ANTIMICROBIAL AGENTs AND CHEMOTHERAPY, June 1976, p. 866-873 Copyright X 1976 American Society for Microbiology
Vol. 9, No. 6 Printed in U.SA.
Multiresistant Plasmids from Pseudomonas aeruginosa Highly Resistant to Either or Both Gentamicin and Carbenicillin POLYXENI KONTOMICHALOU,* EFSTATHIA PAPACHRISTOU, AND FEVRONIA ANGELATOU Department of Clinical Therapeutics, School of Medicine, University ofAthens, Alexandra Hospital, Athens, Greece
Received for publication 29 September 1975
High-level resistance to gentamicin and carbenicillin was found in 30 and 10.7%, respectively, of Pseudomonas aeruginosa strains, especially in isolates from urine. In 23 out of 25 strains tested, these resistances were R mediated and linked to multiresistant plasmids, carrying genes for resistances to five other aminoglycosides, tobramycin, kanamycin, neomycin, streptomycin, and spectinomycin, and for resistances to chloramphenicol, tetracycline, sulfonamides, and mercury chloride. Carbenicillin resistance was unstable in Pseudomonas, and in its presence the multiresistant plasmids had a host range extended to the Enterobacteriaceae (group I plasmids). Otherwise they were transferable intragenerically only (group II plasmids). The extended host range plasmids were, as a rule, in fi- incompatibility class A-C. Segregants incompatible with both class A-C and P plasmids were detected. The 8-lactamase specified by the carbenicillin marker was of the TEM-like type. Multiple linkages of resistance determinants to the aminoglycosides were concomitantly present in most of the plasmids. Results from the bioassay indicated the presence of at least two aminoglycoside-inactivating enzymes.
The study of R-plasmids originating from Pseudomonas aeruginosa was very limited during the first decade (1959-1969) of the history of R-factors (26, 29, 32). The introduction in clinical medicine of carbenicillin (Cb) and gentamicin (Gm), antibiotics with special antipseudomonal activity, and the emergence in Pseudomonas of resistance towards these drugs have stimulated an interest in detecting the episomal nature of these resistances (3, 4, 27). Such studies have revealed that R-plasmids carrying genes coding for enzymes inactivating Cb or the aminoglycosides have evolved in Pseudomonas in several parts of the world (4, 8, 12, 18, 19, 22, 23, 25, 35-37). Most of the Pseudomonas R-plasmids either have been found in Pseudomonas isolated from wounds, after long use of these antipseudomonal drugs in topical applications, or were transferable intragenerically. Reports on Pseudomonas plasmids originating from strains of other sources and displaying a host range extending to the Enterobacteriaceae are still rare (8, 36). The purpose of this study was to deternine whether R-mediated resistance to Gm and Cb would appear in our clinical Pseudomonas isolates and to investigate the properties of the evolving plasmids. We found that resistance to both of these drugs was R mediated in almost
all cases tested and linked to multiresistant plasmids, carrying genes for resistance to many antibacterial drugs and to HgCl2. The compatibility class of plasmids with intergeneric host range was identified. The type of 3-lactaminactivating enzymes was also investigated. Aminoglycoside-inactivating enzymes have been detected by the paper disk bioassay. MATERIALS AND METHODS Bacterial strains and R-plasmids. (i) Clinical isolates. The clinical isolates consisted of: (i) 100 unselected Pseudomonas strains, which represented all P. aeruginosa isolated in our department from 1 October 1972 until 31 January 1974 (the collection of these strains started when Pseudomonas resistant to Gm first appeared in our clinic in September 1972), (ii) 25 unselected Pseudomonas representing all isolates of the laboratory of the Athens Accident Hospital for the first 3 months of the above period, and (iii) five P. aeruginosa that were the survivors of a random number of Pseudomonas isolated in our department in 1971. The sources of the total of 130 P. aeruginosa strains tested were as follows: 83 from urine, 20 from sputum, 15 from pus, and the remaining 12 from blood and other sources. Only one strain from each patient was studied. Identification of clinical isolates was based on conventional methods (13, 33). (ii) Bacterial hosts. The bacterial hosts used in mating experiments were (i) Escherichia coli K-12 866
VoL. 9, 1976
P. AERUGINOSA PLASMIDS RESISTANT TO Cb AND Gm
F-: RC85:W1655 (met-,X-,Xr) (28) made rifampin resistant, J62:RC711 (pro-his-,try-,lac-) (9, 11) made nalidixic acid resistant, J53 (pro-,met-) (9, 11); (ii) E. coli HfrH (thi-) (9); (iii) Pseudomonas: PU21 (FP-,ilv-,leu-,str') (19), wild-type P. aeruginosa P190 (human origin; this report), and PH (animal origin, calf mastitis; this report), the last two made rifampin resistant. (iii) Reference plasmids. The reference plasmids for compatibility experiments were: class P, RP4 (CbKm/NmTc) (10); class A-C, R55 (AmpChlSulGenk) (7), R57b-1 (CSu) (14), and RA-1 (TSu) (14). Antibiotics. The following aminoglycosidic aminocyclitol antibiotics were available commercially: Gm sulfate (Garamycin, Schering), kanamycin (Km) sulfate (Kantrex, Bristol), neomycin (Nm) sulfate (Mycifradin, Upjohn), and streptomycin (Sm) sulfate (Hoechst). The following pure substances were obtained: tobramycin (Tm) from Eli Lilly & Co. and spectinomycin (Sp) sulfate from the Upjohn Co. Cb sodium was commercial (Pyopen, Beecham). Other antibiotics were standard laboratory preparations. Resistance to antibiotics. Minimum inhibitory concentration determinations were performed by the tube dilution method in brain heart infusion broth (Difco), pH 7.0. Overnight cultures were diluted to 2 x 104 bacteria/ml, and 0.5 ml of this inoculum was added to 2 ml of broth containing antibiotics ranging from 0.25 to 4,000 ,ug/ml for the aminoglycosides and 0.5 to 8,000 ug/ml for Cb. Resistance patterns of wild-type strains and R+ cultures were tested by streaking inocula of 104 bacteria/ml on McConkey agar ditch plates containing drugs at the following concentrations (micrograms per milliliter) for E. coli K-12: Cb, 1,000; Gm and Tm, 20; Km, 50 and 500 (KM); Nm, 25 and 500 (NM); Sm, 20; Sp, 50; chloramphenicol and tetracycline, 40; and nalidixic acid and colimycin, 30. For Pseudomonas the concentrations of drugs differed from the above as follows: Sm, 500; Sp, 2,000; and chloramphenicol and tetracycline, 300. Resistance to sulfonamides and trimethoprim was tested on Muller-Hinton agar ditch plates incorporating 1,000 ,ug of sulfamezathine or 25 ug of trimethoprim per ml. Resistance to HgCl2. Constitutive resistance to HgCl2 was tested on Muller-Hinton agar ditch plates containing 0.1 mM HgCl2 (19). Mating experiments. Isogenic transfers between E. coli K-12 derivatives were performed in broth as described previously (24). Mating experiments pairing wild-type Pseudomonas x E. coli K-12, E. coli K-12 x wild-type Pseudomonas, and Pseudomonas x Pseudomonas were performed by a membrane method, the mixed cultures starting as for isogenic transfers in K-12 derivatives; their sediments were incubated on membranes at 37 C for 24 h. Dilutions of the overnight growth from the membranes were plated on McConkey agar containing drugs to select the R+ recipient: Cb, 1,000 Ag/ml, for the K-12 recipient Gm, 15 ,Ag/ml for the K-12 recipient, and 20 ,ugl ml for the Pseudomonas recipient, or tetracycline, 100 ug/ml, for the Pseudomonas recipient. The counterselecting agent was, in most cases, 200 ,ug of rifampin per ml for the K-12 recipient and 500 ug/ml for Pseudomonas PU21, P190, and PH recipients and
867
30 ,ug of colimycin per ml for the wild-type Pseudomonas.
fi character. The plasmids transferred to K-12 were tested for the fi character by their ability to inhibit plating of male-specific phages in HfrR+ strains as described previously (24). Compatibility classification. Compatibility classification was performed for the intergenerically transferable fi- plasmids. As mating pairs the K-12 derivatives J53 x RC85 with the reference plasmids in the donor and the tested plasmids as resident plasmids and J62 x RC85 with the tested plasmids in the donor and the reference plasmids as resident plasmids in the recipient were used. Selection was made for chromosomal markers of the recipient host and drug resistance markers of the incoming plasmid. Single colonies with such characters were grown overnight in drug-free nutrient broth and then plated on drug-free medium. After a 24-h incubation, the descending clones were replicated on drug-containing plates to test the compatibility between the resident and the incoming plasmids. Susceptibility to plasmid-specific phages. The ribonucleic acid-containing somatic phages PR3 and PR4, specific for bacteria carrying class P plasmids, were provided by V. Stanisich (34). Susceptibility to phages was tested by a double-layer method in Trypticase soy broth (BBL), with dilutions of the phages containing 109 to 105 plaque-forming units/ml. fi-Lactamase assays. (8-Lactamase assays were performed on crude enzyme preparations of the cultures, and other properties ofthe /8-lactamases were tested on the shockates of the cultures as described previously (24). Aminoglycoside-inactivating enzymes. A paper disk bioassay was used with substrates of the six aminoglycosides Gm, Tm, Km, Nm, Sm, and Sp in the presence of adenosine. 5'-triphosphate or acetylcoenzyme A (1, 17, 38).
RESULTS Resistance ofPseudomonas to Gm, Tm, and Cb. Table 1 shows that resistance to Gm, Tm, and Cb was found, respectively, in 30, 28.4, and 10.7% of the 130 Pseudomonas clinical isolates. There was almost complete cross-resistance between Gm and Tm, with a difference only in the levels of resistance. Resistance to both Gm and Cb was as a rule of a high level. The strains resistant to Gm, Tm, or Cb were isolates from only one of the two hospitals (our department). Ninety-three percent of the resistant strains were from urine. Resistance plasmids in Pseudomonas. There was a total of 42 resistant strains: 3 1971 isolates resistant to Cb, 11 strains resistant to all three, Gm, Tm, and Cb, and 28 resistant to Gm and Tm but susceptible to Cb. Transferability tests with E. coli K-12 RC85 as recipient gave, in selection by Cb, a positive transfer in the 3 Cb-resistant Pseudomonas and in 9 out of 11 strains resistant to Gm, Tm, and Cb, with frequencies of 10-6 to 10-8 (group I donors).
868
ANTIMICROB. AGEN" CHEMOTHER.
KONTOMICHALOU ET AL.
TABLE 1. Susceptibility of 130 P. aeruginosa strains to Gm, Tm, and Cb Gm
MICa (,g/ ml)
No. of strains
Cb
Tm Resistant strains
No.
MIC (jtg/ ml)
No. of strains
Resistant strains
No.
%
s0.25
81
Vol. 9, No. 6 Printed in U.SA.
Multiresistant Plasmids from Pseudomonas aeruginosa Highly Resistant to Either or Both Gentamicin and Carbenicillin POLYXENI KONTOMICHALOU,* EFSTATHIA PAPACHRISTOU, AND FEVRONIA ANGELATOU Department of Clinical Therapeutics, School of Medicine, University ofAthens, Alexandra Hospital, Athens, Greece
Received for publication 29 September 1975
High-level resistance to gentamicin and carbenicillin was found in 30 and 10.7%, respectively, of Pseudomonas aeruginosa strains, especially in isolates from urine. In 23 out of 25 strains tested, these resistances were R mediated and linked to multiresistant plasmids, carrying genes for resistances to five other aminoglycosides, tobramycin, kanamycin, neomycin, streptomycin, and spectinomycin, and for resistances to chloramphenicol, tetracycline, sulfonamides, and mercury chloride. Carbenicillin resistance was unstable in Pseudomonas, and in its presence the multiresistant plasmids had a host range extended to the Enterobacteriaceae (group I plasmids). Otherwise they were transferable intragenerically only (group II plasmids). The extended host range plasmids were, as a rule, in fi- incompatibility class A-C. Segregants incompatible with both class A-C and P plasmids were detected. The 8-lactamase specified by the carbenicillin marker was of the TEM-like type. Multiple linkages of resistance determinants to the aminoglycosides were concomitantly present in most of the plasmids. Results from the bioassay indicated the presence of at least two aminoglycoside-inactivating enzymes.
The study of R-plasmids originating from Pseudomonas aeruginosa was very limited during the first decade (1959-1969) of the history of R-factors (26, 29, 32). The introduction in clinical medicine of carbenicillin (Cb) and gentamicin (Gm), antibiotics with special antipseudomonal activity, and the emergence in Pseudomonas of resistance towards these drugs have stimulated an interest in detecting the episomal nature of these resistances (3, 4, 27). Such studies have revealed that R-plasmids carrying genes coding for enzymes inactivating Cb or the aminoglycosides have evolved in Pseudomonas in several parts of the world (4, 8, 12, 18, 19, 22, 23, 25, 35-37). Most of the Pseudomonas R-plasmids either have been found in Pseudomonas isolated from wounds, after long use of these antipseudomonal drugs in topical applications, or were transferable intragenerically. Reports on Pseudomonas plasmids originating from strains of other sources and displaying a host range extending to the Enterobacteriaceae are still rare (8, 36). The purpose of this study was to deternine whether R-mediated resistance to Gm and Cb would appear in our clinical Pseudomonas isolates and to investigate the properties of the evolving plasmids. We found that resistance to both of these drugs was R mediated in almost
all cases tested and linked to multiresistant plasmids, carrying genes for resistance to many antibacterial drugs and to HgCl2. The compatibility class of plasmids with intergeneric host range was identified. The type of 3-lactaminactivating enzymes was also investigated. Aminoglycoside-inactivating enzymes have been detected by the paper disk bioassay. MATERIALS AND METHODS Bacterial strains and R-plasmids. (i) Clinical isolates. The clinical isolates consisted of: (i) 100 unselected Pseudomonas strains, which represented all P. aeruginosa isolated in our department from 1 October 1972 until 31 January 1974 (the collection of these strains started when Pseudomonas resistant to Gm first appeared in our clinic in September 1972), (ii) 25 unselected Pseudomonas representing all isolates of the laboratory of the Athens Accident Hospital for the first 3 months of the above period, and (iii) five P. aeruginosa that were the survivors of a random number of Pseudomonas isolated in our department in 1971. The sources of the total of 130 P. aeruginosa strains tested were as follows: 83 from urine, 20 from sputum, 15 from pus, and the remaining 12 from blood and other sources. Only one strain from each patient was studied. Identification of clinical isolates was based on conventional methods (13, 33). (ii) Bacterial hosts. The bacterial hosts used in mating experiments were (i) Escherichia coli K-12 866
VoL. 9, 1976
P. AERUGINOSA PLASMIDS RESISTANT TO Cb AND Gm
F-: RC85:W1655 (met-,X-,Xr) (28) made rifampin resistant, J62:RC711 (pro-his-,try-,lac-) (9, 11) made nalidixic acid resistant, J53 (pro-,met-) (9, 11); (ii) E. coli HfrH (thi-) (9); (iii) Pseudomonas: PU21 (FP-,ilv-,leu-,str') (19), wild-type P. aeruginosa P190 (human origin; this report), and PH (animal origin, calf mastitis; this report), the last two made rifampin resistant. (iii) Reference plasmids. The reference plasmids for compatibility experiments were: class P, RP4 (CbKm/NmTc) (10); class A-C, R55 (AmpChlSulGenk) (7), R57b-1 (CSu) (14), and RA-1 (TSu) (14). Antibiotics. The following aminoglycosidic aminocyclitol antibiotics were available commercially: Gm sulfate (Garamycin, Schering), kanamycin (Km) sulfate (Kantrex, Bristol), neomycin (Nm) sulfate (Mycifradin, Upjohn), and streptomycin (Sm) sulfate (Hoechst). The following pure substances were obtained: tobramycin (Tm) from Eli Lilly & Co. and spectinomycin (Sp) sulfate from the Upjohn Co. Cb sodium was commercial (Pyopen, Beecham). Other antibiotics were standard laboratory preparations. Resistance to antibiotics. Minimum inhibitory concentration determinations were performed by the tube dilution method in brain heart infusion broth (Difco), pH 7.0. Overnight cultures were diluted to 2 x 104 bacteria/ml, and 0.5 ml of this inoculum was added to 2 ml of broth containing antibiotics ranging from 0.25 to 4,000 ,ug/ml for the aminoglycosides and 0.5 to 8,000 ug/ml for Cb. Resistance patterns of wild-type strains and R+ cultures were tested by streaking inocula of 104 bacteria/ml on McConkey agar ditch plates containing drugs at the following concentrations (micrograms per milliliter) for E. coli K-12: Cb, 1,000; Gm and Tm, 20; Km, 50 and 500 (KM); Nm, 25 and 500 (NM); Sm, 20; Sp, 50; chloramphenicol and tetracycline, 40; and nalidixic acid and colimycin, 30. For Pseudomonas the concentrations of drugs differed from the above as follows: Sm, 500; Sp, 2,000; and chloramphenicol and tetracycline, 300. Resistance to sulfonamides and trimethoprim was tested on Muller-Hinton agar ditch plates incorporating 1,000 ,ug of sulfamezathine or 25 ug of trimethoprim per ml. Resistance to HgCl2. Constitutive resistance to HgCl2 was tested on Muller-Hinton agar ditch plates containing 0.1 mM HgCl2 (19). Mating experiments. Isogenic transfers between E. coli K-12 derivatives were performed in broth as described previously (24). Mating experiments pairing wild-type Pseudomonas x E. coli K-12, E. coli K-12 x wild-type Pseudomonas, and Pseudomonas x Pseudomonas were performed by a membrane method, the mixed cultures starting as for isogenic transfers in K-12 derivatives; their sediments were incubated on membranes at 37 C for 24 h. Dilutions of the overnight growth from the membranes were plated on McConkey agar containing drugs to select the R+ recipient: Cb, 1,000 Ag/ml, for the K-12 recipient Gm, 15 ,Ag/ml for the K-12 recipient, and 20 ,ugl ml for the Pseudomonas recipient, or tetracycline, 100 ug/ml, for the Pseudomonas recipient. The counterselecting agent was, in most cases, 200 ,ug of rifampin per ml for the K-12 recipient and 500 ug/ml for Pseudomonas PU21, P190, and PH recipients and
867
30 ,ug of colimycin per ml for the wild-type Pseudomonas.
fi character. The plasmids transferred to K-12 were tested for the fi character by their ability to inhibit plating of male-specific phages in HfrR+ strains as described previously (24). Compatibility classification. Compatibility classification was performed for the intergenerically transferable fi- plasmids. As mating pairs the K-12 derivatives J53 x RC85 with the reference plasmids in the donor and the tested plasmids as resident plasmids and J62 x RC85 with the tested plasmids in the donor and the reference plasmids as resident plasmids in the recipient were used. Selection was made for chromosomal markers of the recipient host and drug resistance markers of the incoming plasmid. Single colonies with such characters were grown overnight in drug-free nutrient broth and then plated on drug-free medium. After a 24-h incubation, the descending clones were replicated on drug-containing plates to test the compatibility between the resident and the incoming plasmids. Susceptibility to plasmid-specific phages. The ribonucleic acid-containing somatic phages PR3 and PR4, specific for bacteria carrying class P plasmids, were provided by V. Stanisich (34). Susceptibility to phages was tested by a double-layer method in Trypticase soy broth (BBL), with dilutions of the phages containing 109 to 105 plaque-forming units/ml. fi-Lactamase assays. (8-Lactamase assays were performed on crude enzyme preparations of the cultures, and other properties ofthe /8-lactamases were tested on the shockates of the cultures as described previously (24). Aminoglycoside-inactivating enzymes. A paper disk bioassay was used with substrates of the six aminoglycosides Gm, Tm, Km, Nm, Sm, and Sp in the presence of adenosine. 5'-triphosphate or acetylcoenzyme A (1, 17, 38).
RESULTS Resistance ofPseudomonas to Gm, Tm, and Cb. Table 1 shows that resistance to Gm, Tm, and Cb was found, respectively, in 30, 28.4, and 10.7% of the 130 Pseudomonas clinical isolates. There was almost complete cross-resistance between Gm and Tm, with a difference only in the levels of resistance. Resistance to both Gm and Cb was as a rule of a high level. The strains resistant to Gm, Tm, or Cb were isolates from only one of the two hospitals (our department). Ninety-three percent of the resistant strains were from urine. Resistance plasmids in Pseudomonas. There was a total of 42 resistant strains: 3 1971 isolates resistant to Cb, 11 strains resistant to all three, Gm, Tm, and Cb, and 28 resistant to Gm and Tm but susceptible to Cb. Transferability tests with E. coli K-12 RC85 as recipient gave, in selection by Cb, a positive transfer in the 3 Cb-resistant Pseudomonas and in 9 out of 11 strains resistant to Gm, Tm, and Cb, with frequencies of 10-6 to 10-8 (group I donors).
868
ANTIMICROB. AGEN" CHEMOTHER.
KONTOMICHALOU ET AL.
TABLE 1. Susceptibility of 130 P. aeruginosa strains to Gm, Tm, and Cb Gm
MICa (,g/ ml)
No. of strains
Cb
Tm Resistant strains
No.
MIC (jtg/ ml)
No. of strains
Resistant strains
No.
%
s0.25
81
