). MostPseudomonas organisms within cardiac vegetations ob-tained from these five animals exhibited amikacin resistance,with resistance ratios (10) ranging from -2.33 to -5.0 (mean± standard error of the mean, -4.24 ± 0.26).Growth characteristics. Growth curves for two amikacin-

resistant variants (PA-83-627 and -83-630) were comparedwith that for the parental strain (PA-96) in antibiotic-free MHbroth. The strains were grown overnight at 37°C in MH brothand adjusted by McFarland nephelometric standards tocontain -106 CFU/ml as the starting inoculum at time zero.Samples (1 ml) were quantitatively cultured in MHA at 0, 2,4, 8, 12, and 24 h to construct the growth curves.

Antibiotic susceptibility testing. The MICs of amikacin,kanamycin, and gentamicin were determined by the standardagar dilution method (23) using MHA. The range of concen-trations tested was 0.5 to 128 jig/ml. Logarithmic-growth-phase cultures were used to prepare inocula of the P.aeruginosa strains at 5 x 107 CFU/ml. A 1-,lI sample of theinoculum was transferred to antibiotic-containing and con-

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AMIKACIN PERMEABILITY IN PSEUDOMONAS ENDOCARDITIS 71

trol plates. The MIC was defined as the lowest antibioticconcentration causing no visible growth on the plates after24 h of incubation at 37°C. Resistance to amikacin andkanamycin was considered to be indicated by an MIC of >32Fjg/ml, and resistance to gentamicin was indicated by an MICof >16 p.g/ml (16).

In vitro passage studies. The stability of amikacin resis-tance induced in vivo was determined by in vitro passage

experiments. One amikacin-resistant variant (PA-83-630),isolated from cardiac vegetations of an animal receivingamikacin-ceftazidime treatment, was studied. This strainwas grown in antibiotic-free MH broth overnight and thenparallel plated onto both antibiotic-free and amikacin (50pg/ml)-containing MHA. The serial passage of this strain inantibiotic-free MH broth, with parallel plating as describedabove, was done 10 more times.

Detection of aminoglycoside-modifying enzymes. Cell-freepreparations of the five selected amikacin-resistant variantsof PA-96 cultured from cardiac vegetations were made byultrasonic disruption as previously described (4, 18). Thepresence of aminoglycoside-modifying enzymes in crudecell-free preparations was demonstrated by a previouslyreported method (4, 18). Briefly, reaction mixtures consistedof the cell-free preparation, the aminoglycoside being tested,and the radiolabeled substrate. [14C]ATP was used for de-tecting the presence of adenyltransferases, 14C-acetyl coen-

zyme A was used for detecting acetyltransferases, and[32P]ATP was used for detecting phosphotransferases. Allradiolabeled enzyme substrates were obtained fromAmersham-Searle, Arlington Heights, Ill. The test aminogly-cosides were amikacin, kanamycin, and gentamicin. Positivecontrols for the various enzyme classes were included inparallel testing. The amount of radioactivity specificallyassociated with each test aminoglycoside was estimated byusing a Tri-Carb liquid scintillation spectrometer (PackardInstrument Co., Inc., Downer's Grove, Ill.).Measurement of intracellular accumulation of [3H]ami-

kacin. Transmembrane permeability defects of amikacintransport were detected by measuring the amount of radio-labeled amikacin accumulating within bacterial cells bypreviously described techniques (9, 17). The isolates testedwere (i) PA-96 (parental strain), (ii) the five amikacin-resistant Pseudomonas variants from cardiac vegetations,(iii) an amikacin-resistant strain (PA-83-630) which revertedto amikacin susceptibility in vitro within five passages

through antibiotic-free MH broth, and (iv) a wild-type,amikacin-susceptible P. aeruginosa reference strain (PA-A9843a) with an amikacin MIC of 0.5 ig/ml. Cells were grownexponentially in MH broth, and the suspensions were stan-dardized to an inoculum of -2 x 109 cells in a ColemanJunior spectrophotometer (Coleman Instruments, May-wood, Ill.) by an optical density of 0.21 at 550 nm.

[3H]amikacin was added to give a radioactivity level of 1

RCi/ml. "Cold" (unlabeled) amikacin was then used toachieve a final antibiotic concentration of 4 pg/ml (sub-MIC)for the amikacin-resistant variants (PA-83-627, -83-630, -20,-43, and -61) and 0.125 pg/ml (sub-MIC) for the amikacin-susceptible strains (PA-96, PA-83-630 [postpassage], andPA-A 9843a). Cold amikacin at sub-MIC produces consistentgrowth curves during the subsequent 120-min samplingperiod (9). Samples of the bacterial suspension were re-

moved at 0, 30, and 120 min, and the optical density was

recorded. Cells were collected by filtration and washed anddried. The [3H]amikacin content of each sample was thenestimated in a Tri-Carb liquid scintillation spectrometer.Uptake of amikacin was expressed as nanograms of amika-

cin activity incorporated by a suspension containing 2 x 109cells over the 120-min sampling period. Organisms havinguptake values of <60% of that of the wild-type, amikacin-susceptible reference strain of P. aeruginosa (PA-A 9843a)were considered to have a significantly impaired transmem-brane permeability for amikacin (17).

Scanning and transmission EM. To determine whether theamikacin-resistant phenotype was associated with any ultra-structural changes in the organisms isolated from cardiacvegetations, scanning and transmission electron microscopy(EM) were performed. Vegetations were preserved in 2.5%glutaraldehyde. Those vegetations from which the amikacin-resistant Pseudomonas variants used in this investigationwere obtained were prepared for transmission EM by stan-dard techniques (12). Tissue blocks of vegetations were alsofreeze fractured and prepared for scanning EM by themethod of Hayat (13).Animal virulence studies. The in vivo virulence of two

amikacin-resistant variants was compared with that of theparental strain (PA-96) in the experimental endocarditismodel. The two variants were the same strains as those usedin the in vitro growth-curve analyses described above(PA-83-627 and -83-630). Briefly, 10 New Zealand Whiterabbits were anesthetized with ketamine hydrochloride andxylazine given intramuscularly and transcarotid-to-transaortic valve catheterization was performed as previ-ously described (1). Twenty-four hours after catheterization,four animals each received -108 CFU of one of the Pseu-domonas variants as an intravenous challenge, and twoanimals were so challenged by the parental strain. Inductionof bacterial endocarditis was confirmed at the time of sacri-fice, 24 h after the intravenous challenge, by quantitativelyculturing homogenates of individual aortic valve vegetations(1). Vegetation homogenates from animals challenged withthe resistant variants were parallel plated into antibiotic-freeand antibiotic (amikacin, 50 pLg/ml)-containing MHA; thiswas to confirm maintenance of the resistance phenotypeduring the 24-h period after induction of endocarditis. Re-sistance ratios, defined as the log10 of the ratio of the numberof amikacin-resistant organisms to the total number of organ-isms, were determined for each vegetation (10).

RESULTS

Antibiotic susceptibility tests. The parental strain (PA-96)used to initially infect cardiac vegetations was susceptible toamikacin and gentamicin (MICs, 2 and 1 pug/ml, respectively[Table 1]); the kanamycin MIC was 32 [kg/ml. In contrast,the five Pseudomonas variants obtained from vegetations onamikacin-containing MHA not only exhibited amikacin re-sistance but were also now cross resistant to gentamicin(MICs, 64 pLg/ml; Table 1). Moreover, the kanamycin MICsfor these five variants had increased at least eightfold (MICs,>128 [ig/ml).

In vitro passage studies. The significantly increased MICsfor PA-83-630 were labile in vitro upon serial passage inantibiotic-free media. This isolate no longer grew on amika-cin (50 ,ig/ml)-containing agar after five passages in antibi-otic-free media. Moreover, this variant reverted to a genta-micin- and amikacin-susceptible state postpassage (MICs, 2and 4 1Lg/ml, respectively). In addition, the kanamycin MICdecreased at least fourfold postpassage (64 pg/ml).Growth characteristics. At the time of initial isolation from

cardiac vegetations (2), surface colonies from the amikacin-resistant Pseudomonas variants produced single colonies of-1 mm at 24 h of incubation (37°C) on amikacin-containing

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TABLE 1. Antimicrobial susceptibility and transmembrane permeability of radiolabeled amikacin for parental and variantP. aeruginosa strains

Isolate TherapY MIC (ug/ml) % Uptake ofAmikacin Kanamycin Gentamicin I3HJamlikacina

PA-96 (parent) 2 32 1 100PA-A 9843a (wild type) 0.5 NDb ND 100PA-83-627 Amikacin 128 >128 64 5PA-43 Amikacin 128 >128 64 21PA-61 Amikacin 32 >128 64 26PA-20 Combinationc 128 >128 64 21PA-83-630 Combinationc 128 >128 64 0PA-83-630 (postpassage) 4 64 2 89

a Uptake expressed as percent of that achieved by wild-type susceptible P. aeruginosa (PA-A 9843a).b ND, Not done.c Amikacin-ceftazidime.

MHA. Also, these colonies failed to produce pyocyaninpigment at the time of initial isolation from cardiac vegeta-tions. This phenotypic trait was labile, and pigment produc-tion was regained after serial passage of the variants onantibiotic-free media. In addition to regaining pigment pro-duction, variants produced larger surface colonies (2 to 3mm) after serial in vitro passage on antibiotic-free media andresembled the parental strain (PA-96) morphologically. Bythe end of 24 h of incubation, the parental strain hadachieved counts of >9 loglo CFU/ml on growth-curve anal-ysis; in contrast, both variants reached counts of -1 logloCFU/ml lower at this point (Fig. 1). Moreover, the genera-tion (doubling) times of the two variants, determined duringa logarithmic growth phase (2- to 4-h period), were 1.5 to 2times longer than for the parental strain (30 and 40 versus 20min).

Aminoglycoside-modifying enzymes. There was no evi-

, PA -96

E

U-

0

(-9

0--J

PA-83-627

sterile

0 2 4 8 12

HOURS24

FIG. 1. Growth curves of parental P. aeruginosa strain (PA-96)and two amikacin-resistant impermeability variants (PA-83-627 and-83-630).

dence of production of any of the eight definable aminogly-coside-modifying enzymes (aminoglycoside acetyltrans-ferase [2', (3) I, (3)II, (3)III, (3)IV, 6'], aminoglycosidenucleotidyltransferase [previously aminoglycose adenyl-transferase; 211, 41], and aminoglycoside phosphotransfer-ase) by the five amikacin-resistant Pseudomonas variants.Amikacin transmembrane permeability. As seen in Table

1, the parental strain (PA-96) exhibited normal transmem-brane permeability to radiolabeled amikacin, identical to thatfor the amikacin-susceptible reference strain of P. aerugi-nosa. In contrast, all five amikacin-resistant variants dem-onstrated significant defects in transmembrane penetrationand uptake of radiolabeled amikacin, ranging from 0 to 26%amikacin uptake in relation to the reference strain (mean,-15%). Of note, amikacin permeability for strain PA-83-630normalized after serial passage in antibiotic-free media,coincident with a greater-than-fourfold decrease in amika-cin, gentamicin, and kanamycin MICs. Prepassage, therewas no detectable uptake of radiolabeled amikacin forPA-83-630, whereas amikacin uptake was 89% of that of thereference strain postpassage.EM. Freeze-fracture preparations of selected cardiac veg-

etations, from which the five amikacin-resistant variantswere obtained, were studied by scanning EM. The scanningEM demonstrated morphologically normal bacilli within thedepths of the vegetations (Fig. 2A). Cell elongation inpreparation for pinching off and division was commonlynoted. None of the frequently observed morphologicchanges seen when P. aeruginosa is exposed in vitro to 1/2MIC of amikacin was noted in these in vivo preparations.These included increased interorganism filamentation andnub and stalked nub formations (22).

Similarly, transmission EM of these same vegetations,containing large numbers of amikacin-resistant variants,revealed normal-appearing bacilli surrounded by an undu-lated, trilamellar wall with a smooth surface (Fig. 2B). Noneof the frequently observed cell wall or intracellular changesinduced in vitro by aminoglycosides at sub-MIC was noted.These include cell wall bleb formation, ribosomal peripher-alization, and reduction in ribosomal numbers (14).Animal virulence studies. Both amikacin-resistant variants

were virulent in vivo, inducing aortic valve endocarditis inall 10 catheterized animals which were challenged intrave-nously with these strains. Of note, the slower growth rate ofthe variants in vitro was apparently reflected in vivo. Meanbacterial densities achieved by the variants within cardiacvegetations were -2.5 log,o CFU/g lower than those seen inanimals challenged with the parental strain (Table 2). Resis-

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AMIKACIN PERMEABILITY IN PSEUDOMONAS ENDOCARDITIS 73

FIG. 2. (A) High-powered (magnification, x7,000) scanning electron micrograph showing numerous bacilli within depths of cardiacvegetation. This vegetation contained -5 log10 CFU of amikacin-resistant P. aeruginosa per g of tissue. Note the normal morphology of thesebacilli, including many undergoing elongation in preparation for cell division (arrows). (B) Transmission electron micrograph of P. aeruginosafrom the same cardiac vegetation as that shown in panel A (magnification, x55,000). Organisms appear normal morphologically.

tance ratios were low (<-2) in each vegetation from animalschallenged with the resistant variants; this indicated a main-tenance of the amikacin-resistant phenotype over the 24-hperiod after induction of endocarditis (10).

DISCUSSION

Amikacin permeability mutants of P. aeruginosa are fre-quently demonstrated in epidemiologic surveys of aminogly-coside-resistant organisms. For example, Price et al. showedthat -90% of aminoglycoside-resistant P. aeruginosa that donot produce aminoglycoside-modifying enzymes exhibit im-paired uptake and permeability of amikacin (18). However,correlation of clinically definable infections with the in vivoisolation of such permeability mutants during amikacin ther-apy has been only sporadically reported in the literature.Meyer found four P. aeruginosa isolates which were resis-

tant to amikacin on the basis of permeability defects amongfive patients in whom amikacin resistance was convincinglyassociated with clinical failures of therapy (15). The trueprevalence of aminoglycoside-resistant variants of the im-

TABLE 2. Mean bacterial density in cardiac vegetations

Organism ~~~~~~~~DensityOrganism lmean logi0 CFU/g ± SEM]-

PA96b....... 9.18 ± 0.47 (2/7)PA-83-627C...... 6.46 ± 0.37 (4/8)PA-83-630C...... 6.32 ± 0.73 (4/6)

a The number of animals sacrificed/number of vegetations cultured is shownin parentheses. P < 0.005 for the amikacin-resistant variants versus theparental strain.

b Parental Pseudomonas strain.c Amikacin-resistant variant.

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permeability phenotype has not been adequately defined inhuman cases of Pseudomonas endocarditis. Routine isola-tion of variants of this type in the clinical laboratory is likelyto be rare because of the high reversion frequencies and slowin vitro growth rates relative to those of wild-type isolates(6). These important data need to be pursued in geographicareas that continue to see large numbers of patients withPseudomonas endocarditis (19, 21). When such patientssuffer bacteriologic relapse or failure, the clinical laboratoryshould routinely plate appropriate cultures onto media con-taining supra-MICs of aminoglycoside in parallel with stan-dard susceptibility testing schemas. Disparities in resultsbetween the two procedures (i.e., the organism testing assusceptible by routine methods, yet exhibiting growth onaminoglycoside-containing media) suggest the presence ofthe impermeability phenotype.

Aminoglycoside uptake and transmembrane permeationhave been shown to represent a complex, multifactorialprocess involving (i) initial ionic interaction with the outercell envelope, (ii) an energy-dependent, electron-transport-requiring process and negative membrane potential, and (iii)binding to specific ribosomal binding sites with a secondary,energy-dependent enhancement of amihoglycoside accumu-lation (7). Moreover, Bryan et al. recently demonstrated thata clinical isolate of P. aeruginosa (from a burn patient) withimpermeability-type amninoglycoside resistance had signifi-cant alterations in the lipopolysaccharide structure of theouter cell membrane as an additional cofactor for this type ofresistance (8). Thus, defects in one or more of the amino-glycoside-uptake pathways may result in the impermeabilityphenotype. The small-colony morphology, relatively slow invitro growth characteristics, labile nature of the imperme-ability defect in vitro and in vivo, and retention of in vivovirulence (albeit with evidence of slower in vivo growthrates) each suggest that the impermeability variants in thisstudy were analogous to the unstable "energy" mutants(microbial persisters) recently described by Bryan et al. (6).Variants of this sort exhibit impaired aminoglycoside uptakebecause of altered electron-transport systems or an impairedelectrical potential (5, 6). Although we did not direcilyeliminate ribosomal-type resistance as a factor in our iso-lates, such resistance to aminoglycosides other than strep-tomycin is not believed to occur in clinical isolates (20).The reason stich impermeability-type aminoglycoside re-

sistance is commonly observed in Pseudomonas strainsisolated from left-sided cardiac vegetations but has not beenseen in organisms obtained from right-sided cardiac vegeta-tions is also not known (2, 3, 11). The major detectabledifference between vegetations on the two sides of the heartinvolve bacterial densities, which tend to be -2 to 4 log10higher in left-sided vegetations (1-3, llj. Also, the EManalysis in this study provided indirect evidence that sub-MICs of aminoglycosides were achieved within the depths ofcardiac vegetations. How a large in vivo inoculum andreduced aminoglycoside penetration into cardiac vegetationsmight interrelate to predispose for the development of im-permeability-type amikacin-resistance in vivo remains to beelucidated.

ACKNOWLEDGMENTS

We thank Peter Kresel (Bristol Mechanisms of Resistance Labo-ratory, Syracuse, N.Y.) for performing the enzyme modification andpermeability assays. We thank Robert Hancock and Thomas Parrfor their critical review of the manuscript.

This study was supported in part by a research grant from BristolResearch Laboratories.

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8. Bryan, L. E.,-K. O'Hara, and S. Wong. 1984. Lipopolysaccha-ride changes in impermeability-type aminoglycoside resistancein Pseudomonas aeruginosa. Antimicrob. Agents Chemother.26:250-255.

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13. Hayat, M. A. 1974. Scanning electron microscopy-biologicalapplications, p. 4-10. Van Nostrand Reinhold Co., New York.

14. Iida, K., and M. Koike. 1974. Cell wall alterations of gram-negative bacteria by aminoglycoside antibiotics. Antimicrob.Agents Chemother. 5:95-97.

15. Meyer, R. D. 1977. Patterns and mechanisms of resistance toamikacin. J. Infect. Dis. 136:449-452.

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17. Price, K. E., M. D. DeFuria, and T. A. Pursiano. 1976. Amika-cin, an aminogylcoside with marked activity against antibiotic-resistant clinical isolates. J. Infect. Dis. 134(Suppl.):249-261.

18. Price, K. E., P. A. Kresel, L. A. Farchione, S. B. Siskin, andS. A. Karpow. 1981. Epidemiologic studies of aminoglyco-side resistance in the USA. J. Antimicrob. Chemother.8(Suppl.):89-105.

19. Reyes, M. P., and A. M. Lerner. 1983. Current problems in thetreatment of infective endocarditis due to Pseudomonas aeru-ginosa. Rev. Infect. Dis. 5:314-321.

20. Shannon, D., and I. Phillips. 1982. Mechanisms of resistance toaminoglycosides in clinical isolates. J. Antimicrob. Chemother.9:91-102.

21. Shekar, R., T. W. Rice, C. H. Zierdt, and C. A. Kallick. 1985.Outbreak of endocarditis caused by Pseudomonas aeruginosaserotype O11 among pentazocine and tripelennamine abusers in

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Chicago. J. Infect. Dis. 151:203-208.22. Waisbren, S. J., D. J. Hurley, and B. A. Waisbren. 1980.

Morphological expressions of antibiotic synergism against Pseu-domonas aeruginosa as observed by scanning electron micros-copy. Antimicrob. Agents Chemother. 18:969-975.

23. Washington, J. A., II, and V. L. Sutter. 1980. Dilution suscep-tibility test: agar and macro-broth dilution procedures, p.453-458. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., andJ. P. Truant (ed.), Manual of clinical microbiology, 3rd ed.American Society for Microbiology, Washington, D.C.

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