ANTIMICROBIAL AGENTS AND CizEMOTHERAPY, Apr. 1977, p. 589-593 Copyright 0 1977 American Society for Microbiology
Vol. 11, No. 4 Printed in U.S.A.
Pseudomonas aeruginosa Strain Isolated in France That Carries a Plasmid Determining Carbenicillin Resistance Y. MICHEL-BRIAND,* VILMA A. STANISICH,1
AND
M. JOUVENOT
Laboratoire de Bacteriologie-Virologie, Faculte de Medecine et de Pharmacie, Place Saint-Jacques, 25030 Besancon, France,* and Department of Bacteriology, Bristol University Medical School, Bristol BS8 1 TD, England Received for publication 8 October 1976
A plasmid, R56Be, derived from a clinical strain of Pseudomonas aeruginosa isolated in France in 1973 has been studied. This plasmid confers resistance only to carbenicillin, and although freely transferable between unrelated strains of P. aeruginosa, its transfer from P. aeruginosa to Escherichia coli K-12 was undetectable. R56Be could not be isolated as covalently closed circular deoxyribonucleic acid, but the plasmid could be transduced intact by phage F116L. This suggests that its molecular weight does not exceed that of the phage genome (ca. 40 x 106). In terms of its interaction with male and female sex-specific phages, its exclusion characteristics, and the main properties of (-lactamase determined by them, R56Be is similar to the previously characterized plasmid RP1-1.
Pseudomonas aeruginosa is an important opportunistic pathogen that has a marked resistance to many antibiotics, but it is usually sensitive to carbenicillin. However, in 1969, carbenicillin-resistant strains of P. aeruginosa were isolated from patients in a Burns Unit in Birmingham, England, and the resistance was shown to be plasmid mediated (6, 16). Similarly resistant strains have subsequently been isolated in different parts of the world, and the plasmids carried by them have been shown to comprise at least five incompatibility groups (2, 12-14, 26, 29). However, with the exception of RP1-1 (9) and R91 (27), both of which were derived from the Birmingham strains and confer resistance only to carbenicillin, these various plasmids confer a multiple resistance phenotype. This study examines the nature of carbenicillin resistance in a strain of P. aeruginosa isolated from a patient in France. We show that resistance is determined by a conjugative plasmid whose transfer is confined to strains of P. aeruginosa. Moreover, a comparison of the properties of this new plasmid with those conferred by other carbenicillin resistance plasmids -suggests that it is similar to the previously described plasmid RPl-1.
medium was that described by Stanisich and Holloway (28). Antibiotic supplements to nutrient agar and minimal medium were as follows: carbenicillin, 500 ,Lg/ml; streptomycin sulfate, 250 ,ug/ml; rifampin, 100 ,ug/ml. Bacterial strains and plasmids. The properties and origin of the bacterial strains and plasmids used are shown in Table 1. Psl9 is a clinical isolate of P.
aeruginosa that is prototrophic and does not carry resistance plasmids (N. A. C. Curtis, personal communication). Plasmid-carrying derivatives of various PAO strains were also used. R91-21 is a transfer-derepressed (drd) mutant of 1R91 (27). Such mutants also express an Spp, phenotype. R18-1-1 and R91-16, respectively, are carbenicillin-susceptible mutants of R18-1 and a drd mutant (R91-5) of R91. RP1-2 is a Cbo mutant of RP1 (4). General methods. All bacterial strains were used in exponential growth phase. In mating experiments in nutrient broth, donor and recipient bacteria were mixed in a ratio of 5:1 and maintained at 37°C for the mating periq4, i+id samples of the mixture were plated onto the appropriate selective medium. Spot plate matings, spot assays for the determination of efficiency of phage plating, and the method for F116L-mediated transduction have been described previously (29). Phages. The origins and properties of the phages used have been described previously (29). They were the RP1 donor-specific phages PRR1, PR4, PRD1, and Pf3, the temperate phages B3, B33, B39, and F116L, and the virulent phage E79. The clear plaque mutant GlOlc was also used. Determination of substrate profile of ,B-lactamase and the isolation of plasmid DNA. The procedures for the growth of bacteria, induction of the (3lactamase, and the determination of its substrate profile were those previously described (17, 18).-The isolation of plasmid deoxyribonucleic acid (DNA) as
MATERIALS AND METHODS Media. Oxoid Muller-Hinton medium was used as the nutrient agar and Oxoid heart brain infusion as the nutrient broth. The composition of the minimal 1 Present address: Department of Microbiology, La Trobe University, Bundoora 3083, Victoria, Australia.
589
590
MICHEL-BRIAND, STANISICH, AND JOUVENOT
ANTiMICROB. AGENTS CHEMOTHER.
TABLE 1. Bacterial strains and plasmids Strain or plasmid
P. aeruginosa Ps19 PAT900 PAO8 PA0303 PA0307 PA0315 PA0366
PA0381 PA01670 PAO2001 PA02003 PA02637 E. coli W3110 Plasmids RP1 RP1-2 RP1-1 R18-1-1 R91 R91-16 R91-21
Genotype or phenotypea
Reference or sourceb
Prototroph his-404 str-1100 met-28 ilv-202 str-1 argB18 chl-2 argC54 chl-2 argG9 chl-2 argA36 chl-2 leu-38 str-13 FP2+ ade-136 leu-8 chl-3 rif-1 argH32 str-39 chl-2 argH32 str-39 chl-2 rec-2 Prototroph, chl-2 fon-100
N. A. C. Curtis 28 10 11 11 11 11 28 15 15 15 29
Prototroph, rif
M. H. Richmond
Cbr Nmr/Kmr Tcr Spp1+ Tra+ Inc P 6, 24 4 Cbs Nmr/Kmr Tcr Sppl+ Tra+ Inc P Cbr Spp,,,+ Tra+ 9, 24 P. M. Chandler Cb' Spp,l,+ Tra+ 27 Cbr Tra+ P. M. Chandler Cbs Sppl+ Tradrd+ 27 Cbr Sppl+ Tradrd+ a Resistance to phage F116L. Plasmid et al. follow those Demerec Genotypic symbols proposed by (5). fon, phenotypic designations follow those proposed by Novick et al. (20). Spp+, Reduced plating of phage G101 (29); SppA,', reduced plating of phages B33 and B39 (19). b N. A. C. Curtis and M. H. Richmond are from the Department of Microbiology, Bristol University, Bristol U.K.; P. M. Chandler is from the Department of Genetics, Monash University, Monash, Australia.
the closed covalent circular form was carried out using the procedures of Grinsted et al. (6). [3H]adenine was used to label the DNA of R+ derivatives of PA01670. The subline carrying RP1 was used as a control.
RESULTS Derivation of R56Be. P. aeruginosa strain 56Be was isolated in 1973 from a patient in the hospital in Besangon, France. The strain was highly* resistant to carbenicillin (>8 mg/ml), and also to terramycin (100 ,ug/ml), kanamycin (500 ,ug/ml), streptomycin (100 ,ug/ml), and mercuric chloride (25 ug/ml), but was susceptible to rifampin. To determine whether the carbenicillin resistance (Cbr) phenotype of this strain was transferable, matings were carried out with the recipient PA01670 (Rifr). Cbr transconjugants were selected on nutrient agar containing carbenicillin and rifampin and occurred at a frequency of about 104/donor per 3-h mating. The transconjugants displayed only the Cbr phenotype of the donor parent (20 colonies tested), and one derivative from each mating was chosen for further study after ensuring that it had not been lysogenized by phages carried by the donor strain.
The possession by this PA01670 derivative of a conjugative plasmid was confirmed in subsequent matings with PA02001 (arg-32 str-39) and its recombination-deficient subline PA02003 (arg-32 str-39 rec-2). Cb' transconjugants were recovered from all matings at frequencies of between 1.2 x 10-2 and 3.8 x 10-4/ donor per 30-min mating, but transfer of the chromosomal allele arg-32+ was not observed (
Vol. 11, No. 4 Printed in U.S.A.
Pseudomonas aeruginosa Strain Isolated in France That Carries a Plasmid Determining Carbenicillin Resistance Y. MICHEL-BRIAND,* VILMA A. STANISICH,1
AND
M. JOUVENOT
Laboratoire de Bacteriologie-Virologie, Faculte de Medecine et de Pharmacie, Place Saint-Jacques, 25030 Besancon, France,* and Department of Bacteriology, Bristol University Medical School, Bristol BS8 1 TD, England Received for publication 8 October 1976
A plasmid, R56Be, derived from a clinical strain of Pseudomonas aeruginosa isolated in France in 1973 has been studied. This plasmid confers resistance only to carbenicillin, and although freely transferable between unrelated strains of P. aeruginosa, its transfer from P. aeruginosa to Escherichia coli K-12 was undetectable. R56Be could not be isolated as covalently closed circular deoxyribonucleic acid, but the plasmid could be transduced intact by phage F116L. This suggests that its molecular weight does not exceed that of the phage genome (ca. 40 x 106). In terms of its interaction with male and female sex-specific phages, its exclusion characteristics, and the main properties of (-lactamase determined by them, R56Be is similar to the previously characterized plasmid RP1-1.
Pseudomonas aeruginosa is an important opportunistic pathogen that has a marked resistance to many antibiotics, but it is usually sensitive to carbenicillin. However, in 1969, carbenicillin-resistant strains of P. aeruginosa were isolated from patients in a Burns Unit in Birmingham, England, and the resistance was shown to be plasmid mediated (6, 16). Similarly resistant strains have subsequently been isolated in different parts of the world, and the plasmids carried by them have been shown to comprise at least five incompatibility groups (2, 12-14, 26, 29). However, with the exception of RP1-1 (9) and R91 (27), both of which were derived from the Birmingham strains and confer resistance only to carbenicillin, these various plasmids confer a multiple resistance phenotype. This study examines the nature of carbenicillin resistance in a strain of P. aeruginosa isolated from a patient in France. We show that resistance is determined by a conjugative plasmid whose transfer is confined to strains of P. aeruginosa. Moreover, a comparison of the properties of this new plasmid with those conferred by other carbenicillin resistance plasmids -suggests that it is similar to the previously described plasmid RPl-1.
medium was that described by Stanisich and Holloway (28). Antibiotic supplements to nutrient agar and minimal medium were as follows: carbenicillin, 500 ,Lg/ml; streptomycin sulfate, 250 ,ug/ml; rifampin, 100 ,ug/ml. Bacterial strains and plasmids. The properties and origin of the bacterial strains and plasmids used are shown in Table 1. Psl9 is a clinical isolate of P.
aeruginosa that is prototrophic and does not carry resistance plasmids (N. A. C. Curtis, personal communication). Plasmid-carrying derivatives of various PAO strains were also used. R91-21 is a transfer-derepressed (drd) mutant of 1R91 (27). Such mutants also express an Spp, phenotype. R18-1-1 and R91-16, respectively, are carbenicillin-susceptible mutants of R18-1 and a drd mutant (R91-5) of R91. RP1-2 is a Cbo mutant of RP1 (4). General methods. All bacterial strains were used in exponential growth phase. In mating experiments in nutrient broth, donor and recipient bacteria were mixed in a ratio of 5:1 and maintained at 37°C for the mating periq4, i+id samples of the mixture were plated onto the appropriate selective medium. Spot plate matings, spot assays for the determination of efficiency of phage plating, and the method for F116L-mediated transduction have been described previously (29). Phages. The origins and properties of the phages used have been described previously (29). They were the RP1 donor-specific phages PRR1, PR4, PRD1, and Pf3, the temperate phages B3, B33, B39, and F116L, and the virulent phage E79. The clear plaque mutant GlOlc was also used. Determination of substrate profile of ,B-lactamase and the isolation of plasmid DNA. The procedures for the growth of bacteria, induction of the (3lactamase, and the determination of its substrate profile were those previously described (17, 18).-The isolation of plasmid deoxyribonucleic acid (DNA) as
MATERIALS AND METHODS Media. Oxoid Muller-Hinton medium was used as the nutrient agar and Oxoid heart brain infusion as the nutrient broth. The composition of the minimal 1 Present address: Department of Microbiology, La Trobe University, Bundoora 3083, Victoria, Australia.
589
590
MICHEL-BRIAND, STANISICH, AND JOUVENOT
ANTiMICROB. AGENTS CHEMOTHER.
TABLE 1. Bacterial strains and plasmids Strain or plasmid
P. aeruginosa Ps19 PAT900 PAO8 PA0303 PA0307 PA0315 PA0366
PA0381 PA01670 PAO2001 PA02003 PA02637 E. coli W3110 Plasmids RP1 RP1-2 RP1-1 R18-1-1 R91 R91-16 R91-21
Genotype or phenotypea
Reference or sourceb
Prototroph his-404 str-1100 met-28 ilv-202 str-1 argB18 chl-2 argC54 chl-2 argG9 chl-2 argA36 chl-2 leu-38 str-13 FP2+ ade-136 leu-8 chl-3 rif-1 argH32 str-39 chl-2 argH32 str-39 chl-2 rec-2 Prototroph, chl-2 fon-100
N. A. C. Curtis 28 10 11 11 11 11 28 15 15 15 29
Prototroph, rif
M. H. Richmond
Cbr Nmr/Kmr Tcr Spp1+ Tra+ Inc P 6, 24 4 Cbs Nmr/Kmr Tcr Sppl+ Tra+ Inc P Cbr Spp,,,+ Tra+ 9, 24 P. M. Chandler Cb' Spp,l,+ Tra+ 27 Cbr Tra+ P. M. Chandler Cbs Sppl+ Tradrd+ 27 Cbr Sppl+ Tradrd+ a Resistance to phage F116L. Plasmid et al. follow those Demerec Genotypic symbols proposed by (5). fon, phenotypic designations follow those proposed by Novick et al. (20). Spp+, Reduced plating of phage G101 (29); SppA,', reduced plating of phages B33 and B39 (19). b N. A. C. Curtis and M. H. Richmond are from the Department of Microbiology, Bristol University, Bristol U.K.; P. M. Chandler is from the Department of Genetics, Monash University, Monash, Australia.
the closed covalent circular form was carried out using the procedures of Grinsted et al. (6). [3H]adenine was used to label the DNA of R+ derivatives of PA01670. The subline carrying RP1 was used as a control.
RESULTS Derivation of R56Be. P. aeruginosa strain 56Be was isolated in 1973 from a patient in the hospital in Besangon, France. The strain was highly* resistant to carbenicillin (>8 mg/ml), and also to terramycin (100 ,ug/ml), kanamycin (500 ,ug/ml), streptomycin (100 ,ug/ml), and mercuric chloride (25 ug/ml), but was susceptible to rifampin. To determine whether the carbenicillin resistance (Cbr) phenotype of this strain was transferable, matings were carried out with the recipient PA01670 (Rifr). Cbr transconjugants were selected on nutrient agar containing carbenicillin and rifampin and occurred at a frequency of about 104/donor per 3-h mating. The transconjugants displayed only the Cbr phenotype of the donor parent (20 colonies tested), and one derivative from each mating was chosen for further study after ensuring that it had not been lysogenized by phages carried by the donor strain.
The possession by this PA01670 derivative of a conjugative plasmid was confirmed in subsequent matings with PA02001 (arg-32 str-39) and its recombination-deficient subline PA02003 (arg-32 str-39 rec-2). Cb' transconjugants were recovered from all matings at frequencies of between 1.2 x 10-2 and 3.8 x 10-4/ donor per 30-min mating, but transfer of the chromosomal allele arg-32+ was not observed (
