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.)
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. AND METHODS MATERIALS Resistance transfer factor was sought be standard The strains studied were isolated from hospitalized methods for conjugation (29) and replica plating (16). patients and identified by standard techniques in the For recombination, overnight cultures of donor and Microbiology Servcie Laboratory. Tests used to speci- recipient were diluted 1:10 in TS broth and incubated ate P. aeruginosa were Gram stain, motility, oxidase, at 37 C for 4 h with shaking (125 rpm). Two milliliters fluorescence, oxidative reaction on glucose (OF me- 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 dium), and reactions in Seller agar. The donor, recipient, and exconjugant strains were without agitation. Aliquots of 0.1 ml of a 10- 5 characterized further: all produced pyocyanin, grew dilution were spread on TS plates and incubated at at 42 C, and had polar monotrichous flagella by 37 C overnight. TS replica plates containing 50 Ag of Leifson's staining method (17). Donor strain 1109 had gentamicin and 1,000 Mg of carbenicillin per ml were a minimal bactericidal concentration (MBC) of 100 incubated overnight at 37 C for selection of exconjugg/ml to gentamicin and gentamicin C1, and C2; it gants. For curing of resistance, ethidium bromide was was 400 gg/ml to gentamicin C,; the minimum used at 6 x 10-6 M (27), and acridine orange and inhibitory concentration was onefold dilution less acriflavine neutral in a range of 1.5 to 100 Ag/ml were than the MBC. Recipient strain 954 was selected used (30). The dyes were used both with and without because of its natural resistance to carbenicillin 15-s exposure to ultraviolet light. Sodium dodecyl (MBC of 12,800 ug/ml). The strains were kept at room sulfate was used at a concentration of 10% with and temperature in cystine-Trypticase agar (Baltimore without 17.5% sucrose (1). Adenylyltransferase activity against gentamicin Biological Laboratories) until subcultured. Disk susceptibility tests to gentamicin were performed on the was measured by the method of Benveniste and primary isolates by the Kirby-Bauer method, using Davis (3) and by use of their positive control, 415
416
MALIWAN, GRIEBLE, AND BIRD
ANTIMICROB. AGENTS CHEMOTHER.
Escherichia coli JR 762. The specific activity of TABLE 1. Gentamicin tube dilution susceptibility of 464 hospital strains of P. aeruginosa in TS broth adenosine 5'-triphosphate was 50 mCi/mM (catalog no. 541, New England Nuclear Corp.). Gentamicin 1972 1971 1970 1969 substrate concentrations used were 0.78, 1.56, 3.12, and 6.25 pg/ml. Osmotic shockate from donor strain MBC (ug/ml, No. (%) No. (%) No. (%) No. (%) 1109, containing 0.12 pg of protein per ml as measured by the Lowry method (20), was assayed for adenylyla0 0 0 1 (0.8) 0.39 tion, as was a shockate preparation sonicated for 1 0 0 0 1 (0.8) 0.78 min in an ice water bath (Branson sonifier, model 185) 3 (2.3) 1 (0.7) 4 (5.9) 6 (4.9) 1.56 prior to centrifugation at 4 C (20,000 x g). Inactiva38 (31.1) 12 (17.2) 27 (20.6) 25 (17.4) 3.12 tion of aminoglycosides by the resistant donor strain 59 (48.4) 26 (38.8) 64 (48.8) 62 (43.0) 6.2 TS broth Overnight (19). a bioassay by studied was 16 (13.1) 15 (22.4) 30 (22.4) 40 (27.8)a 12.5 culture supernatants or sonicates from 108 organisms, 7 (5.3) 16 (JJ.1)a 1 (1.5) 1 (0.8) 25.0 clarified by centrifugation, were passed through a 0.45-pcm membrane filter and incubated overnight at a Fifty-four of 56 resistant strains isolated in 1972 37 C with 10 pg of aminoglycosides per ml, without were tested for pyocin type. They could be differentiand with adenosine 5'-triphosphate added at concen- ated into 46 groups, with no predominance of any trations of 0.45 and 1.0 uM (Calbiochem, lot 201131, particular type. A grade). Bacillus subtilis (ATCC 6633) in MuellerHinton agar was the assay organism. resistant Beta-lactamase, with or without induction by peni- and wounds, and the recovery of cillin or carbenicillin, was measured by the modified strains, was not clustered among patients in any Novick procedure (23) and expressed in units per particular location of the hospital. Analyses of 54 out of 56 resistant strains from milligram of protein (20). Beta-lactamase substrate profiles were calculated relative to penicillin G. Pyo- 1972 showed all but two to be pyocin producers. cin typing was done without mitomycin induction by No particular pyocin type predominated, and a spot-plate technique (14) using eight indicator the 54 strains could be classified into 47 groups. strains (12) obtained from the Center for Disease Resistance to gentamicin in nutrient and TS Control, Atlanta, Ga; 18 indicator strains provided by broth was compared. With TS broth, only 20% the Department of Microbiology, University of Ala- of 93 strains were killed at a concentration of 3.1 bama ("ALA Indicator Strains") (9), and 11 Mayo Clinic (Zabransky) indicators (35). Types were ,gg/ml or less, whereas this was the case for 99% with nutrient broth. The MBC of gentamicin in grouped according to Bobo et al. (4). Antisera to donor and recipient somatic antigens TS as compared to nutrient broth was four times or greater in 90/93 strains. The cation and were produced in rabbits. Heat-killed organisms were suspended in 0.3% formalinized normal saline to phosphorus content of nutrient and TS broth contain 109 organisms/ml (McFarland scale). Intrave- was, respectively: calcium, 0.08 and 3.9 mg/100 nous injections were given at 4-day intervals in doses ml; magnesium, 0.052 and 3.37 mg/100 ml; of 0.5, 1.0, 2.0, and 3.0 ml. Serum was harvested 2 to 3 phosphorus, 6.4 and 54.5 mg/100 ml; sodium, weeks after the last injection. Agglutinin titers were 16.8 and 125.3 meq/liter; potassium, 6.0 and determined by a standard technique (18). RESULTS to Resistance 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,
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-
P. AERUGINOSA GENTAMICIN RESISTANCE
VOL. 8, 1975
417
TABLE 2. Aminoglycoside resistance transfer factor-differential characteristics of donor, recipient, and exconjugant Tests
MBC (gg/ml) Gentamicin Tobramycin Amikacin Kanamycin Streptomycin Neomycin Carbenicillin Ampicillin BL-P 1654a Vancomycin Nalidixic acid Pyocintype' Pyocin typec
Agglutination titer to 1109 anti-O 954 anti-O Beta-lactamase production (Pollock units/mg of protein) against Penicillin Carbenicillin
Donor (1109)
Recipient (954)
Exconjugant (Hines R-1)
100 25 100 1,600 > 25,600 1,600 200 6,400 400 > 50,000 100
3.1 1.5 25 200 6,400 50 12,800 25,600 1,600 25,000 400
50 6.2 50 1,600 25,600 1,600 6,400 12,800 1,600 50,000 400
2,4,5,8 4,7,9,14
2, 4, 5,8 4,7, 9,14
< 1:20 1:640
< 1:20 1:640
5 9,11,15 1:320
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.)
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. AND METHODS MATERIALS Resistance transfer factor was sought be standard The strains studied were isolated from hospitalized methods for conjugation (29) and replica plating (16). patients and identified by standard techniques in the For recombination, overnight cultures of donor and Microbiology Servcie Laboratory. Tests used to speci- recipient were diluted 1:10 in TS broth and incubated ate P. aeruginosa were Gram stain, motility, oxidase, at 37 C for 4 h with shaking (125 rpm). Two milliliters fluorescence, oxidative reaction on glucose (OF me- 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 dium), and reactions in Seller agar. The donor, recipient, and exconjugant strains were without agitation. Aliquots of 0.1 ml of a 10- 5 characterized further: all produced pyocyanin, grew dilution were spread on TS plates and incubated at at 42 C, and had polar monotrichous flagella by 37 C overnight. TS replica plates containing 50 Ag of Leifson's staining method (17). Donor strain 1109 had gentamicin and 1,000 Mg of carbenicillin per ml were a minimal bactericidal concentration (MBC) of 100 incubated overnight at 37 C for selection of exconjugg/ml to gentamicin and gentamicin C1, and C2; it gants. For curing of resistance, ethidium bromide was was 400 gg/ml to gentamicin C,; the minimum used at 6 x 10-6 M (27), and acridine orange and inhibitory concentration was onefold dilution less acriflavine neutral in a range of 1.5 to 100 Ag/ml were than the MBC. Recipient strain 954 was selected used (30). The dyes were used both with and without because of its natural resistance to carbenicillin 15-s exposure to ultraviolet light. Sodium dodecyl (MBC of 12,800 ug/ml). The strains were kept at room sulfate was used at a concentration of 10% with and temperature in cystine-Trypticase agar (Baltimore without 17.5% sucrose (1). Adenylyltransferase activity against gentamicin Biological Laboratories) until subcultured. Disk susceptibility tests to gentamicin were performed on the was measured by the method of Benveniste and primary isolates by the Kirby-Bauer method, using Davis (3) and by use of their positive control, 415
416
MALIWAN, GRIEBLE, AND BIRD
ANTIMICROB. AGENTS CHEMOTHER.
Escherichia coli JR 762. The specific activity of TABLE 1. Gentamicin tube dilution susceptibility of 464 hospital strains of P. aeruginosa in TS broth adenosine 5'-triphosphate was 50 mCi/mM (catalog no. 541, New England Nuclear Corp.). Gentamicin 1972 1971 1970 1969 substrate concentrations used were 0.78, 1.56, 3.12, and 6.25 pg/ml. Osmotic shockate from donor strain MBC (ug/ml, No. (%) No. (%) No. (%) No. (%) 1109, containing 0.12 pg of protein per ml as measured by the Lowry method (20), was assayed for adenylyla0 0 0 1 (0.8) 0.39 tion, as was a shockate preparation sonicated for 1 0 0 0 1 (0.8) 0.78 min in an ice water bath (Branson sonifier, model 185) 3 (2.3) 1 (0.7) 4 (5.9) 6 (4.9) 1.56 prior to centrifugation at 4 C (20,000 x g). Inactiva38 (31.1) 12 (17.2) 27 (20.6) 25 (17.4) 3.12 tion of aminoglycosides by the resistant donor strain 59 (48.4) 26 (38.8) 64 (48.8) 62 (43.0) 6.2 TS broth Overnight (19). a bioassay by studied was 16 (13.1) 15 (22.4) 30 (22.4) 40 (27.8)a 12.5 culture supernatants or sonicates from 108 organisms, 7 (5.3) 16 (JJ.1)a 1 (1.5) 1 (0.8) 25.0 clarified by centrifugation, were passed through a 0.45-pcm membrane filter and incubated overnight at a Fifty-four of 56 resistant strains isolated in 1972 37 C with 10 pg of aminoglycosides per ml, without were tested for pyocin type. They could be differentiand with adenosine 5'-triphosphate added at concen- ated into 46 groups, with no predominance of any trations of 0.45 and 1.0 uM (Calbiochem, lot 201131, particular type. A grade). Bacillus subtilis (ATCC 6633) in MuellerHinton agar was the assay organism. resistant Beta-lactamase, with or without induction by peni- and wounds, and the recovery of cillin or carbenicillin, was measured by the modified strains, was not clustered among patients in any Novick procedure (23) and expressed in units per particular location of the hospital. Analyses of 54 out of 56 resistant strains from milligram of protein (20). Beta-lactamase substrate profiles were calculated relative to penicillin G. Pyo- 1972 showed all but two to be pyocin producers. cin typing was done without mitomycin induction by No particular pyocin type predominated, and a spot-plate technique (14) using eight indicator the 54 strains could be classified into 47 groups. strains (12) obtained from the Center for Disease Resistance to gentamicin in nutrient and TS Control, Atlanta, Ga; 18 indicator strains provided by broth was compared. With TS broth, only 20% the Department of Microbiology, University of Ala- of 93 strains were killed at a concentration of 3.1 bama ("ALA Indicator Strains") (9), and 11 Mayo Clinic (Zabransky) indicators (35). Types were ,gg/ml or less, whereas this was the case for 99% with nutrient broth. The MBC of gentamicin in grouped according to Bobo et al. (4). Antisera to donor and recipient somatic antigens TS as compared to nutrient broth was four times or greater in 90/93 strains. The cation and were produced in rabbits. Heat-killed organisms were suspended in 0.3% formalinized normal saline to phosphorus content of nutrient and TS broth contain 109 organisms/ml (McFarland scale). Intrave- was, respectively: calcium, 0.08 and 3.9 mg/100 nous injections were given at 4-day intervals in doses ml; magnesium, 0.052 and 3.37 mg/100 ml; of 0.5, 1.0, 2.0, and 3.0 ml. Serum was harvested 2 to 3 phosphorus, 6.4 and 54.5 mg/100 ml; sodium, weeks after the last injection. Agglutinin titers were 16.8 and 125.3 meq/liter; potassium, 6.0 and determined by a standard technique (18). RESULTS to Resistance 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,
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-
P. AERUGINOSA GENTAMICIN RESISTANCE
VOL. 8, 1975
417
TABLE 2. Aminoglycoside resistance transfer factor-differential characteristics of donor, recipient, and exconjugant Tests
MBC (gg/ml) Gentamicin Tobramycin Amikacin Kanamycin Streptomycin Neomycin Carbenicillin Ampicillin BL-P 1654a Vancomycin Nalidixic acid Pyocintype' Pyocin typec
Agglutination titer to 1109 anti-O 954 anti-O Beta-lactamase production (Pollock units/mg of protein) against Penicillin Carbenicillin
Donor (1109)
Recipient (954)
Exconjugant (Hines R-1)
100 25 100 1,600 > 25,600 1,600 200 6,400 400 > 50,000 100
3.1 1.5 25 200 6,400 50 12,800 25,600 1,600 25,000 400
50 6.2 50 1,600 25,600 1,600 6,400 12,800 1,600 50,000 400
2,4,5,8 4,7,9,14
2, 4, 5,8 4,7, 9,14
< 1:20 1:640
< 1:20 1:640
5 9,11,15 1:320
