Characterization of Impermeability Variants of Pseudomonas aeruginosa Isolated during Unsuccessful Therapy of Experimental Endocarditis

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 31, No. 1 Characterization of Impermeability Variants of Pseudomonas aeruginosa Isolated during Unsuccessful Therapy of Experimental Endocarditis ARNOLD 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 *; Research and Medical Services, Wadsworth Veterans Administration Medical Center, Los Angeles, California ; and School of Medicine, University of California, Los Angeles, Los Angeles, California Received 7 July 1986/Accepted 21 October 1986 We characterized five amikacin-resistant variants of Pseudomonas aeruginosa isolated from aortic valve vegetations during unsuccessful therapy of experimental endocarditis. These organisms were cross resistant to other aminoglycosides. No aminoglycoside-modifying enzymes were produced by these strains. However, all five variants demonstrated significant defects in permeability and intracellular uptake of [3lHlamikacin when compared 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-free media. The variants grew as nonpigmented, small-colony types, with in vitro generation times -1.5 to 2 times longer than that of the parental strain (30 to 40 versus 20 min, respectively). Two impermeability variants were compared with the parental strain for ability to induce experimental endocarditis in rabbits with aortic catheters. Both variants were virulent in vivo; however, mean bacterial densities in vegetations were -2.5 logl0 CFU/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 in humans with Pseudomonas aeruginosa endocarditis has been the development of antibiotic resistance in vivo (19). This has been most prominent and prevalent with infection of left-sided cardiac valves and has usually involved a P-lactam agent (e.g., piperacillin [19]). We recently reported on the development of aminoglycoside-3-lactam resistance in vivo during amikacin-ceftazidime therapy of rabbits with experimentally induced aortic valve endocarditis caused by P. aeruginosa (3). In that study, the emergence of multiply resistant isolates within cardiac vegetations was associated with poor bacteriologic efficacy in the model. The present investigation (i) examined the in vitro characteristics of the amikacin resistance manifested by the P. aeruginosa variants from the latter study, and (ii) delineated the in vivo virulence of these variants in the experimental endocarditis model. (This investigation was presented in part at the 23rd Interscience Conference on Antimicrobial Agents and Chemotherapy, 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. Agents Chemother., abstr. no. 370, 1983] and the 85th Annual Meeting of the American Society for Microbiology, Las Vegas, 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 to originally 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 aortic valve endocarditis was a clinical isolate used in several earlier efficacy studies in this laboratory (2, 3, 11). Its identification, serotyping, and resistance to rabbit serum were previously described (11). The details of the induction of endocarditis, antimicrobial therapy, and animal sacrifices were recently given elsewhere (3). Five amikacin-resistant variants of PA-96 from the latter study (PA , , -20, -43, and -61) were originally isolated on amikacin (50,ug/ml)-containing Mueller-Hinton agar (MHA; BBL Microbiology Systems, Cockeysville, Md.) pour plates during quantitative culturing of cardiac vegetations. Three of these five variants (PA , -43, and -61) were recovered from animals at day 14 of amikacin monotherapy, and two (PA and -20) were isolated at day 14 of amikacinceftazidime treatment (3). PA and PA-20 exhibited resistance in vitro only to amikacin (not ceftazidime). Most Pseudomonas organisms within cardiac vegetations obtained from these five animals exhibited amikacin resistance, with resistance ratios (10) ranging from to -5.0 (mean ± standard error of the mean, ± 0.26). Growth characteristics. Growth curves for two amikacinresistant variants (PA and ) were compared with that for the parental strain (PA-96) in antibiotic-free MH broth. The strains were grown overnight at 37 C in MH broth and adjusted by McFarland nephelometric standards to 70 contain -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 standard agar dilution method (23) using MHA. The range of concentrations tested was 0.5 to 128 jig/ml. Logarithmic-growthphase cultures were used to prepare inocula of the P. aeruginosa strains at 5 x 107 CFU/ml. A 1-,lI sample of the inoculum was transferred to antibiotic-containing and con-

2 VOL. 31, 1987 AMIKACIN PERMEABILITY IN PSEUDOMONAS ENDOCARDITIS 71 trol plates. The MIC was defined as the lowest antibiotic concentration causing no visible growth on the plates after 24 h of incubation at 37 C. Resistance to amikacin and kanamycin was considered to be indicated by an MIC of >32 Fjg/ml, and resistance to gentamicin was indicated by an MIC of >16 p.g/ml (16). In vitro passage studies. The stability of amikacin resistance induced in vivo was determined by in vitro passage experiments. One amikacin-resistant variant (PA ), isolated from cardiac vegetations of an animal receiving amikacin-ceftazidime treatment, was studied. This strain was grown in antibiotic-free MH broth overnight and then parallel plated onto both antibiotic-free and amikacin (50 pg/ml)-containing MHA. The serial passage of this strain in antibiotic-free MH broth, with parallel plating as described above, was done 10 more times. Detection of aminoglycoside-modifying enzymes. Cell-free preparations of the five selected amikacin-resistant variants of PA-96 cultured from cardiac vegetations were made by ultrasonic disruption as previously described (4, 18). The presence of aminoglycoside-modifying enzymes in crude cell-free preparations was demonstrated by a previously reported method (4, 18). Briefly, reaction mixtures consisted of the cell-free preparation, the aminoglycoside being tested, and the radiolabeled substrate. [14C]ATP was used for detecting the presence of adenyltransferases, 14C-acetyl coenzyme A was used for detecting acetyltransferases, and [32P]ATP was used for detecting phosphotransferases. All radiolabeled enzyme substrates were obtained from Amersham-Searle, Arlington Heights, Ill. The test aminoglycosides were amikacin, kanamycin, and gentamicin. Positive controls for the various enzyme classes were included in parallel testing. The amount of radioactivity specifically associated with each test aminoglycoside was estimated by using a Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downer's Grove, Ill.). Measurement of intracellular accumulation of [3H]amikacin. Transmembrane permeability defects of amikacin transport were detected by measuring the amount of radiolabeled amikacin accumulating within bacterial cells by previously described techniques (9, 17). The isolates tested were (i) PA-96 (parental strain), (ii) the five amikacinresistant Pseudomonas variants from cardiac vegetations, (iii) an amikacin-resistant strain (PA ) which reverted to amikacin susceptibility in vitro within five passages through antibiotic-free MH broth, and (iv) a wild-type, amikacin-susceptible P. aeruginosa reference strain (PA-A 9843a) with an amikacin MIC of 0.5 ig/ml. Cells were grown exponentially in MH broth, and the suspensions were standardized to an inoculum of -2 x 109 cells in a Coleman Junior spectrophotometer (Coleman Instruments, Maywood, 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 to achieve a final antibiotic concentration of 4 pg/ml (sub-mic) for the amikacin-resistant variants (PA , , -20, -43, and -61) and pg/ml (sub-mic) for the amikacinsusceptible strains (PA-96, PA [postpassage], and PA-A 9843a). Cold amikacin at sub-mic produces consistent growth curves during the subsequent 120-min sampling period (9). Samples of the bacterial suspension were removed at 0, 30, and 120 min, and the optical density was recorded. Cells were collected by filtration and washed and dried. The [3H]amikacin content of each sample was then estimated in a Tri-Carb liquid scintillation spectrometer. Uptake of amikacin was expressed as nanograms of amikacin activity incorporated by a suspension containing 2 x 109 cells over the 120-min sampling period. Organisms having uptake values of <60% of that of the wild-type, amikacinsusceptible reference strain of P. aeruginosa (PA-A 9843a) were considered to have a significantly impaired transmembrane permeability for amikacin (17). Scanning and transmission EM. To determine whether the amikacin-resistant phenotype was associated with any ultrastructural changes in the organisms isolated from cardiac vegetations, scanning and transmission electron microscopy (EM) were performed. Vegetations were preserved in 2.5% glutaraldehyde. Those vegetations from which the amikacinresistant Pseudomonas variants used in this investigation were obtained were prepared for transmission EM by standard techniques (12). Tissue blocks of vegetations were also freeze fractured and prepared for scanning EM by the method of Hayat (13). Animal virulence studies. The in vivo virulence of two amikacin-resistant variants was compared with that of the parental strain (PA-96) in the experimental endocarditis model. The two variants were the same strains as those used in the in vitro growth-curve analyses described above (PA and ). Briefly, 10 New Zealand White rabbits were anesthetized with ketamine hydrochloride and xylazine given intramuscularly and transcarotid-totransaortic valve catheterization was performed as previously described (1). Twenty-four hours after catheterization, four animals each received -108 CFU of one of the Pseudomonas variants as an intravenous challenge, and two animals were so challenged by the parental strain. Induction of bacterial endocarditis was confirmed at the time of sacrifice, 24 h after the intravenous challenge, by quantitatively culturing homogenates of individual aortic valve vegetations (1). Vegetation homogenates from animals challenged with the resistant variants were parallel plated into antibiotic-free and antibiotic (amikacin, 50 plg/ml)-containing MHA; this was to confirm maintenance of the resistance phenotype during the 24-h period after induction of endocarditis. Resistance ratios, defined as the log10 of the ratio of the number of amikacin-resistant organisms to the total number of organisms, were determined for each vegetation (10). RESULTS Antibiotic susceptibility tests. The parental strain (PA-96) used to initially infect cardiac vegetations was susceptible to amikacin 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 on amikacin-containing MHA not only exhibited amikacin resistance but were also now cross resistant to gentamicin (MICs, 64 plg/ml; Table 1). Moreover, the kanamycin MICs for these five variants had increased at least eightfold (MICs, >128 [ig/ml). In vitro passage studies. The significantly increased MICs for PA were labile in vitro upon serial passage in antibiotic-free media. This isolate no longer grew on amikacin (50,ig/ml)-containing agar after five passages in antibiotic-free media. Moreover, this variant reverted to a gentamicin- and amikacin-susceptible state postpassage (MICs, 2 and 4 1Lg/ml, respectively). In addition, the kanamycin MIC decreased at least fourfold postpassage (64 pg/ml). Growth characteristics. At the time of initial isolation from cardiac vegetations (2), surface colonies from the amikacinresistant Pseudomonas variants produced single colonies of -1 mm at 24 h of incubation (37 C) on amikacin-containing

3 72 BAYER ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Antimicrobial susceptibility and transmembrane permeability of radiolabeled amikacin for parental and variant P. aeruginosa strains Isolate TherapY MIC (ug/ml) % Uptake of Amikacin Kanamycin Gentamicin I3HJamlikacina PA-96 (parent) PA-A 9843a (wild type) 0.5 NDb ND 100 PA Amikacin 128 > PA-43 Amikacin 128 > PA-61 Amikacin 32 > PA-20 Combinationc 128 > PA Combinationc 128 > PA (postpassage) 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 pyocyanin pigment at the time of initial isolation from cardiac vegetations. This phenotypic trait was labile, and pigment production was regained after serial passage of the variants on antibiotic-free media. In addition to regaining pigment production, variants produced larger surface colonies (2 to 3 mm) after serial in vitro passage on antibiotic-free media and resembled the parental strain (PA-96) morphologically. By the end of 24 h of incubation, the parental strain had achieved counts of >9 loglo CFU/ml on growth-curve analysis; in contrast, both variants reached counts of -1 loglo CFU/ml lower at this point (Fig. 1). Moreover, the generation (doubling) times of the two variants, determined during a logarithmic growth phase (2- to 4-h period), were 1.5 to 2 times longer than for the parental strain (30 and 40 versus 20 min). Aminoglycoside-modifying enzymes. There was no evi- E U- 0 ( J sterile, PA -96 PA HOURS FIG. 1. Growth curves of parental P. aeruginosa strain (PA-96) and two amikacin-resistant impermeability variants (PA and ). dence of production of any of the eight definable aminoglycoside-modifying enzymes (aminoglycoside acetyltransferase [2', (3) I, (3)II, (3)III, (3)IV, 6'], aminoglycoside nucleotidyltransferase [previously aminoglycose adenyltransferase; 211, 41], and aminoglycoside phosphotransferase) by the five amikacin-resistant Pseudomonas variants. Amikacin transmembrane permeability. As seen in Table 1, the parental strain (PA-96) exhibited normal transmembrane permeability to radiolabeled amikacin, identical to that for the amikacin-susceptible reference strain of P. aeruginosa. In contrast, all five amikacin-resistant variants demonstrated significant defects in transmembrane penetration and 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 normalized after serial passage in antibiotic-free media, coincident with a greater-than-fourfold decrease in amikacin, gentamicin, and kanamycin MICs. Prepassage, there was no detectable uptake of radiolabeled amikacin for PA , whereas amikacin uptake was 89% of that of the reference strain postpassage. EM. Freeze-fracture preparations of selected cardiac vegetations, from which the five amikacin-resistant variants were obtained, were studied by scanning EM. The scanning EM demonstrated morphologically normal bacilli within the depths of the vegetations (Fig. 2A). Cell elongation in preparation for pinching off and division was commonly noted. None of the frequently observed morphologic changes seen when P. aeruginosa is exposed in vitro to 1/2 MIC of amikacin was noted in these in vivo preparations. These included increased interorganism filamentation and nub 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 undulated, trilamellar wall with a smooth surface (Fig. 2B). None of the frequently observed cell wall or intracellular changes induced in vitro by aminoglycosides at sub-mic was noted. These include cell wall bleb formation, ribosomal peripheralization, and reduction in ribosomal numbers (14). Animal virulence studies. Both amikacin-resistant variants were virulent in vivo, inducing aortic valve endocarditis in all 10 catheterized animals which were challenged intravenously with these strains. Of note, the slower growth rate of the variants in vitro was apparently reflected in vivo. Mean bacterial densities achieved by the variants within cardiac vegetations were -2.5 log,o CFU/g lower than those seen in animals challenged with the parental strain (Table 2). Resis-

4 VOL. 31, 1987 AMIKACIN PERMEABILITY IN PSEUDOMONAS ENDOCARDITIS 73 ts. 0 Downloaded from FIG. 2. (A) High-powered (magnification, x7,000) scanning electron micrograph showing numerous bacilli within depths of cardiac vegetation. This vegetation contained -5 log10 CFU of amikacin-resistant P. aeruginosa per g of tissue. Note the normal morphology of these bacilli, including many undergoing elongation in preparation for cell division (arrows). (B) Transmission electron micrograph of P. aeruginosa from 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 animals challenged with the resistant variants; this indicated a maintenance of the amikacin-resistant phenotype over the 24-h period after induction of endocarditis (10). Amikacin permeability mutants of P. aeruginosa are frequently demonstrated in epidemiologic surveys of aminoglycoside-resistant organisms. For example, Price et al. showed that -90% of aminoglycoside-resistant P. aeruginosa that do not produce aminoglycoside-modifying enzymes exhibit impaired uptake and permeability of amikacin (18). However, correlation of clinically definable infections with the in vivo isolation of such permeability mutants during amikacin therapy has been only sporadically reported in the literature. Meyer found four P. aeruginosa isolates which were resistant to amikacin on the basis of permeability defects among five patients in whom amikacin resistance was convincingly associated with clinical failures of therapy (15). The true prevalence of aminoglycoside-resistant variants of the im- on October 12, 2018 by guest DISCUSSION TABLE 2. Organism Mean bacterial density in cardiac vegetations ~~~~~~~~Density lmean logi0 CFU/g ± SEM]- PA96b ± 0.47 (2/7) PA C ± 0.37 (4/8) PA C ± 0.73 (4/6) a The number of animals sacrificed/number of vegetations cultured is shown in parentheses. P < for the amikacin-resistant variants versus the parental strain. b Parental Pseudomonas strain. c Amikacin-resistant variant.

5 74 BAYER ET AL. permeability phenotype has not been adequately defined in human cases of Pseudomonas endocarditis. Routine isolation of variants of this type in the clinical laboratory is likely to be rare because of the high reversion frequencies and slow in vitro growth rates relative to those of wild-type isolates (6). These important data need to be pursued in geographic areas that continue to see large numbers of patients with Pseudomonas endocarditis (19, 21). When such patients suffer bacteriologic relapse or failure, the clinical laboratory should routinely plate appropriate cultures onto media containing supra-mics of aminoglycoside in parallel with standard susceptibility testing schemas. Disparities in results between the two procedures (i.e., the organism testing as susceptible by routine methods, yet exhibiting growth on aminoglycoside-containing media) suggest the presence of the impermeability phenotype. Aminoglycoside uptake and transmembrane permeation have been shown to represent a complex, multifactorial process involving (i) initial ionic interaction with the outer cell envelope, (ii) an energy-dependent, electron-transportrequiring process and negative membrane potential, and (iii) binding to specific ribosomal binding sites with a secondary, energy-dependent enhancement of amihoglycoside accumulation (7). Moreover, Bryan et al. recently demonstrated that a clinical isolate of P. aeruginosa (from a burn patient) with impermeability-type amninoglycoside resistance had significant alterations in the lipopolysaccharide structure of the outer cell membrane as an additional cofactor for this type of resistance (8). Thus, defects in one or more of the aminoglycoside-uptake pathways may result in the impermeability phenotype. The small-colony morphology, relatively slow in vitro growth characteristics, labile nature of the impermeability defect in vitro and in vivo, and retention of in vivo virulence (albeit with evidence of slower in vivo growth rates) each suggest that the impermeability variants in this study were analogous to the unstable "energy" mutants (microbial persisters) recently described by Bryan et al. (6). Variants of this sort exhibit impaired aminoglycoside uptake because of altered electron-transport systems or an impaired electrical potential (5, 6). Although we did not direcily eliminate ribosomal-type resistance as a factor in our isolates, such resistance to aminoglycosides other than streptomycin is not believed to occur in clinical isolates (20). The reason stich impermeability-type aminoglycoside resistance is commonly observed in Pseudomonas strains isolated from left-sided cardiac vegetations but has not been seen in organisms obtained from right-sided cardiac vegetations is also not known (2, 3, 11). The major detectable difference between vegetations on the two sides of the heart involve bacterial densities, which tend to be -2 to 4 log10 higher in left-sided vegetations (1-3, llj. Also, the EM analysis in this study provided indirect evidence that sub- MICs of aminoglycosides were achieved within the depths of cardiac vegetations. How a large in vivo inoculum and reduced aminoglycoside penetration into cardiac vegetations might interrelate to predispose for the development of impermeability-type amikacin-resistance in vivo remains to be elucidated. ACKNOWLEDGMENTS We thank Peter Kresel (Bristol Mechanisms of Resistance Laboratory, Syracuse, N.Y.) for performing the enzyme modification and permeability assays. We thank Robert Hancock and Thomas Parr for their critical review of the manuscript. ANTIMICROB. AGENTS CHEMOTHER. This study was supported in part by a research grant from Bristol Research Laboratories. LITERATURE CITED 1. Archer, G. L., and F. R. Fekety Experimental endocarditis due to Pseudomonas aeruginosa-description of a model. J. Infect. Dis. 134: Bayer, A. S., K. Lam, D. Norman, K. S. Kim, and J. 0. Morrison Amikacin + ceftazidime therapy of experimental right-sided Pseudomonas aeruginosa endocarditis in rabbits. Chemotherapy (Basel) 31: Bayer, A. S., D. Norman, and K. S. Kim Efficacy of amikacin and ceftazidime in experimental aortic valve endocarditis due to Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 28: Beneviste, R., and J. Davies R-factor mediated gentamicin resistance-a new enzyme which modifies aminoglycoside antibiotics. FEBS Lett. 14: Bryan, L. E Antimicrobial drug resistance, p Academic Press, Inc., New York. 6. Bryan, L. E., A. J. Godfrey, and T. 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