Vol. 35, No. 4
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1991, p. 753-755
0066-4804/91/040753-03$02.00/0 Copyright C 1991, American Society for Microbiology
Imipenem Resistance in Pseudomonas aeruginosa PAO: Mapping of the OprD2 Gene JOHN P. QUINN,'* ALDIS DARZINS,2 DEBORAH MIYASHIRO,1 STEVEN RIPP,3 AND ROBERT V. MILLER3 Section of Infectious Diseases, Department of Medicine, Michael Reese Hospital, Chicago, Illinois 606161; Department of Microbiology, Ohio State University, Columbus, Ohio 432102; and Department of Molecular and Cellular Biochemistry and Program in Molecular Biology, Loyola University of Chicago, Maywood, Illinois 601533 Received 16 October 1990/Accepted 2 February 1991
Carbapenem antibiotics have been shown to penetrate the outer membrane of Pseudomonas aeruginosa through a unique porin protein, OprD2. We mapped the OprD2 gene by conjugation using plasmid FP2 and by transduction using phage F116L. This gene maps between 71 and 75 min on the PAO1 chromosome.
Imipenem resistance (Impr) in Pseudomonas aeruginosa is usually dissociated from resistance to other ,-lactams (6, 13). We (6, 7) and others (1, 4, 10) have shown that imipenem-specific resistance is due to loss of a specific outer membrane protein (Opr) with a molecular weight of 45,000 to 47,000. This Opr, recently identified by Trias and Nikaido as porin D2, is a porin which facilitates the penetration of carbapenem antibiotics across the outer membrane of P. aeruginosa (11). The purpose of this study was to map the chromosomal genes responsible for imipenem resistance in a genetically well-characterized strain of P. aeruginosa, PAO1. (This work was presented in part at the 28th Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, Calif. [J. P. Quinn, A. Darzins, D. Miyashiro, and M. Casadaban, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 488, 1988].) Bacterial strains, bacteriophages, and plasmids. All bacterial strains used in this study are listed in Table 1. Phage F116L was used for transduction (3). Plasmid FP2 was used in conjugation experiments (3). Media. Bacteria were grown in Luria-Bertani (LB) medium (Difco, Detroit, Mich.). Vogel-Bonner minimal medium (12) without citrate was used to select recombinants in conjugation experiments. A stock solution of glucose was added at a final concentration of 50 mM. The concentration of amino acids was 1 mM. Pseudomonas minimal medium supplemented with 0.4% glucose (5) was used for transduction. Amino acids were used at a concentration of 25 ,ug/ml. Isolation of mutants. Spontaneous imipenem-resistant mutants were obtained by plating 109 cells each of strains PAO1 and PA0381 onto LB plates containing 10 ,ug of imipenem per ml. Genetic crosses. Plate matings were performed as described by Haas et al. (2). Transductions were carried out by the method of Miller and Ku (5). Chemicals. Imipenem was a gift from Merck Sharp and Dohme (Rahway, N.J.). MICs. MICs were determined by an automated method (Vitek Systems, St. Louis, Mo.) (9). Table 2 lists the P-lactam susceptibilities of the parent strains (PA0381 and PAO1), four impenem-resistant mutants (PAO1.1, PAO1.2, *
PAO1.3, and PAO1.4) picked at random for further study and then cloned on imipenem-containing agar, and PJQ382. Four of these five mutants were selectively resistant to imipenem, while one (PAO1.2) was broadly resistant. Outer membrane proteins. Outer membrane proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (6). As seen in Fig. 1, the imipenem-resistant mutants (lanes 3 and 5 through 8) show reduction or deletion of a 47-kDa outer membrane protein compared with the parent PA0381 and PAO1 strains. Mapping of mutations conferring imipenem resistance. Impr variants (resistance frequency, 10-7) of P. aeruginosa PAO381 [leu-10 FP2+(Hgr)] were isolated by plating on overnight culture on LB agar containing imipenem (10 ,ug/ml). When imipenem-resistant PJQ382 was mated with recipient PA0222 (ilv-226 his4 lys-12 met-28 trp-6 proA82 FP-), 50% of the ilv+ selected recombinants were imipenem resistant. When the same donor was mated with recipient PA01042 (pur-67 cys-59 thr-9001 proB65 FP-), 100% of the proB+ recombinants were imipenem resistant. Other chromosomal markers tested (e.g., his4, met-28, trp-6, proA, and cys-59) did not yield significant numbers of imipenemresistant colonies. Thus, imipenem resistance appears to map in a late region of the PAO1 chromosome, linked to the proB locus (Fig. 2). Cell-free lysates of the generalized transducing phage F116L were prepared with PJQ382 and used to transduce PA0222 and PA01042. The transfer of amino acid prototroTABLE 1. Strains of P. aeruginosa used in this study Strain
Wild type Impr Impr Impr Impr PA0381 leu-10 FP2+ (Hgr) PJQ382 leu-10 FP2+ (Hgr) Impr PA0222 ilv-226 his4 lys-12 met-28 trp-6 proA82 FPPA01042 pur-67 cys-59 thr-9001 proB65 FP-
PAO1 PAO1.1 PAO1.2 PAO1.3 PAO1.4
Corresponding author. 753
Reference or derivation
Genotype or phenotype
3
Impr derivative of PAO1 Impr derivative of PAO1 Impr derivative of PAO1 Impr derivative of PAO1 3
Impr derivative of PA0381 3 3
754
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. 1-Lactam MICs for P. aeruginosa strains
~~~~~~MIC
Strain Strain
70-
AZTR
CARB
CFTZ
IMP
PIP
PAO1 PAO1.1 PAO1.2 PAO1.3
C8 C8 >32 C8
C8 C8 >32 C8
PA0381 PJQ382
C8 C8
16 16 >256 16 16 64 16
C4 >16 >16 >16 >16 C4 >16
8 8 >256 8 8 C8 8
PAO1.4
C8
C8
C8 C8
xcp-l' -ned-YXJ3
(,ug/ml)"
71'----pro B
73'+- val-2
a AZTR, Aztreonam; CARB, carbenicillin; CFTZ, ceftazidime; IMP, imipenem; PIP, piperacillin.
phy was used as the primary criterion of selection for transductants. At least 100 and usually 200 transductants were screened for coinheritance of imipenem resistance by replica plating on LB plates containing 10 ,ug of imipenem per ml. The frequency of cotransduction of Impr with proB65 was 7.2%, and the frequency of cotransduction with ilv-226 was 1.5%. No cotransduction with met-28 or thr-9001 was detected. These data indicate that the Impr gene lies between proB and ilv-226 and is more closely linked to proB than to ilv-226. Since our initial report describing loss of a specific outer membrane protein in clinical isolates of P. aeruginosa displaying selective resistance to imipenem (6), a compelling body of evidence has emerged to support the hypothesis that carbapenems traverse the outer membrane of this organism through a unique porin. This evidence includes the followng points. (i) Experiments in other laboratories have produced similar data on outer membrane protein profiles in clinical isolates (4) and laboratory mutants (1) of imipenem-resistant P. aeruginosa. (ii) Two groups of investigators have described an imipenem-specific permeability barrier in resistant strains (8, 11). (iii) Trias and Nikaido recently identified the 47-kDa outer membrane protein as porin D2 (11). The normal substrate of this porin may be basic amino acids (11). The purpose of this study was to map the gene controlling imipenem resistance on the PAO chromosome. To our
oP D2 thi-800l
7Z-
7cagi A ilv B. S
pgi-9A01
1'- i car-9 orul
\Jjtol A
-narE -
3.-
pur-Al vtue
-gin-2020
-hisIF) ser.33
go a
5-J FIG. 2. Map of the late region of the P. aeruginosa PAO1 chromosome. OprD2 maps near proB.
knowledge, this is the first outer membrane protein gene in this organism to be mapped. This work was supported in part by a grant from Merck Sharp and Dohme and by cooperative agreements CR815234 and CR815282 from the Gulf Breeze Environment Research Laboratory of the U.S. Environmental Protection Agency.
REFERENCES 1. Buscher, K.-H., W. Cullmann, W. Dick, and W. Opferkuch.
2. 3.
4. 5.
FIG.
1.
SDS-PAGE
gel
of
outer
membrane
proteins
from
PA0381 (lane 2), PJQ382 (lane 3), PAO1 (lane 4) and Impr PA01.1, PAO1.2, PAO1.3, and PAO1.4 (lanes 5 through 8, respectively). Molecular mass markers, expressed in kilodaltons on the left, are in lane 1.
1987. Imipenem resistance in Pseudomonas aeruginosa resulting from diminished expression of an outer membrane protein. Antimicrob. Agents Chemother. 31:703-708. Haas, D., and B. W. Holloway. 1978. Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics. Mol. Gen. Genet. 158:229-237. Holloway, B. W., V. Krishnapillai, and A. F. Morgan. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43: 73-102. Lynch, M. J., G. L. Drusano, and H. L. T. Mobley. 1987. Emergence of resistance to imipenem in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:1892-1896. Miller, R. V., and C.-M. C. Ku. 1978. Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny. J. Bacteriol. 134:875-883.
6. Quinn, J. P., E. J. Dudek, C. A. DiVincenzo, D. A. Lucks, and S. A. Lerner. 1986. Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections. J. Infect. Dis.
154:289-294. 7. Quinn, J. P., A. E. Studemeister, C. A. DiVincenzo, and S. A. Lerner. 1988. Resistance to imipenem in Pseudomonas aerugi-
VOL. 35, 1991 nosa: clinical observations and biochemical mechanism. Rev. Infect. Dis. 10:892-898. 8. Studemeister, A. E., and J. P. Quinn. 1988. Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability. Antimicrob. Agents Chemother. 32:1267-1268. 9. Thornsberry, C. 1985. Automated procedures for antimicrobial susceptibility tests, p. 1015-1018. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 10. Trias, J., J. Dufresne, R. C. Levesque, and H. Nikaido. 1989. Decreased outer membrane permeability in imipenem-resistant
NOTES
755
mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 33:1201-1206. 11. Trias, J., and H. Nikaido. 1990. Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 34:52-57. 12. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of E. coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 13. Watanabe, M., S. lyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1991, p. 753-755
0066-4804/91/040753-03$02.00/0 Copyright C 1991, American Society for Microbiology
Imipenem Resistance in Pseudomonas aeruginosa PAO: Mapping of the OprD2 Gene JOHN P. QUINN,'* ALDIS DARZINS,2 DEBORAH MIYASHIRO,1 STEVEN RIPP,3 AND ROBERT V. MILLER3 Section of Infectious Diseases, Department of Medicine, Michael Reese Hospital, Chicago, Illinois 606161; Department of Microbiology, Ohio State University, Columbus, Ohio 432102; and Department of Molecular and Cellular Biochemistry and Program in Molecular Biology, Loyola University of Chicago, Maywood, Illinois 601533 Received 16 October 1990/Accepted 2 February 1991
Carbapenem antibiotics have been shown to penetrate the outer membrane of Pseudomonas aeruginosa through a unique porin protein, OprD2. We mapped the OprD2 gene by conjugation using plasmid FP2 and by transduction using phage F116L. This gene maps between 71 and 75 min on the PAO1 chromosome.
Imipenem resistance (Impr) in Pseudomonas aeruginosa is usually dissociated from resistance to other ,-lactams (6, 13). We (6, 7) and others (1, 4, 10) have shown that imipenem-specific resistance is due to loss of a specific outer membrane protein (Opr) with a molecular weight of 45,000 to 47,000. This Opr, recently identified by Trias and Nikaido as porin D2, is a porin which facilitates the penetration of carbapenem antibiotics across the outer membrane of P. aeruginosa (11). The purpose of this study was to map the chromosomal genes responsible for imipenem resistance in a genetically well-characterized strain of P. aeruginosa, PAO1. (This work was presented in part at the 28th Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, Calif. [J. P. Quinn, A. Darzins, D. Miyashiro, and M. Casadaban, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 488, 1988].) Bacterial strains, bacteriophages, and plasmids. All bacterial strains used in this study are listed in Table 1. Phage F116L was used for transduction (3). Plasmid FP2 was used in conjugation experiments (3). Media. Bacteria were grown in Luria-Bertani (LB) medium (Difco, Detroit, Mich.). Vogel-Bonner minimal medium (12) without citrate was used to select recombinants in conjugation experiments. A stock solution of glucose was added at a final concentration of 50 mM. The concentration of amino acids was 1 mM. Pseudomonas minimal medium supplemented with 0.4% glucose (5) was used for transduction. Amino acids were used at a concentration of 25 ,ug/ml. Isolation of mutants. Spontaneous imipenem-resistant mutants were obtained by plating 109 cells each of strains PAO1 and PA0381 onto LB plates containing 10 ,ug of imipenem per ml. Genetic crosses. Plate matings were performed as described by Haas et al. (2). Transductions were carried out by the method of Miller and Ku (5). Chemicals. Imipenem was a gift from Merck Sharp and Dohme (Rahway, N.J.). MICs. MICs were determined by an automated method (Vitek Systems, St. Louis, Mo.) (9). Table 2 lists the P-lactam susceptibilities of the parent strains (PA0381 and PAO1), four impenem-resistant mutants (PAO1.1, PAO1.2, *
PAO1.3, and PAO1.4) picked at random for further study and then cloned on imipenem-containing agar, and PJQ382. Four of these five mutants were selectively resistant to imipenem, while one (PAO1.2) was broadly resistant. Outer membrane proteins. Outer membrane proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (6). As seen in Fig. 1, the imipenem-resistant mutants (lanes 3 and 5 through 8) show reduction or deletion of a 47-kDa outer membrane protein compared with the parent PA0381 and PAO1 strains. Mapping of mutations conferring imipenem resistance. Impr variants (resistance frequency, 10-7) of P. aeruginosa PAO381 [leu-10 FP2+(Hgr)] were isolated by plating on overnight culture on LB agar containing imipenem (10 ,ug/ml). When imipenem-resistant PJQ382 was mated with recipient PA0222 (ilv-226 his4 lys-12 met-28 trp-6 proA82 FP-), 50% of the ilv+ selected recombinants were imipenem resistant. When the same donor was mated with recipient PA01042 (pur-67 cys-59 thr-9001 proB65 FP-), 100% of the proB+ recombinants were imipenem resistant. Other chromosomal markers tested (e.g., his4, met-28, trp-6, proA, and cys-59) did not yield significant numbers of imipenemresistant colonies. Thus, imipenem resistance appears to map in a late region of the PAO1 chromosome, linked to the proB locus (Fig. 2). Cell-free lysates of the generalized transducing phage F116L were prepared with PJQ382 and used to transduce PA0222 and PA01042. The transfer of amino acid prototroTABLE 1. Strains of P. aeruginosa used in this study Strain
Wild type Impr Impr Impr Impr PA0381 leu-10 FP2+ (Hgr) PJQ382 leu-10 FP2+ (Hgr) Impr PA0222 ilv-226 his4 lys-12 met-28 trp-6 proA82 FPPA01042 pur-67 cys-59 thr-9001 proB65 FP-
PAO1 PAO1.1 PAO1.2 PAO1.3 PAO1.4
Corresponding author. 753
Reference or derivation
Genotype or phenotype
3
Impr derivative of PAO1 Impr derivative of PAO1 Impr derivative of PAO1 Impr derivative of PAO1 3
Impr derivative of PA0381 3 3
754
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. 1-Lactam MICs for P. aeruginosa strains
~~~~~~MIC
Strain Strain
70-
AZTR
CARB
CFTZ
IMP
PIP
PAO1 PAO1.1 PAO1.2 PAO1.3
C8 C8 >32 C8
C8 C8 >32 C8
PA0381 PJQ382
C8 C8
16 16 >256 16 16 64 16
C4 >16 >16 >16 >16 C4 >16
8 8 >256 8 8 C8 8
PAO1.4
C8
C8
C8 C8
xcp-l' -ned-YXJ3
(,ug/ml)"
71'----pro B
73'+- val-2
a AZTR, Aztreonam; CARB, carbenicillin; CFTZ, ceftazidime; IMP, imipenem; PIP, piperacillin.
phy was used as the primary criterion of selection for transductants. At least 100 and usually 200 transductants were screened for coinheritance of imipenem resistance by replica plating on LB plates containing 10 ,ug of imipenem per ml. The frequency of cotransduction of Impr with proB65 was 7.2%, and the frequency of cotransduction with ilv-226 was 1.5%. No cotransduction with met-28 or thr-9001 was detected. These data indicate that the Impr gene lies between proB and ilv-226 and is more closely linked to proB than to ilv-226. Since our initial report describing loss of a specific outer membrane protein in clinical isolates of P. aeruginosa displaying selective resistance to imipenem (6), a compelling body of evidence has emerged to support the hypothesis that carbapenems traverse the outer membrane of this organism through a unique porin. This evidence includes the followng points. (i) Experiments in other laboratories have produced similar data on outer membrane protein profiles in clinical isolates (4) and laboratory mutants (1) of imipenem-resistant P. aeruginosa. (ii) Two groups of investigators have described an imipenem-specific permeability barrier in resistant strains (8, 11). (iii) Trias and Nikaido recently identified the 47-kDa outer membrane protein as porin D2 (11). The normal substrate of this porin may be basic amino acids (11). The purpose of this study was to map the gene controlling imipenem resistance on the PAO chromosome. To our
oP D2 thi-800l
7Z-
7cagi A ilv B. S
pgi-9A01
1'- i car-9 orul
\Jjtol A
-narE -
3.-
pur-Al vtue
-gin-2020
-hisIF) ser.33
go a
5-J FIG. 2. Map of the late region of the P. aeruginosa PAO1 chromosome. OprD2 maps near proB.
knowledge, this is the first outer membrane protein gene in this organism to be mapped. This work was supported in part by a grant from Merck Sharp and Dohme and by cooperative agreements CR815234 and CR815282 from the Gulf Breeze Environment Research Laboratory of the U.S. Environmental Protection Agency.
REFERENCES 1. Buscher, K.-H., W. Cullmann, W. Dick, and W. Opferkuch.
2. 3.
4. 5.
FIG.
1.
SDS-PAGE
gel
of
outer
membrane
proteins
from
PA0381 (lane 2), PJQ382 (lane 3), PAO1 (lane 4) and Impr PA01.1, PAO1.2, PAO1.3, and PAO1.4 (lanes 5 through 8, respectively). Molecular mass markers, expressed in kilodaltons on the left, are in lane 1.
1987. Imipenem resistance in Pseudomonas aeruginosa resulting from diminished expression of an outer membrane protein. Antimicrob. Agents Chemother. 31:703-708. Haas, D., and B. W. Holloway. 1978. Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics. Mol. Gen. Genet. 158:229-237. Holloway, B. W., V. Krishnapillai, and A. F. Morgan. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43: 73-102. Lynch, M. J., G. L. Drusano, and H. L. T. Mobley. 1987. Emergence of resistance to imipenem in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:1892-1896. Miller, R. V., and C.-M. C. Ku. 1978. Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny. J. Bacteriol. 134:875-883.
6. Quinn, J. P., E. J. Dudek, C. A. DiVincenzo, D. A. Lucks, and S. A. Lerner. 1986. Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections. J. Infect. Dis.
154:289-294. 7. Quinn, J. P., A. E. Studemeister, C. A. DiVincenzo, and S. A. Lerner. 1988. Resistance to imipenem in Pseudomonas aerugi-
VOL. 35, 1991 nosa: clinical observations and biochemical mechanism. Rev. Infect. Dis. 10:892-898. 8. Studemeister, A. E., and J. P. Quinn. 1988. Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability. Antimicrob. Agents Chemother. 32:1267-1268. 9. Thornsberry, C. 1985. Automated procedures for antimicrobial susceptibility tests, p. 1015-1018. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 10. Trias, J., J. Dufresne, R. C. Levesque, and H. Nikaido. 1989. Decreased outer membrane permeability in imipenem-resistant
NOTES
755
mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 33:1201-1206. 11. Trias, J., and H. Nikaido. 1990. Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 34:52-57. 12. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of E. coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 13. Watanabe, M., S. lyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.
