ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1975, p. 227-230 Copyright 0 1975 American Society for Microbiology
Vol. 8, No. 3 Printed in U.S.A.
Third Type of Plasmid Conferring Gentamicin Resistance Pseudomonas aeruginosa
in
DAVID I. SMITH, R GOMEZ LUS, M. C. RUBIO CALVO, NAOMI DAITA, A. E. JACOB, AND R. W. HEDGES* Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706; Departmento de Microbiologia e Higiene, Facultad de Medicina, Universidad de Zaragoza, Spain; and Bacteriology Department, Royal Postgraduate Medical School, London W12 OHS, United Kingdom*
Received for publication 5 May 1975
R1033 is a plasmid of compatibility group P (= P1) transferred from a wild strain of Pseudomonas aeruginosa. It confers resistance to gentamicin by gentamicin acetyl-transferase 1 and to kanamycin and neomycin by neomycin phosphotransferase 1.
The self-transmissible R factors of Pseudomonas aeruginosa clinical isolates have been assigned to three compatibility groups by Bryan and his collaborators (2-4). Group P1 contains the R factors that in Escherichia coli K-12 constitute group P (13). Group P2 plasmids are not transmissible to E. coli, and plasmids of group P3 are those assigned to group C (12) or com6 (6) in the E. coli plasmid classifications. Since gentamicin is useful in the treatment of P. aeruginosa infections, attention has been concentrated on mechanisms by which resistance to this drug can be attained. Isolates of P. aeruginosa carrying plasmids conferring gentamicin resistance have been reported (3, 17, 21). Bryan et al. (3) found among North American isolates gentamicin resistance conferred by plasmids of groups P2 and P3, and Jacoby (17) has observed such R factors of group P2. In France, gentamicin R factors of group P3 have been observed in P. aeruginosa (21). We report here the isolation, in Spain, of a strain resistant to gentamicin (and several other antibiotics) by virtue of an R factor of group P1. MATERIALS AND METHODS Bacteria. The bacteria used were P. aeruginosa CRT and E. coli K-12 strains J53-2 (F-, pro,met,rif), J62 (F-, pro,his,trp,lac), and CR34Thy (F-, thr,leu,
thi,thy,lac). R factors. The R factors used were Plac, a plasmid
of compatibility group C (= P3) which confers lactose fermentation ability and sulfonamide resistance (15), and R751, a plasmid of compatibility group P (= P1) which confers resistance to trimethoprim (18). Phage. The phage used was PRR1, which adsorbs to bacteria carrying plasmids of group P (= P1) and not to plasmid-free becteria or cells carrying plasmids of other groups (11, 19). Conjugal plasmid transfer and plasmid compatibility properties. Procedures for conjugal plasmid
transfer and the determination of plasmid compatibility properties were as described previously (9, 13).
Minimal inhibitory concentrations. The drugs used and their minimal inhibitory concentrations are listed in Table 1. Radiolabeling and lysis of CR34Thy (R1033) and isolation of plasmid DNA. Rabiolabeling and lysis of CR34Thy (R1033) and isolation of plasmid deoxyribonucleic acid (DNA) by cesium chloride-ethidium bromide density gradient centrifugation were as described previously (7). Neutral sucrose gradient analysis and calculation of plasmid molecular weight. Neutral sucrose gradient analysis and calculation of plasmid molecular weight were as described previously (1). Preparation of cell-free extracts. R1033 in E. coli was grown to late log phage in yeast extract-tryptone medium (100 ml). The cells were harvested by centrifugation and washed twice with a solution of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.8), 50 mM NH4C1, 10 mM MgCl2, and 0.64 mM 2-mercaptoethanol. The pellet was resuspended in 4 ml of the same buffer and disrupted by sonic treatment. The cell debris was removed by centrifugation at 100,000 x g for 90 min. The resulting supernatant was designated the sonic extract and is the preparation that was used in all radioactive assays. Enzymatic assays. Phosphorylation of aminoglycoside antibiotics was assayed by the phosphocellulose paper binding assay, as described previously (20). Acetylation of aminoglycoside antibiotics was also assayed by the phosphocellulose paper binding assay, as described previously (5).
RESULTS
Properties of P. aeruginosa CRT. The strain was pigmented and showed the typical characters of P. aeruginosa (10). The minimal inhibitory concentrations of relevant antibiotics are shown in Table 1. Strain CRT is unstable, producing segregants with increased susceptibility to gentamicin (kanamycin, carbenicillin, tetracycline, strep227
228
ANTIMICROB. AGENTS CHEMOTHER.
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TABLE 1. Minimal inhibitory concentrations (MIC) of antibiotics for P. aeruginosa CRT and E. coli K-12, with and without R factor R1033 MICa
Strain
P. aeruginosa CRT P. aeruginosa CRTSb E. coli K-12 strain J53 E. coli K-12 strain J53 (R1033)
Genta
Tobra
Kana
Chlor
500 1.5 0.1 12.5
0.8 0.4 0.2 0.2
1,000
> 200
25 0.2 50
25 5 25
Carb 300
40 2.5 > 5,000
a The figures show the lowest concentrations of drug (micrograms per milliliter) in Oxoid DST (direct sensitivity test) agar with added 4% lysed horse blood that prevented visible growth from inocula that gave dense, but not confluent, growth on drug-free control plates. The MIC of butirosin was not measured, but the diameters of the zones of inhibition of E. coli K-12 R- and R1033+ round filter paper disks containing 20 ,g of butirosin were identical. Abbreviations: Genta, gentamicin; Tobra, tobramycin; Kana, kanamycin; Chlor, chloramphenicol; Carb, carbenicillin. bP. aeruginosa CRTS is the wild strain after spontaneous loss of resistance (see text).
tomycin, chloramphenicol, and sulfonamides). Such segregants have, presumably, lost the R factor. One of these, strain CRTS, is included in Table 1. When strain CRT was grown with E. coli J53-2, resistance to gentamicin, kanamycin, ampicillin, tetracycline, streptomycin, chloramphenicol, and sulfonamides was transferred. Since all resistances were regularly co-transferred and in view of the compatibility properties and molecular characteristics of the resistant strains, it was concluded that all of the resistances were determined by a single plasmid, R1033. Properties of R1033 in E. coli K-12. In E. coli, R1033 conferred resistance to gentamicin,
kanamycin, ampicillin, tetracycline, and chloramphenicol, and to very low levels of streptomycin and sulfonamides. It confers sensitivity to phage PRR1. J62 (R1033) was mated with CR34Thy(Plac). Transfer in both directions was observed without elimination, and both plasmids were stable in each strain. Thus, R1033 is not a member of group C. When R751 was transferred into J53-2 (R1033), all the resistance markers of the resident plasmid were eliminated. When R1033 was transferred into J62 (R751), trimethoprim resistance was eliminated (whether selection was made for transfer of ampicillin, gentamicin, or kanamycin). Thus, R1033 is a member of group P (= P1). Antibiotic-modifying enzymes found in R1033+ in E. coli. When sonic extracts of R1033 were assayed for phosphorylating activity, it was found that neomycin and lividomycin were good acceptors for the phosphate moiety but that butirosin was not. This indicates the presence of a phosphotransferase that fits into the
neomycin phosphotransferase 1 category (Table 2). When sonic extracts of R1033 in E. coli were assayed for acetylating activity, it was found that gentamicin C antibiotics and sisomicin were good acceptors for the acetate moiety (Table 3). On the basis of substrate specificities, we have assigned the acetylator found in RI033 in E. coli to the gentamicin acetyltransferase 1 category (5). R1033 determines production of a TEM f6-lactamase and this enzyme is responsible for the resistance to ,8-lactam antibiotics (M. Matthews and R. W. Hedges, submitted for publication). Molecular properties of R1033. The molecular weight of R1033, isolated as the covalently closed circular DNA tertiary form by cesium chloride-ethidium bromide density centrifugation, was determined by comparing its sedimentation rate through a neutral sucrose gradient with covalently closed circular Rl plasmid DNA. Figure 1 shows the sedimentation of 3H-labeled R1033 DNA together with "4Clabeled Rl DNA. Sedimentation was from right to left. The covalently closed circular DNA forms of R1033 and Rl peaked at fractions 19 and 15, respectively, and their own circular forms (formed by spontaneous degradation of the covalently closed circular DNA) peaked at 30 and 28, respectively. By using the value 60 x 10' for the molecular weight of Rl (8; P. T. Barth, unpublished data), the molecular weight of R1033 was calculated to be 45 x 106.
DISCUSSION R1033 in E. coli is found to code for two antibiotic-modifying enzymes. The neomycin phosphotransferase 1 confers resistance to neomycin, kanamycin, and lividomycin, and the gentamicin acetyltransferase 1 confers re-
229
PLASMID CONFERRING GENTAMICIN RESISTANCE
VOL. 8, 1975
TABLE 2. Efficiency of different antibiotics as substrates for neomycin phosphotransferase found in R1033 in E. colia Antibiotic
%
I
Paromamycin ..................... Ribostamycin .....................
100 80 63 48 15
Kanamycin B ..................... Butirosin A ....................... Butirosin B .13 162 Lividomycin A ......... 149 Lividomycin B .................... 53 Gentamicin A ..................... 45 Gentamicin B ...................... 3 Tobramycin ...................... aEach compotund was incubated with the sonic extract obtained from R1033 in E. coli. Incubation was for 30 min, and results are expressed relative to the phosphorylation of neomycin B (100%). TABLE 3. Efficiency of different antibiotics as substrates for gentamicin acetyltransferase found in R1033 in E. colia Antibiotic
%
Gentamicin C1 ..................... Gentamicin B ..................... Sisomicin ......................... Neomycin B ...................... Tobramycin ...................... Kanamycin A ..................... Kanamycin B ..................... Kanamycin C ..................... Butirosin A ........................ Lividomicin A .................... Amikacin .......................
100 47 175 0 30 2 53 5 0 0 8
a Each compound was incubated with the sonic extract obtained from R1033 in E. coli. Incubation was for 30 min. Results are expressed relative to the acetylation of gentamicin Cia (100%).
sistance to gentamicin C compounds, sisomicin, and kanamycin B. An interesting result is that the acetylation enzyme found in R1033 in E. coli will utilize tobramycin as a substrate, but that the cells are found to be susceptible to tobramycin in vitro. This is not surprising in view of the fact that many acetylated aminoglycoside antibiotics are still potent antibiotics (M. J. Haas, personal communication). R1033, of molecular weight 45 x 10, is larger than any P plasmid previously reported. RP4 has a molecular weight of 34 x 106 (14), that of R906 is 35 x 106 (16), that of R751 is 30 x 106 (P. T. Barth and N. J. Grinter, personal communication), and that of RW4a is 38 x 106 (A. E. J. and R. W. H., unpublished data). R1033 is the first plasmid of group P1 shown to
III
I2
I
0
1 01
I
I
2 i
I , I 0 1 1
1
1
-1
Neomycin B ......................
3
0
O' 0I
I
R
1
it
I
I
I
1
1 I
I
Z:
2
i
I I
p I
I
0.
I
,
1
1 I
O
0I
L
40
Fraction number
FIG. 1. Neutral sucrose gradient analysis of piasmid R1033 DNA. 3H-labeled R1033 DNA was mixed with "4C-labeled Rl DNA and sedimented through a 5 to 20%o sucrose gradient at 100,000 x g and 20 C for 90 min. Fractions (0.1 ml) were collected onto glassfiber disks and, after being dried, washed, and redried, were assayed for radioactivity. Symbols: 0, 3H;
0, 14C.
confer resistance to gentamicin or chloramphenicol. Perhaps it carries a DNA sequence derived from some unknown source, which confers these resistances and augments the molecular weight of the plasmid. ACKNOWLEDGMENTS A. E. Jacob was supported by a grant to Naomi Datta from the Medical Research Council. LITERATURE CITED 1. Barth, P. T., and N. J. Grinter. 1974. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonomides. J. Bacteriol. 120:618-630. 2. Bryan, L. E., S. D. Semaka, H. M. van den Elzen, J. E. Kinnear, and R. L. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob. Agents Chemother. 3:625-637. 3. Bryan, L. E., M. S. Shahrabadi, and H. M. van den Elzen. 1974. Gentamicin resistance in Pseudomonas aeruginosa: R factor-mediated resistance. Antimicrob. Agents Chemother. 6:191-199. 4. Bryan, L. E., H. M. van den Elzen, and J. T. Tseng. 1972. Transferable drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1:22-29. 5. Brzezinska, M., R. Benveniste, J. Davies, P. Daniels, and J. Weinstein. 1972. Gentamicin resistance in strains of Pseudomonas aeruginosa mediated by enzymatic Nacetylation of the deoxystreptamine moiety. Biochemistry 11:761-766. 6. Chabbert, Y. A., M. R. Scavizzi, J. L. Witchitz, G. R. Gerbaud, and D. H. Bouanchaud. 1972. Incompatibility groups and the classification of fi- resistance factors. J. Bacteriol. 112:666-675. 7. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to the open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166. 8. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 9. Coetzee, J. N., N. Datta, R. W. Hedges, and P. C. Appelbaum. 1973. Transduction of R factors in Proteus mirabilis and P. rettgeri. J. Gen. Microbiol. 76:355368.
230
ANTIMICROB. AGENTS CHEMOTHER.
SMITH ET AL.
10. Cowan, S. T., and K. J. Steel. 1974. Manual for the identification of medical bacteria, 2nd ed., revised by S. T. Cowan. Cambridge University Press, Cambridge. 11. Datta, N. 1975. Epidemiology and classification of plasmids, p. 9-15. In D. Schlessinger (ed.), Microbiology1974. American Society for Microbiology, Washington, D.C. 12. Datta, N., and R. W. Hedges. 1972. Host ranges of R factors. J. Gen. Microbiol. 70:453-460. 13. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. 14. Falkow, S., P. Guerry, R. W. Hedges, and N. Datta. 1974. Polynucleotide sequence relationships among plasmids of the I compatibility complex. J. Gen. Microbiol. 85:65-76. 15. Hedges, R. W. 1974. R factors from Providence. J. Gen. Microbiol. 81:171-181. 16. Hedges, R. W., A. E. Jacob, and J. T. Smith. 1974.
17.
18. 19. 20.
21.
Properties of an R factor from Bordetella bronchiseptics. J. Gen. Microbiol. 84:199-204. Jacoby, G. A. 1974. Properties of R plasmids determining gentamicin resistance by acetylation in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6:239-252. Jobanputra, R. S. and N. Datta. 1974. Trimethoprim resistance factors in enterobacteria from clinical specimens. J. Med. Microbiol. 7:169-177. Olsen, R. H., and P. Shipley. 1973. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J. Bacteriol. 113:772-780. Ozanne, B., R. Benveniste, D. Tipper, and J. Davies. 1969. Aminoglycoside antibiotics: inactivation by phosph6rylation in Escherichia coli carrying R factors. J. Bacteriol. 100:1144-1146. Witchitz, J. L., and G. R. Gerbaud. 1972. Classification de plasmides conferant la resistance a la gentamicine. Ann. Inst. Pasteur Paris 123:333-339.
Vol. 8, No. 3 Printed in U.S.A.
Third Type of Plasmid Conferring Gentamicin Resistance Pseudomonas aeruginosa
in
DAVID I. SMITH, R GOMEZ LUS, M. C. RUBIO CALVO, NAOMI DAITA, A. E. JACOB, AND R. W. HEDGES* Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706; Departmento de Microbiologia e Higiene, Facultad de Medicina, Universidad de Zaragoza, Spain; and Bacteriology Department, Royal Postgraduate Medical School, London W12 OHS, United Kingdom*
Received for publication 5 May 1975
R1033 is a plasmid of compatibility group P (= P1) transferred from a wild strain of Pseudomonas aeruginosa. It confers resistance to gentamicin by gentamicin acetyl-transferase 1 and to kanamycin and neomycin by neomycin phosphotransferase 1.
The self-transmissible R factors of Pseudomonas aeruginosa clinical isolates have been assigned to three compatibility groups by Bryan and his collaborators (2-4). Group P1 contains the R factors that in Escherichia coli K-12 constitute group P (13). Group P2 plasmids are not transmissible to E. coli, and plasmids of group P3 are those assigned to group C (12) or com6 (6) in the E. coli plasmid classifications. Since gentamicin is useful in the treatment of P. aeruginosa infections, attention has been concentrated on mechanisms by which resistance to this drug can be attained. Isolates of P. aeruginosa carrying plasmids conferring gentamicin resistance have been reported (3, 17, 21). Bryan et al. (3) found among North American isolates gentamicin resistance conferred by plasmids of groups P2 and P3, and Jacoby (17) has observed such R factors of group P2. In France, gentamicin R factors of group P3 have been observed in P. aeruginosa (21). We report here the isolation, in Spain, of a strain resistant to gentamicin (and several other antibiotics) by virtue of an R factor of group P1. MATERIALS AND METHODS Bacteria. The bacteria used were P. aeruginosa CRT and E. coli K-12 strains J53-2 (F-, pro,met,rif), J62 (F-, pro,his,trp,lac), and CR34Thy (F-, thr,leu,
thi,thy,lac). R factors. The R factors used were Plac, a plasmid
of compatibility group C (= P3) which confers lactose fermentation ability and sulfonamide resistance (15), and R751, a plasmid of compatibility group P (= P1) which confers resistance to trimethoprim (18). Phage. The phage used was PRR1, which adsorbs to bacteria carrying plasmids of group P (= P1) and not to plasmid-free becteria or cells carrying plasmids of other groups (11, 19). Conjugal plasmid transfer and plasmid compatibility properties. Procedures for conjugal plasmid
transfer and the determination of plasmid compatibility properties were as described previously (9, 13).
Minimal inhibitory concentrations. The drugs used and their minimal inhibitory concentrations are listed in Table 1. Radiolabeling and lysis of CR34Thy (R1033) and isolation of plasmid DNA. Rabiolabeling and lysis of CR34Thy (R1033) and isolation of plasmid deoxyribonucleic acid (DNA) by cesium chloride-ethidium bromide density gradient centrifugation were as described previously (7). Neutral sucrose gradient analysis and calculation of plasmid molecular weight. Neutral sucrose gradient analysis and calculation of plasmid molecular weight were as described previously (1). Preparation of cell-free extracts. R1033 in E. coli was grown to late log phage in yeast extract-tryptone medium (100 ml). The cells were harvested by centrifugation and washed twice with a solution of 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.8), 50 mM NH4C1, 10 mM MgCl2, and 0.64 mM 2-mercaptoethanol. The pellet was resuspended in 4 ml of the same buffer and disrupted by sonic treatment. The cell debris was removed by centrifugation at 100,000 x g for 90 min. The resulting supernatant was designated the sonic extract and is the preparation that was used in all radioactive assays. Enzymatic assays. Phosphorylation of aminoglycoside antibiotics was assayed by the phosphocellulose paper binding assay, as described previously (20). Acetylation of aminoglycoside antibiotics was also assayed by the phosphocellulose paper binding assay, as described previously (5).
RESULTS
Properties of P. aeruginosa CRT. The strain was pigmented and showed the typical characters of P. aeruginosa (10). The minimal inhibitory concentrations of relevant antibiotics are shown in Table 1. Strain CRT is unstable, producing segregants with increased susceptibility to gentamicin (kanamycin, carbenicillin, tetracycline, strep227
228
ANTIMICROB. AGENTS CHEMOTHER.
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TABLE 1. Minimal inhibitory concentrations (MIC) of antibiotics for P. aeruginosa CRT and E. coli K-12, with and without R factor R1033 MICa
Strain
P. aeruginosa CRT P. aeruginosa CRTSb E. coli K-12 strain J53 E. coli K-12 strain J53 (R1033)
Genta
Tobra
Kana
Chlor
500 1.5 0.1 12.5
0.8 0.4 0.2 0.2
1,000
> 200
25 0.2 50
25 5 25
Carb 300
40 2.5 > 5,000
a The figures show the lowest concentrations of drug (micrograms per milliliter) in Oxoid DST (direct sensitivity test) agar with added 4% lysed horse blood that prevented visible growth from inocula that gave dense, but not confluent, growth on drug-free control plates. The MIC of butirosin was not measured, but the diameters of the zones of inhibition of E. coli K-12 R- and R1033+ round filter paper disks containing 20 ,g of butirosin were identical. Abbreviations: Genta, gentamicin; Tobra, tobramycin; Kana, kanamycin; Chlor, chloramphenicol; Carb, carbenicillin. bP. aeruginosa CRTS is the wild strain after spontaneous loss of resistance (see text).
tomycin, chloramphenicol, and sulfonamides). Such segregants have, presumably, lost the R factor. One of these, strain CRTS, is included in Table 1. When strain CRT was grown with E. coli J53-2, resistance to gentamicin, kanamycin, ampicillin, tetracycline, streptomycin, chloramphenicol, and sulfonamides was transferred. Since all resistances were regularly co-transferred and in view of the compatibility properties and molecular characteristics of the resistant strains, it was concluded that all of the resistances were determined by a single plasmid, R1033. Properties of R1033 in E. coli K-12. In E. coli, R1033 conferred resistance to gentamicin,
kanamycin, ampicillin, tetracycline, and chloramphenicol, and to very low levels of streptomycin and sulfonamides. It confers sensitivity to phage PRR1. J62 (R1033) was mated with CR34Thy(Plac). Transfer in both directions was observed without elimination, and both plasmids were stable in each strain. Thus, R1033 is not a member of group C. When R751 was transferred into J53-2 (R1033), all the resistance markers of the resident plasmid were eliminated. When R1033 was transferred into J62 (R751), trimethoprim resistance was eliminated (whether selection was made for transfer of ampicillin, gentamicin, or kanamycin). Thus, R1033 is a member of group P (= P1). Antibiotic-modifying enzymes found in R1033+ in E. coli. When sonic extracts of R1033 were assayed for phosphorylating activity, it was found that neomycin and lividomycin were good acceptors for the phosphate moiety but that butirosin was not. This indicates the presence of a phosphotransferase that fits into the
neomycin phosphotransferase 1 category (Table 2). When sonic extracts of R1033 in E. coli were assayed for acetylating activity, it was found that gentamicin C antibiotics and sisomicin were good acceptors for the acetate moiety (Table 3). On the basis of substrate specificities, we have assigned the acetylator found in RI033 in E. coli to the gentamicin acetyltransferase 1 category (5). R1033 determines production of a TEM f6-lactamase and this enzyme is responsible for the resistance to ,8-lactam antibiotics (M. Matthews and R. W. Hedges, submitted for publication). Molecular properties of R1033. The molecular weight of R1033, isolated as the covalently closed circular DNA tertiary form by cesium chloride-ethidium bromide density centrifugation, was determined by comparing its sedimentation rate through a neutral sucrose gradient with covalently closed circular Rl plasmid DNA. Figure 1 shows the sedimentation of 3H-labeled R1033 DNA together with "4Clabeled Rl DNA. Sedimentation was from right to left. The covalently closed circular DNA forms of R1033 and Rl peaked at fractions 19 and 15, respectively, and their own circular forms (formed by spontaneous degradation of the covalently closed circular DNA) peaked at 30 and 28, respectively. By using the value 60 x 10' for the molecular weight of Rl (8; P. T. Barth, unpublished data), the molecular weight of R1033 was calculated to be 45 x 106.
DISCUSSION R1033 in E. coli is found to code for two antibiotic-modifying enzymes. The neomycin phosphotransferase 1 confers resistance to neomycin, kanamycin, and lividomycin, and the gentamicin acetyltransferase 1 confers re-
229
PLASMID CONFERRING GENTAMICIN RESISTANCE
VOL. 8, 1975
TABLE 2. Efficiency of different antibiotics as substrates for neomycin phosphotransferase found in R1033 in E. colia Antibiotic
%
I
Paromamycin ..................... Ribostamycin .....................
100 80 63 48 15
Kanamycin B ..................... Butirosin A ....................... Butirosin B .13 162 Lividomycin A ......... 149 Lividomycin B .................... 53 Gentamicin A ..................... 45 Gentamicin B ...................... 3 Tobramycin ...................... aEach compotund was incubated with the sonic extract obtained from R1033 in E. coli. Incubation was for 30 min, and results are expressed relative to the phosphorylation of neomycin B (100%). TABLE 3. Efficiency of different antibiotics as substrates for gentamicin acetyltransferase found in R1033 in E. colia Antibiotic
%
Gentamicin C1 ..................... Gentamicin B ..................... Sisomicin ......................... Neomycin B ...................... Tobramycin ...................... Kanamycin A ..................... Kanamycin B ..................... Kanamycin C ..................... Butirosin A ........................ Lividomicin A .................... Amikacin .......................
100 47 175 0 30 2 53 5 0 0 8
a Each compound was incubated with the sonic extract obtained from R1033 in E. coli. Incubation was for 30 min. Results are expressed relative to the acetylation of gentamicin Cia (100%).
sistance to gentamicin C compounds, sisomicin, and kanamycin B. An interesting result is that the acetylation enzyme found in R1033 in E. coli will utilize tobramycin as a substrate, but that the cells are found to be susceptible to tobramycin in vitro. This is not surprising in view of the fact that many acetylated aminoglycoside antibiotics are still potent antibiotics (M. J. Haas, personal communication). R1033, of molecular weight 45 x 10, is larger than any P plasmid previously reported. RP4 has a molecular weight of 34 x 106 (14), that of R906 is 35 x 106 (16), that of R751 is 30 x 106 (P. T. Barth and N. J. Grinter, personal communication), and that of RW4a is 38 x 106 (A. E. J. and R. W. H., unpublished data). R1033 is the first plasmid of group P1 shown to
III
I2
I
0
1 01
I
I
2 i
I , I 0 1 1
1
1
-1
Neomycin B ......................
3
0
O' 0I
I
R
1
it
I
I
I
1
1 I
I
Z:
2
i
I I
p I
I
0.
I
,
1
1 I
O
0I
L
40
Fraction number
FIG. 1. Neutral sucrose gradient analysis of piasmid R1033 DNA. 3H-labeled R1033 DNA was mixed with "4C-labeled Rl DNA and sedimented through a 5 to 20%o sucrose gradient at 100,000 x g and 20 C for 90 min. Fractions (0.1 ml) were collected onto glassfiber disks and, after being dried, washed, and redried, were assayed for radioactivity. Symbols: 0, 3H;
0, 14C.
confer resistance to gentamicin or chloramphenicol. Perhaps it carries a DNA sequence derived from some unknown source, which confers these resistances and augments the molecular weight of the plasmid. ACKNOWLEDGMENTS A. E. Jacob was supported by a grant to Naomi Datta from the Medical Research Council. LITERATURE CITED 1. Barth, P. T., and N. J. Grinter. 1974. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonomides. J. Bacteriol. 120:618-630. 2. Bryan, L. E., S. D. Semaka, H. M. van den Elzen, J. E. Kinnear, and R. L. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob. Agents Chemother. 3:625-637. 3. Bryan, L. E., M. S. Shahrabadi, and H. M. van den Elzen. 1974. Gentamicin resistance in Pseudomonas aeruginosa: R factor-mediated resistance. Antimicrob. Agents Chemother. 6:191-199. 4. Bryan, L. E., H. M. van den Elzen, and J. T. Tseng. 1972. Transferable drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1:22-29. 5. Brzezinska, M., R. Benveniste, J. Davies, P. Daniels, and J. Weinstein. 1972. Gentamicin resistance in strains of Pseudomonas aeruginosa mediated by enzymatic Nacetylation of the deoxystreptamine moiety. Biochemistry 11:761-766. 6. Chabbert, Y. A., M. R. Scavizzi, J. L. Witchitz, G. R. Gerbaud, and D. H. Bouanchaud. 1972. Incompatibility groups and the classification of fi- resistance factors. J. Bacteriol. 112:666-675. 7. Clewell, D. B., and D. R. Helinski. 1969. Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to the open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166. 8. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 9. Coetzee, J. N., N. Datta, R. W. Hedges, and P. C. Appelbaum. 1973. Transduction of R factors in Proteus mirabilis and P. rettgeri. J. Gen. Microbiol. 76:355368.
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