ANTIMICROBIAL AGoNTs AND CHEmoTHEmtPY, May 1978, p. 716-725 0066-4804/78/0013-0716$02.00/0 Copyright C) 1978 American Society for Microbiology
Vol. 13, No.5
Printed in U.S.A.
Plasmid-Mediated Mechanisms of Resistance to Aminoglycoside-Aminocyclitol Antibiotics and to Chloramphenicol in Group D Streptococci PATRICE M. COURVALIN,' WILLLAM V. SHAW2 AND ALAN E. JACOBt * Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin, 537061; Department of Biochemistry, University of Leicester, Leicester, LEI 7RH, England'; and Department ofBacteriology, Royal Postgraduate Medical School, DuCane Road, London, W12 OHS, England
Received for publication 18 July 1977
Genes conferring resistance to aminoglycoside-aminocyclitol antibiotics in three group D streptococcal strains, Streptococcus faecalis JH1 and JH6 and S. faecium JH7, and to chloramphenicol in JH6 are carried by plasmids that can transfer to other S faecalis celLs. The aminoglycoside resistance is mediated by constitutively
synthesized phosphotransferase enzymes that have substrate profiles very similar to those of aminoglycoside phosphotransferases found in gram-negative bacteria. Phosphorylation probably occurs at the aminoglycoside 3'-hydroxyl group. Plasmid-borne streptomycin resistance is due to production of the enzyme streptomycin adenylyltransferase, which, as in staphylococci and in contrast to that detected in gram-negative bacteria, is less effective against spectinomycin as substrate. Resistance to chloramphenicol is by enzymatic acetylation. The chloramphenicol acetyltransferase is inducible and bears a close resemblance to the type D chloramphenicol acetyltransferase variant from staphylococci.
In recent years it has become clear that many of the genes conferring resistance to antibiotics in group D streptococci are plasmid borne (4, 5, 13). The plaids can transfer between group D
METHODS Bacterial strains. The bacterial strains used are
listed in Table 1. Each wild strain was isolated from a separate patient in Hammersmith Hospital and charstreptococcal strains; some plasmids are conju- acterized by biochemical and serological tests (12, 13). gative (13), and nonconjugative plasmids can be Derivative strains were isolated by selection of spontaneous mutants (13). mobilized by conjugative plasmids (9). Media. Brain heart infusion broth and agar (Oxoid) In gram-negative bacteria and Staphylococcus the mechanism of plasmid-mediated resistance were used, except where stated. All incubations were 370C. involves the production of enzymes that inacti- at MIC The method of Steers et al. vate the antibiotic by either modification or (18) wasdeterminations. used to determine antibiotic minimal inhibidestruction of the antibiotic structure (8). This tory concentrations (MICs). is in contrast to chromosomally detennined anRadiolabeling, isolation, and characterization tibiotic resistance, which is more usually due of plasmid DNA. Plasmid DNA was isolated by either to prevention of entry of the antibiotic cesium chloride-ethidium bromide density gradient into the cell or to a specific alteration of the ultracentrifugation and characterized by neutral suantibiotic target site (8). In this report it is shown crose gradient analysis as described previously (12, that, like the plasmid-borne resistance in the 13). Calculation of plasmid molecular weight. The gram-negative bacteria and staphylococci, plas- empirical formulae of Barth and Grinter (1) were used mid-linked resistances to aminoglycoside-ami- to calculate plasmid molecular weights. nocyclitol antibiotics and chloramphenicol in Transfer of antibiotic resistance traits. The group D streptococci are also mediated by en- technique described by Jacob and Hobbs (13) was zymatic modification of the antibiotic. The prop- used in transferring antibiotic resistance traits. erties of the Streptococcus faecalis enzymes are Enzyme assays. (i) Assays of aminoglycoside compared with the similar enzymes of staphy- phosphorylation and streptomycin adenylylation. The extracts for assay were prepared as follows: lococci and the gram-negative bacteria. 100 ml of an exponential culture in broth was hart Prent addres: Department of Bacteriology and Virol- vested and washed once in 10 mM tris(hydroxymethyl) ogy, The Medical School, University of Manchester, Man- aminomethane-hydrochloride (pH 7.2)-i mM ethylchester, M13 9PT, England. enedianminetetraacetic acid, then resuspended in 1 ml 716
VOL. 13, 1978
GROUP D STREPTOCOCCI RESISTANCE MECHANISMS
of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.2)-10 mM MgC1r-25 mM NH4C-0.6 mM fi-mercaptoethanol. The celLs were treated for 60 min at 370C with 500,ug of lysozyme (Sigma) per ml and then subjected to five 60-s bursts from a sonicator at 70W output. The resulting suspension was centrifuged at 105,000 x g for 45 min, and the supernatant was removed and used in assays performed as described previously (11). (ii) Assays of chloramphenicol acetylation. The techniques employed for the detection of [14C]chloramphenicol acetylation by bacterial cell suspensions have been described previously (16). Chloramphenicol acetyltransferase activity in cell-free extracts was determined by the spectrophotometric method of Shaw and Brodsky (17). The induction of chloramphenicol acetyltransferase by the parent antibiotic and by gratuitous inducers has been described (21). In the present experiments, S. faecalis strains were grown for 18 h in brain heart infusion broth at 370C without shaking. The overnight cultures were then diluted with 4 volumes of fresh medium, after which they were TABLE 1. Bacterial strains used Strain
S. faecalis JH1
Relevant phenotype'
717
allowed to grow for an additional 4 h under the same conditions, before addition of inducers at the concentrations specified. Cells were collected by centrifugation after 2 h of further incubation and were suspended for lysis by sonic disruption as previously described (16). The immunological techniques for the characterization of chloramphenicol acetyltransferase employed both agar diffusion tests with and neutralization by antisera prepared against chloramphenicol acetyltransferase from Staphylococcus aureus or Escherichia coli (17, 21). Controls run in each instance with pre-immunization sera failed to yield either precipitation lines or inactivation of enzyme. The techniques used for polyacrylamide gel electrophoresis and for the localization of enzyme activity on cylindrical gels have been reported previously (17). Antibiotics. The antibiotics were provided by the following laboratories: neomycin B and spectinomycin, Upjohn; kanamycins A, B, and C and amikacin, Bristol; paromomycin, butirosin, and chloramphenicol, Parke-Davis; lividomycin A, Kowa; ribostamycin, Meiji; tobramycin, Lilly; streptomycin, Pfizer; erythromycin, Abbott; fusidic acid, Leo; tetracycline, Lederle; rifampin, Ciba-Geigy.
Reference
RESULTS
Location of the genes determining antibiotic resistance in S. faecalis JH1 and JH6 and S. faecium JH7. The genes determining 12 JH2 resistance to kanamycin (Kan), streptomycin 13 JH2-7 (Str), erythromycin (Em), and tetracycline (Tc) in strain JH1 have previously been shown to be JH6 carried by a conjugative plasmid, pJH1 (13). S. faecium The MICs of these antibiotics for inhibition of Wild strain, Kan, Str, Em JH7 strain JH1 and of a recipient strain, JH2-7, that a Abbreviations: Kan, kanamycin; Str, streptomycin; has received plasmid pJH1 (strain JH2-15) are Em, erythromycin; Tc, tetracycline; Fus, fusidic acid; shown in Table 2. The genes determining resistance to chlorRif, Rifampin; Thy, thymine. Wild strain, Kan, Str, Em, Tc Wild strain JH2 derivative, Thy-, Fus, Rif Wild strain, Kan, Str, Cm, Tc, Em
13
TABLE 2. MIC of antibiotics for inhibition of group D streptococci MIC of antibiotics
Strain
Resistances determined by wild strains
(lAg/mi)a: Introduced chromosomal resistancesb
Str Cm Tc Em Fus Rif Str Karn 4 1
Vol. 13, No.5
Printed in U.S.A.
Plasmid-Mediated Mechanisms of Resistance to Aminoglycoside-Aminocyclitol Antibiotics and to Chloramphenicol in Group D Streptococci PATRICE M. COURVALIN,' WILLLAM V. SHAW2 AND ALAN E. JACOBt * Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin, 537061; Department of Biochemistry, University of Leicester, Leicester, LEI 7RH, England'; and Department ofBacteriology, Royal Postgraduate Medical School, DuCane Road, London, W12 OHS, England
Received for publication 18 July 1977
Genes conferring resistance to aminoglycoside-aminocyclitol antibiotics in three group D streptococcal strains, Streptococcus faecalis JH1 and JH6 and S. faecium JH7, and to chloramphenicol in JH6 are carried by plasmids that can transfer to other S faecalis celLs. The aminoglycoside resistance is mediated by constitutively
synthesized phosphotransferase enzymes that have substrate profiles very similar to those of aminoglycoside phosphotransferases found in gram-negative bacteria. Phosphorylation probably occurs at the aminoglycoside 3'-hydroxyl group. Plasmid-borne streptomycin resistance is due to production of the enzyme streptomycin adenylyltransferase, which, as in staphylococci and in contrast to that detected in gram-negative bacteria, is less effective against spectinomycin as substrate. Resistance to chloramphenicol is by enzymatic acetylation. The chloramphenicol acetyltransferase is inducible and bears a close resemblance to the type D chloramphenicol acetyltransferase variant from staphylococci.
In recent years it has become clear that many of the genes conferring resistance to antibiotics in group D streptococci are plasmid borne (4, 5, 13). The plaids can transfer between group D
METHODS Bacterial strains. The bacterial strains used are
listed in Table 1. Each wild strain was isolated from a separate patient in Hammersmith Hospital and charstreptococcal strains; some plasmids are conju- acterized by biochemical and serological tests (12, 13). gative (13), and nonconjugative plasmids can be Derivative strains were isolated by selection of spontaneous mutants (13). mobilized by conjugative plasmids (9). Media. Brain heart infusion broth and agar (Oxoid) In gram-negative bacteria and Staphylococcus the mechanism of plasmid-mediated resistance were used, except where stated. All incubations were 370C. involves the production of enzymes that inacti- at MIC The method of Steers et al. vate the antibiotic by either modification or (18) wasdeterminations. used to determine antibiotic minimal inhibidestruction of the antibiotic structure (8). This tory concentrations (MICs). is in contrast to chromosomally detennined anRadiolabeling, isolation, and characterization tibiotic resistance, which is more usually due of plasmid DNA. Plasmid DNA was isolated by either to prevention of entry of the antibiotic cesium chloride-ethidium bromide density gradient into the cell or to a specific alteration of the ultracentrifugation and characterized by neutral suantibiotic target site (8). In this report it is shown crose gradient analysis as described previously (12, that, like the plasmid-borne resistance in the 13). Calculation of plasmid molecular weight. The gram-negative bacteria and staphylococci, plas- empirical formulae of Barth and Grinter (1) were used mid-linked resistances to aminoglycoside-ami- to calculate plasmid molecular weights. nocyclitol antibiotics and chloramphenicol in Transfer of antibiotic resistance traits. The group D streptococci are also mediated by en- technique described by Jacob and Hobbs (13) was zymatic modification of the antibiotic. The prop- used in transferring antibiotic resistance traits. erties of the Streptococcus faecalis enzymes are Enzyme assays. (i) Assays of aminoglycoside compared with the similar enzymes of staphy- phosphorylation and streptomycin adenylylation. The extracts for assay were prepared as follows: lococci and the gram-negative bacteria. 100 ml of an exponential culture in broth was hart Prent addres: Department of Bacteriology and Virol- vested and washed once in 10 mM tris(hydroxymethyl) ogy, The Medical School, University of Manchester, Man- aminomethane-hydrochloride (pH 7.2)-i mM ethylchester, M13 9PT, England. enedianminetetraacetic acid, then resuspended in 1 ml 716
VOL. 13, 1978
GROUP D STREPTOCOCCI RESISTANCE MECHANISMS
of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.2)-10 mM MgC1r-25 mM NH4C-0.6 mM fi-mercaptoethanol. The celLs were treated for 60 min at 370C with 500,ug of lysozyme (Sigma) per ml and then subjected to five 60-s bursts from a sonicator at 70W output. The resulting suspension was centrifuged at 105,000 x g for 45 min, and the supernatant was removed and used in assays performed as described previously (11). (ii) Assays of chloramphenicol acetylation. The techniques employed for the detection of [14C]chloramphenicol acetylation by bacterial cell suspensions have been described previously (16). Chloramphenicol acetyltransferase activity in cell-free extracts was determined by the spectrophotometric method of Shaw and Brodsky (17). The induction of chloramphenicol acetyltransferase by the parent antibiotic and by gratuitous inducers has been described (21). In the present experiments, S. faecalis strains were grown for 18 h in brain heart infusion broth at 370C without shaking. The overnight cultures were then diluted with 4 volumes of fresh medium, after which they were TABLE 1. Bacterial strains used Strain
S. faecalis JH1
Relevant phenotype'
717
allowed to grow for an additional 4 h under the same conditions, before addition of inducers at the concentrations specified. Cells were collected by centrifugation after 2 h of further incubation and were suspended for lysis by sonic disruption as previously described (16). The immunological techniques for the characterization of chloramphenicol acetyltransferase employed both agar diffusion tests with and neutralization by antisera prepared against chloramphenicol acetyltransferase from Staphylococcus aureus or Escherichia coli (17, 21). Controls run in each instance with pre-immunization sera failed to yield either precipitation lines or inactivation of enzyme. The techniques used for polyacrylamide gel electrophoresis and for the localization of enzyme activity on cylindrical gels have been reported previously (17). Antibiotics. The antibiotics were provided by the following laboratories: neomycin B and spectinomycin, Upjohn; kanamycins A, B, and C and amikacin, Bristol; paromomycin, butirosin, and chloramphenicol, Parke-Davis; lividomycin A, Kowa; ribostamycin, Meiji; tobramycin, Lilly; streptomycin, Pfizer; erythromycin, Abbott; fusidic acid, Leo; tetracycline, Lederle; rifampin, Ciba-Geigy.
Reference
RESULTS
Location of the genes determining antibiotic resistance in S. faecalis JH1 and JH6 and S. faecium JH7. The genes determining 12 JH2 resistance to kanamycin (Kan), streptomycin 13 JH2-7 (Str), erythromycin (Em), and tetracycline (Tc) in strain JH1 have previously been shown to be JH6 carried by a conjugative plasmid, pJH1 (13). S. faecium The MICs of these antibiotics for inhibition of Wild strain, Kan, Str, Em JH7 strain JH1 and of a recipient strain, JH2-7, that a Abbreviations: Kan, kanamycin; Str, streptomycin; has received plasmid pJH1 (strain JH2-15) are Em, erythromycin; Tc, tetracycline; Fus, fusidic acid; shown in Table 2. The genes determining resistance to chlorRif, Rifampin; Thy, thymine. Wild strain, Kan, Str, Em, Tc Wild strain JH2 derivative, Thy-, Fus, Rif Wild strain, Kan, Str, Cm, Tc, Em
13
TABLE 2. MIC of antibiotics for inhibition of group D streptococci MIC of antibiotics
Strain
Resistances determined by wild strains
(lAg/mi)a: Introduced chromosomal resistancesb
Str Cm Tc Em Fus Rif Str Karn 4 1
