Hospital Pseudomonas aeruginosa: Surveillance of Resistance

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1975, p. 415-420 Copyright i 1975 American Society for Microbiology Vol. 8, No. 4 Printed in U.S.A. Hospital Pseudomonas aeruginosa: Surveillance of Resistance to Gentamicin and Transfer of Aminoglycoside R Factor NITAYA MALIWAN, HANS G. GRIEBLE,* AND THOMAS J. BIRD Veterans Administration Hospital, Hines, Illinois 60141, and The Chicago Medical School, Chicago, Illinois 60612 Received for publication 19 May 1975 Tube dilution susceptibility tests in Trypticase soy broth showed that resistance to gentamicin (minimum bactericidal concentration 2 12.5 ug/ml) among hospital isolates of Pseudomonas aeruginosa increased from 13.9% in 1969 to 38.9% in 1972. Transfer of drug resistance to six aminoglycosides from one wild Pseudomonas strain to another was accomplished in recombination experiments. A carbenicillin-resistant, beta-lactamase-producing strain served as the recipient. The exconjugant was resistant not only to aminoglycosides, including amikacin, but also to all clinically employed antimicrobials. Aminoglycoside resistance in the exconjugant was cured by sodium dodecyl sulfate. This transferable aminoglycoside resistance was not mediated by adenylylation or, as judged by bioassay, by other antibiotic-inactivating or -modifying processes. The monitoring of gentamicin susceptibility in clinical isolates of Pseudomonas aeruginosa was designed to detect evolution of resistance. Because of the great potential impact of infectious gentamicin resistance on the ecology of P. aeruginosa in the hospital, a search for drug resistance factor and attempts at its transfer from one wild Pseudomonas strain to another were made. (We reported such drug resistance and its unique linkage pattern to other aminoglycosides at the 1973 Annual Meeting of the American Society for Microbiology, Miami Beach, Fla.) MATERIALS AND METHODS The strains studied were isolated from hospitalized patients and identified by standard techniques in the Microbiology Servcie Laboratory. Tests used to speciate P. aeruginosa were Gram stain, motility, oxidase, fluorescence, oxidative reaction on glucose (OF medium), and reactions in Seller agar. The donor, recipient, and exconjugant strains were characterized further: all produced pyocyanin, grew at 42 C, and had polar monotrichous flagella by Leifson's staining method (17). Donor strain 1109 had a minimal bactericidal concentration (MBC) of 100 gg/ml to gentamicin and gentamicin C1, and C2; it was 400 gg/ml to gentamicin C,; the minimum inhibitory concentration was onefold dilution less than the MBC. Recipient strain 954 was selected because of its natural resistance to carbenicillin (MBC of 12,800 ug/ml). The strains were kept at room temperature in cystine-trypticase agar (Baltimore Biological Laboratories) until subcultured. Disk susceptibility tests to gentamicin were performed on the primary isolates by the Kirby-Bauer method, using 10-,gg disks and a 105 inoculum size (2). Tube dilution susceptibility to gentamicin was determined in Trypticase soy (TS) (lot no. G5DAGU) and nutrient broth (lot no. 006652) (Baltimore Biological Laboratories). The other antimicrobials were tested in TS broth only. The strains were grown for 18 h and subcultured for 4 h before inoculation of 104 organisms into 1.0 ml of medium containing antibiotic. After overnight incubation, the tubes were observed for minimum inhibitory concentration. Nonturbid dilutions were streaked on TS agar plates, incubated, and observed for growth 18 h later. Susceptible Pseudomonas strains (Ellsworth and Hines no. 954) and a resistant strain (Hines no. 1109) were used as susceptibility test controls. All transfer experiments were done in TS media. Resistance transfer factor was sought be standard methods for conjugation (29) and replica plating (16). 415 For recombination, overnight cultures of donor and recipient were diluted 1:10 in TS broth and incubated at 37 C for 4 h with shaking (125 rpm). Two milliliters of donor and 0.1 ml of recipient cultures were mixed in a 50-ml Erlenmeyer flask and incubated at 37 C for 4 h without agitation. Aliquots of 0.1 ml of a 10-5 dilution were spread on TS plates and incubated at 37 C overnight. TS replica plates containing 50 Ag of gentamicin and 1,000 Mg of carbenicillin per ml were incubated overnight at 37 C for selection of exconjugants. For curing of resistance, ethidium bromide was used at 6 x 10-6 M (27), and acridine orange and acriflavine neutral in a range of 1.5 to 100 Ag/ml were used (30). The dyes were used both with and without 15-s exposure to ultraviolet light. Sodium dodecyl sulfate was used at a concentration of 10% with and without 17.5% sucrose (1). Adenylyltransferase activity against gentamicin was measured by the method of Benveniste and Davis (3) and by use of their positive control,

416 MALIWAN, GRIEBLE, AND BIRD Escherichia coli JR 762. The specific activity of adenosine 5'-triphosphate was 50 mci/mm (catalog no. 541, New England Nuclear Corp.). Gentamicin substrate concentrations used were 0.78, 1.56, 3.12, and 6.25 pg/ml. Osmotic shockate from donor strain 1109, containing 0.12 pg of protein per ml as measured by the Lowry method (20), was assayed for adenylylation, as was a shockate preparation sonicated for 1 min in an ice water bath (Branson sonifier, model 185) prior to centrifugation at 4 C (20,000 x g). Inactivation of aminoglycosides by the resistant donor strain was studied by a bioassay (19). Overnight TS broth culture supernatants or sonicates from 108 organisms, clarified by centrifugation, were passed through a 0.45-pcm membrane filter and incubated overnight at 37 C with 10 pg of aminoglycosides per ml, without and with adenosine 5'-triphosphate added at concentrations of 0.45 and 1.0 um (Calbiochem, lot 201131, A grade). Bacillus subtilis (ATCC 6633) in Mueller- Hinton agar was the assay organism. Beta-lactamase, with or without induction by penicillin or carbenicillin, was measured by the modified Novick procedure (23) and expressed in units per milligram of protein (20). Beta-lactamase substrate profiles were calculated relative to penicillin G. Pyocin typing was done without mitomycin induction by a spot-plate technique (14) using eight indicator strains (12) obtained from the Center for Disease Control, Atlanta, Ga; 18 indicator strains provided by the Department of Microbiology, University of Alabama ("ALA Indicator Strains") (9), and 11 Mayo Clinic (Zabransky) indicators (35). Types were grouped according to Bobo et al. (4). Antisera to donor and recipient somatic antigens were produced in rabbits. Heat-killed organisms were suspended in 0.3% formalinized normal saline to contain 109 organisms/ml (McFarland scale). Intravenous injections were given at 4-day intervals in doses of 0.5, 1.0, 2.0, and 3.0 ml. Serum was harvested 2 to 3 weeks after the last injection. Agglutinin titers were determined by a standard technique (18). RESULTS Resistance to gentamicin was examined in 709 strains isolated during the months of October and November for the years 1969 through 1972. After exclusion of repeat isolates from the same patient, 464 strains remained for analyses. The sources of cultures were similar for each year, and the average for the 4-year period was: urine, 38.3%; sputum, 35.1%; and wounds, 26.5%. Burn wounds did not materially contribute to the culture collection. If resistant strains were defined by an MBC s 12.5 Ag/ml in TS broth, the respective numbers for the years 1969 through 1972 were 17/122 or 13.9%, 16/67 or 23.9%, 37/131 or 28.2%, and 56/144 or 38.9% (Table 1). The yearly increases for 1970 over 1969 and for 1972 over 1971 were significant (P < 0.05). The rise in incidence of resistance was similar among strains from urine, sputum, TABLE 1. Gentamicin tube dilution susceptibility of 464 hospital strains of P. aeruginosa in TS broth MBC (ug/ml, ANTIMICROB. AGENTS CHEMOTHER. 1969 1970 1971 1972 No. (%) No. (%) No. (%) No. (%) 0.39 1 (0.8) 0 0 0 0.78 1 (0.8) 0 0 0 1.56 6 (4.9) 4 (5.9) 3 (2.3) 1 (0.7) 3.12 38 (31.1) 12 (17.2) 27 (20.6) 25 (17.4) 6.2 59 (48.4) 26 (38.8) 64 (48.8) 62 (43.0) 12.5 16 (13.1) 15 (22.4) 30 (22.4) 40 (27.8)a 25.0 1 (0.8) 1 (1.5) 7 (5.3) 16 (JJ.1)a a Fifty-four of 56 resistant strains isolated in 1972 were tested for pyocin type. They could be differentiated into 46 groups, with no predominance of any particular type. and wounds, and the recovery of resistant strains, was not clustered among patients in any particular location of the hospital. Analyses of 54 out of 56 resistant strains from 1972 showed all but two to be pyocin producers. No particular pyocin type predominated, and the 54 strains could be classified into 47 groups. Resistance to gentamicin in nutrient and TS broth was compared. With TS broth, only 20% of 93 strains were killed at a concentration of 3.1,gg/ml or less, whereas this was the case for 99% with nutrient broth. The MBC of gentamicin in TS as compared to nutrient broth was four times or greater in 90/93 strains. The cation and phosphorus content of nutrient and TS broth was, respectively: calcium, 0.08 and 3.9 mg/100 ml; magnesium, 0.052 and 3.37 mg/100 ml; phosphorus, 6.4 and 54.5 mg/100 ml; sodium, 16.8 and 125.3 meq/liter; potassium, 6.0 and 36.0 meq/liter. The osmolality of nutrient broth was 50 and of TS broth 340 mosmol/liter. Recombination occurred at a frequency of 27 exconjugant cells per 100 donor cells. Table 2 summarizes the differential characteristics of the donor, the recipient, and the exconjugant, Hines R-1. The antibiogram revealed that, with the exception of amikacin, the MBC to aminoglycosides of the exconjugant differed from the recipient by at least a fourfold dilution. The exconjugant retained the resistance pattern of the recipient for carbenicillin, ampicillin, BL-P 1654 (Bristol Laboratories), and malidixic acid. Antibiograms to 12 other agents, not shown in Table 2, revealed no dissimilarities between the three stains, and these MBCs were as follows: polymyxin B, 25 U/ml; penicillin G, 25,600 U/ml; cephalothin, 51,200 Ag/ml; methicillin, 6,400 gg/ml; tetracycline, 200 gg/ml; erythromycin, 3,200 Ag/ml; chloramphenicol, 800,gg/ml; sulfadiazine, >50,000 gg/ml; actinomy-

VOL. 8, 1975 TABLE 2. P. AERUGINOSA GENTAMICIN RESISTANCE 417 Aminoglycoside resistance transfer factor-differential characteristics of donor, recipient, and exconjugant Tests Donor Recipient Exconjugant (1109) (954) (Hines R-1) MBC (gg/ml) Gentamicin 100 3.1 50 Tobramycin 25 1.5 6.2 Amikacin 100 25 50 Kanamycin 1,600 200 1,600 Streptomycin > 25,600 6,400 25,600 Neomycin 1,600 50 1,600 Carbenicillin 200 12,800 6,400 Ampicillin 6,400 25,600 12,800 BL-P 1654a 400 1,600 1,600 Vancomycin > 50,000 25,000 50,000 Nalidixic acid 100 400 400 Pyocintype' 5 2,4,5,8 2, 4, 5,8 Pyocin typec 9,11,15 4,7,9,14 4,7, 9,14 Agglutination titer to 1109 anti-o 1:320 < 1:20 < 1:20 954 anti-o <1:20 1:640 1:640 Beta-lactamase production (Pollock units/mg of protein) against Penicillin 0.39 51.3 57.3 Carbenicillin 0 25 30 a Bristol Laboratories. t Gillies and Govan Indicators (12). c Alabama indicators (9). cin D, 400 ug/ml; and cycloserine, 400 gg/ml. The exconjugant retained other characteristics of the recipient, including pyocin specificity (Table 2). The "O" antigen marker in the recipient was also retained in Hines R-1, and no cross-reaction with the donor was observed. In addition, beta-lactamase production clearly separated recipient and exconjugant from the donor strain; the substrate profiles of recipient and exconjugant were similar and quite distinct from those of the donor (Fig. 1). We can conclude that Hines R-1 descended from strain 954 and acquired R factor to aminoglycosides from donor strain 1109. Sodium dodecyl sulfate, but none of the other agents, was effective in curing the exconjugant of acquired resistance to gentamicin. The "cure rate" after 24-h exposure to sodium dodecyl sulfate alone was 33%, and it rose to 60% using sodium dodecyl sulfate and sucrose. Tube dilution susceptibility tests of eight of the cured Hines R-1 strains indicated complete loss of resistance. The MBC to gentamicin declined from 25 or 50 gg/ml to 3.1 or 6.2 ug/ml. Hines R-1 was also cured of resistance to the five other aminoglycosides. Storage of Hines R-1 in holding 120 100 80 0 a - 60-0 - 40 20 Pea icillin A mpdci.l'n Catrbeicillin ri U III hohicillir ai CephaJloth. l_ Donor - 1109 Recipient - 954 Recombinant Hines R-1 FIG. 1. Beta-lactamase substrate profiles of donor, recipient, and recombinant (exconjugant) after penicillin induction, relative to penicillin (100%) in units per milligram of protein. media at room temperature resulted in spontaneous loss of resistance to gentamicin from the original MBC of 50 jg/ml to 25 at 8 weeks and 6.2,g/ml at 12 weeks. Resistance in the excon-

418 MALIWAN, GRIEBLE, AND BIRD jugant to the other aminoglycosides was likewise completely lost on storage. In contrast, 1-year storage or use of curing agents produced no change in the donor and recipient MBCs to gentamicin, the other aminoglycosides, or the other antimicrobials enumerated above. Enzymatic adenylylation of gentamicin was not detectable with supernatants or lysates from the donor strain. In the bioassay, these preparations were also not effective in inactivating gentamicin, tobramycin, amikacin, kanamycin, streptomycin, and neomycin; the bioassays were not influenced by the addition of adenosine 5'-triphosphate. DISCUSSION Among 413 strains of P. aeruginosa isolated from 194 patients hospitalized at Hines Veterans Administration (VA) Hospital during 1968 and 1969, none were resistant to gentamicin and all but one had an MBC of 1.56 gig/ml or less (Clin. Res. 19:454, 1971), i.e., if tube dilution susceptibility tests were performed in nutrient broth. With the use of TS broth, examination of representative isolates for the years 1969 through 1972 yielded a grossly different result (Table 1). After the therapeutic introduction of gentamicin to Hines VA hospital in May 1969, resistance to gentamicin rose significantly. Routine disk susceptibility testing of P. aeruginosa indicated resistance in only 5/269 or 1.8% of strains for the year 1971, 17/298 or 5.7% for 1972, and 18/335 or 5.8% for 1973. The increase over 1971 is significant (P < 0.05). Resistance by the disk method was most comparable to results with TS broth tube dilutions if the critieria for resistance were extended to an MBC 2 25 gg/ml, i.e., 5.3% in 1971 and 11.1% in 1972 (Table 1). Using different techniques and culture media, Christol et al. observed a rise in gentamicin resistance of P. aeruginosa from 13% in 1967 to 66% in 1970 (6). The medium-dependent antimicrobial action of gentamicin on P. aeruginosa was descerned by Garrod and Waterworth (10), Zimelis and Jackson (36), Gilbert et al. (11), and Medeiros et al. (21). Even though Mueller-Hinton has been recommended as the standard broth for routine susceptibility tests (8), a recent report invalidates such practice if Ca and Mg concentrations are not controlled (25). Other marked differences in electrolyte composition between TS and nutrient broth include Na, K, and phosphorus. Total osmolality was not physiological in nutrient ANTIMICROB. AGENTS CHEMOTHER. broth (50 mosmol/liter) and hyperosmolar in TS broth (340 mosmol/liter). A closer approximation to a physiological composition of culture media is desirable to improve the extrapolation from in vitro to in vivo bactericidal action of gentamicin. In one study, the 50% protective dose of gentamicin in mice challenged with P. aeruginosa correlated fairly well with resistance in tryptose-phosphate broth and less so in Mueller-Hinton broth (31). Without regard to definition of resistance and irrespective of method-dependent values, we can conclude that there was a significant emergence of strains with decreased in vitro susceptibility to gentamicin after its first clinical use in 1969, accompanied by a fourfold increase in amounts dispensed during the three subsequent years. Use of topical gentamicin was not a contributing factor, since it was never stocked in our hospital pharmacy. R factor to gentamicin in P. aeruginosa was described by Witchitz and Chabbert (32-34). The transfer was made from a naturally resistant P. aeruginosa to a mutant of E. coli K-12, and a linked transfer of resistance to ampicillin, carbenicillin, sulfonamide, and kanamycin was observed. The factor we report on differs and is unique in several respects. The 27% transfer rate is highly suggestive of conjugation as the responsible mechanism. Our donor and the recipient were wild strains isolated from hospitalized patients. Transfer of resistance occurred en bloc to gentamicin, tobramycin, kanamycin, streptomycin, and neomycin and, incompletely or questionably, to amikacin. The recipient strain was highly resistant to ampicillin, carbenicillin, and sulfonamide; therefore, no linked transfer to any of these agents could be examined. The exconjugant Hines R-1 was resistant to all clinically used antimicrobials. Neomycin resistance was not part of the resistance pattern transferred by Witchitz and Chabbert. Knothe et al. (15) reported the transfer of gentamicin resistance from a wild-type P. aeruginosa to a laboratory derived P. aeruginosa mutant, but linkage of resistance to other aminoglycosides was not mentioned. Resistance to carbenicillin was also present in their donor strains, but could not be transferred. Our attempts to transfer carbenicillinase-mediated resistance from P. aeruginosa to P. aeruginosa were likewise unsuccessful, but curing by several agents was observed (20a). Beta-lactamase production was a distinctive marker (Table 2), as was the substrate profile for these enzymes (Fig. 1). Distinctive beta-lactamases produced by strains of Enterobacteriaceae and Pseudomonas were previously described (26, 28). Limited attempts to relate

VOL. 8, 1975 inactivation of aminoglycosides to resistance in the donor strain were unsuccessful. Genetically determined enzymatic resistance to aminoglycosides is well recognized (7), and resistance to gentamicin was shown to be mediated by acetylation, adenylylation, or phosphorylation (5, 22, 24) Ṫhe natural emergence of aminoglycoside R factor as well as beta-lactamase-mediated resistance to carbenicillin signals a potentially dangerous mechanism for dissemination of drug resistance among hospital P. aeruginosa and enteric bacilli. High-degree resistance to gentamicin and carbenicillin coexisted in the exconjugant Hines R-1, but such a naturally occurring combination has, so far, been rarely encountered (13, 15). ACKNOWLEDGMENTS This study was supported by Veterans Administration grant no. 3283-02. We wish to thank Adeline Hess, J. T. Hickerson, and V. M. Olexy for expert technical assistance. LITERATURE CITED 1. Adachi, H., M. Nakano, M. Inuzuka, and M. Tomeda. 1972. 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