ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1978, p. 634-636 0066-4804/78/0013-0634$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 13, No. 4 Printed in U.S.A.
Plasmid-Determined Resistance to Hexachlorophene in Pseudomonas aeruginosa LORRAINE SUTTON AND GEORGE A. JACOBY* Massachusetts General Hospital, Boston, Massachusetts 02114 Received for publication 2 December 1977
Plasmid-containing strains of Pseudomonas aeruginosa were screened for resistance to disinfectants. Two plasmids, pMG1 and pMG2, were found to determine hexachlorophene resistance. Clinical isolates of Pseudomonas aeruginosa are usually distinguished for epidemiological purposes by serological, bacteriophage, or bacteriocin reactions (2), but can also be differentiated by susceptibility to antibiotics or to antiseptics. Delmotte et al. found variations in susceptibility to disinfectants which were reproducible and which allowed an "antiseptogram" for clinical isolates to be determined (5, 6). R plasmids in P. aeruginosa are well known to affect susceptibility to antibiotics and also to organomercurial antiseptics (3, 11). The objective of this study was to determine whether known R factor-containing strains have enhanced resistance to various disinfectants. We have found that resistance to hexachlorophene is a plasmiddetermined property in P. aeruginosa.
MATERIALS AND METHODS Bacterial strains and plasmids. P. aeruginosa strains PU21 (FP- ilvB112 leu-1 str-1 rif'), PUl (FP2+, prototrophic), and PA0303 (FP- arg-18) were used (11). The properties of the plasmids have been described previously (12, 13). Chemicals. Compounds were obtained from the following sources: American Hospital Supply (o-chlorophenol); Benzol Products (chlorobutanol); Eastman Organic Chemicals (2,4,5-trichlorophenol); K & K Laboratories (triclocarban); Phaltz and Bauer, Inc. (cetrimide, chloramine-T, 4-chloro-m-cresol, dichlorophene, hexachlorophene, tetrachlorophene); Stuart Pharmaceuticals (chlorhexidine); West Chemical Products, Inc. (Wescodyne); and Winthrop Laboratories (benzalkonium chloride). Determination of susceptibility to disinfectants. Brain heart infusion agar (Difco) plates containing graded concentrations of inhibitors were spotted with 104 to 105 organisms from an overnight culture in nutrient broth (Difco) containing 4 mg of potassium nitrate per ml (NNB). The minimal inhibitory concentration was determined as the lowest concentration of inhibitor preventing growth after overnight incubation
at 370C.
Kinetics of resistance transfer. PU1(pMG1) at 2 x 108 and PU21 at 5 x 108 cells per ml were incubated together for 5 min, diluted 100-fold, and sampled pe634
riodically for resistant transconjugants. Mating was performed in NNB at 37°C. Sulfonamide- or gentamicin-resistant transconjugants were selected on plates (11) containing the amino acids required by the recipient, 5 mg of sulfadiazine or 20 jig of gentamicin per ml, and 100 jg of rifampin per ml for counterselection. Hexachlorophene-resistant transconjugants were selected on brain heart infusion agar plates containing 100 ug of rifampin per ml and 0.5 mM hexachlorophene.
RESULTS Susceptibility to disinfectants. Derivatives of P. aeruginosa PU21 containing the following R plasmids were tested for susceptibility to antiseptics: RP1, RP4, R30, and R68 (Inc [incompatibility group] P-1); pMG1, pMG2, pMG5, RPL11, R38, R39, R931, R3108, and CAM (IncP2); RIP64 (IncP-3); FP2 (IncP-8); R2 (IncP-9); R46 (IncN); Sa (IncW); and RP8, R40c, and R91 (unclassified). The disinfectants chosen included six found by Delmotte et al. (6) to differentiate clinical isolates of P. aeruginosa. With one exception, the inhibitors failed to discriminate among the P. aeruginosa strains. Table 1 lists those compounds that either failed to distinguish between R- and R+ bacteria or that were not inhibitory at the concentrations tested. Resistance to hexachlorophene. Two plasmids, pMG1 and pMG2, both belonging to Inc P-2, provided resistance to hexachlorophene. RPU21 and derivatives carrying other plasmids had a minimal inhibitory concentration of 0.1 mM hexachlorophene. PU21 carrying pMG1 or pMG2 demonstrated a fivefold increase in hexachlorophene resistance. Kinetics of hexachlorophene transfer. Figure 1 shows the kinetics of transfer of hexachlorophene resistance in a mating between PU1(pMG1) and PU21. Transfer of resistance to hexachlorophene and to gentamicin was delayed as compared with transfer of sulfonamide resistance, but transconjugants selected with sulfadiazine at 70 min or with gentamicin or
HEXACHLOROPHENE RESISTANCE
VOL. 13, 1978
hexachlorophene at later times were uniformly resistant to the other agents, indicating that all three markers were transferred together and that there was a phenotypic lag in the expression of hexachlorophene resistance. Resistance ofother plasmids to hexachlorophene. PU21 and PA0303 derivatives carrying the following additional plasmids were tested for hexachlorophene susceptibility: RP4-pMG2, R527, and R1033 (IncP-1); pMG6, kR61, and Rmsl59 (IncP-2); pMG7 (IncP-3); R679, R1162, and R5265 (IncP-4); Rmsl63 (IncP-5); Rmsl49 (IncP-6); Rmsl48 (IncP-7); and RP1-1, pVS1, FP5, and R716 (unclassified). None gave enhanced hexachlorophene resistance.
25 0
20
aeruginosa Compound Noninhibitorya Chloramine-T ......... Chlorobutanol ......... o-Chlorophenol ........ Triclocarban ........... Tetrachlorophene ....
Nondiscriminatoryb Benzalkonium chloride Cetrimide ............. Chlorhexidine .......... 4-Chloro-m-cresol ...... Dichlorophene ......... 2,4,5-Trichlorophenol ...
*
SulfodiazineR
A
GentomicinR
o Hexachlorophene
15
-
Izi
0o
0
20
40
60
80
MAT/ING TIME (minutes)
FIG. 1. Kinetics of transfer of sulfadiazine (0), gentamicin (A), and hexachlorophene (EJ) resistance in a mating between PUI(pMG1) and PU21.
Concn 10 mM 10 mM 3 x 10-3 dilution 10 mM 1 mM 4 x 10-4 dilution 10 mM 0.2 mM
10 mM 1 mM 0.5 mM
10-1 dilution Compounds that failed to inhibit R- or R+ P. PU21 at the indicated concentrations. aeruginosa " Compounds that inhibited R- and R+ PU21 equally. Concentration figures indicate miniimal inhibitory concentration.
Wescodyne ............
-
~4
DISCUSSION K Hexachlorophene [2,2'-methylene bis-(3,4,6trichlorophenol)] is a bactericidal agent with multiple effects on gram-positive and gram-negative bacteria (10). Studies by Comer, Gerhardt, and co-workers suggest that its primary site of (In action is inhibition of respiration by the mem- I4* brane-bound portion of the electron transport chain (4, 9, 14, 16). How R plasmids interfere with hexachlorophene action is not known, although they could affect either permeability to the inhibitor or an alteration at its site of action. No growth has been observed with PU21, PU21(pMG1), or PU21(pMG2) with hexachlorophene as a sole carbon source, nor do these plasmids allow growth on diphenylmethane, which is metabolized by some soil Pseudomonas species (8). The designation Hex is proposed to TABLE 1. Disinfectants tested on R+ and R- P.
635
describe this phenotype (15). In this survey Hex was found on only 2 of 38 Pseudomonas plasmids tested. Delmotte et al. (6) found considerable variation in the susceptibility of clinical isolates of P. aeruginosa to hexachlorophene. P. aeruginosa is known to be one of the gram-negative bacteria that emerges following hexachlorophene treatment of skin (7) and can also contaminate stocks of hexachlorophene-containing soaps and detergents (1). Whether plasmids play a role in the resistance of natural isolates of P. aeruginosa to hexachlorophene and other antiseptics appears worthy of further study.
a
ACKNOWLEDGMENT This work was supported by a grant from the National Science Foundation (PCM75-03932).
636
SUTTON AND JACOBY LITERATURE CITED
1. Anderson, K. 1962. The contamination of hexachloro-
2.
3. 4.
5.
6.
7. 8.
phene soap with Pseudomonas pyocyanea. Med. J. Aust. 49:463. Bergan, T. 1975. Epidemiological typing of Pseudomonas aeruginosa, p. 189-235. In M. R. W. Brown (ed.), Resistance of Pseudomonas aeruginosa. John Wiley & Sons, London. Clark, D. L., A. A. Weiss, and S. Silver. 1977. Mercury and organomercurial resistances determined by plasmids in Pseudomonas. J. Bacteriol. 132:186-196. Corner, T. R., H. L. Joswick, J. N. Silvernale, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: lysis and fixation of bacterial proptoplasts. J. Bacteriol. 108:501-507. Delmotte, A., J. Beumer, E. Cotton, N. DekeyserDelmotte, M. Millet, K. G. Vanden Abbeele, W. Von Grunigen, and E. Yourassowsky. 1971. Etude sur la sensibilit6 du bacille pyocyanique (Pseudomonas aeruginosa) aux antiseptiques et aux antibiotiques. I. Variations de la sensibilite du bacille pyocyanique aux antiseptiques et antibiotiques au cours du temps et reproductibilite des resultats. Therapie 26:629-644. Delmotte, A., J. Beumer, E. Cotton, N. DekeyserDelmotte, M. Millet, K. G. Vanden Abbeele, W. Von Grunigen, and E. Yourassowsky. 1971. Etude sur la sensibilite du bacille pyocyanique (Pseudomonas aeruginosa) aux antiseptiques et aux antibiotiques. II. De la notion statistique de la sensibilite du bacille pyocyanique aux antiseptiques a la conception de 1'antiseptogramme. Therapie 26:645-651. Evans, Z. A., R. C. Rendtorff, H. Robinson, and E. W. Rosenberg. 1973. Ecological influence of hexachlorophene on skin bacteria. J. Invest. Dermatol. 60:207-214. Francis, A. J., R. J. Spanggord, G. I. Ouchi, R. Bram-
ANTIMICROB. AGENTS CHEMOTHER. hall, and N. Bohonos. 1976. Metabolism of DDT analogues by a Pseudomonas sp. Appl. Environ. Microbiol. 32:213-216. 9. Frederick, J. J., T. R. Corner, and P. Gerhardt. 1974. Antimicrobial actions of hexachlorophene: inhibition of respiration in Bacillus megaterium. Antimicrob. Agents Chemother. 6:712-721. 10. Gump, W. S., and G. R. Walter. 1968. The bis-phenols, p. 257-277. In C. A. Lawrence and S. S. Block (ed.), Disinfection, sterilization, and preservation. Lea & Febiger, Philadelphia. 11. Jacoby, G. A. 1974. Properties of R plasmids determining gentamicin resistance by acetylation in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6:239-252. 12. Jacoby, G. A. 1977. Classification of plasmids in Pseudomonas aeruginosa, p. 119-126. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 13. Jacoby, G. A., and J. A. Shapiro. 1977. Plasmids studied in Pseudomonas aeruginosa and other pseudomonads, p. 639-656. In A. I. Bukhari, J. A. Shapiro, and S. Adhya (ed.), DNA insertion elements, plasmids, and episomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 14. Joswick, H. L., T. R. Corner, J. N. Silvernale, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: release of cytoplasmic materials. J. Bacteriol. 108:492-500. 15. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III, N. Datta, and S. Falkow. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. 16. Silvernale, J. N., H. L. Joswick, T. R. Corner, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: cytological manifestations. J. Bacteriol. 108:482-491.
Vol. 13, No. 4 Printed in U.S.A.
Plasmid-Determined Resistance to Hexachlorophene in Pseudomonas aeruginosa LORRAINE SUTTON AND GEORGE A. JACOBY* Massachusetts General Hospital, Boston, Massachusetts 02114 Received for publication 2 December 1977
Plasmid-containing strains of Pseudomonas aeruginosa were screened for resistance to disinfectants. Two plasmids, pMG1 and pMG2, were found to determine hexachlorophene resistance. Clinical isolates of Pseudomonas aeruginosa are usually distinguished for epidemiological purposes by serological, bacteriophage, or bacteriocin reactions (2), but can also be differentiated by susceptibility to antibiotics or to antiseptics. Delmotte et al. found variations in susceptibility to disinfectants which were reproducible and which allowed an "antiseptogram" for clinical isolates to be determined (5, 6). R plasmids in P. aeruginosa are well known to affect susceptibility to antibiotics and also to organomercurial antiseptics (3, 11). The objective of this study was to determine whether known R factor-containing strains have enhanced resistance to various disinfectants. We have found that resistance to hexachlorophene is a plasmiddetermined property in P. aeruginosa.
MATERIALS AND METHODS Bacterial strains and plasmids. P. aeruginosa strains PU21 (FP- ilvB112 leu-1 str-1 rif'), PUl (FP2+, prototrophic), and PA0303 (FP- arg-18) were used (11). The properties of the plasmids have been described previously (12, 13). Chemicals. Compounds were obtained from the following sources: American Hospital Supply (o-chlorophenol); Benzol Products (chlorobutanol); Eastman Organic Chemicals (2,4,5-trichlorophenol); K & K Laboratories (triclocarban); Phaltz and Bauer, Inc. (cetrimide, chloramine-T, 4-chloro-m-cresol, dichlorophene, hexachlorophene, tetrachlorophene); Stuart Pharmaceuticals (chlorhexidine); West Chemical Products, Inc. (Wescodyne); and Winthrop Laboratories (benzalkonium chloride). Determination of susceptibility to disinfectants. Brain heart infusion agar (Difco) plates containing graded concentrations of inhibitors were spotted with 104 to 105 organisms from an overnight culture in nutrient broth (Difco) containing 4 mg of potassium nitrate per ml (NNB). The minimal inhibitory concentration was determined as the lowest concentration of inhibitor preventing growth after overnight incubation
at 370C.
Kinetics of resistance transfer. PU1(pMG1) at 2 x 108 and PU21 at 5 x 108 cells per ml were incubated together for 5 min, diluted 100-fold, and sampled pe634
riodically for resistant transconjugants. Mating was performed in NNB at 37°C. Sulfonamide- or gentamicin-resistant transconjugants were selected on plates (11) containing the amino acids required by the recipient, 5 mg of sulfadiazine or 20 jig of gentamicin per ml, and 100 jg of rifampin per ml for counterselection. Hexachlorophene-resistant transconjugants were selected on brain heart infusion agar plates containing 100 ug of rifampin per ml and 0.5 mM hexachlorophene.
RESULTS Susceptibility to disinfectants. Derivatives of P. aeruginosa PU21 containing the following R plasmids were tested for susceptibility to antiseptics: RP1, RP4, R30, and R68 (Inc [incompatibility group] P-1); pMG1, pMG2, pMG5, RPL11, R38, R39, R931, R3108, and CAM (IncP2); RIP64 (IncP-3); FP2 (IncP-8); R2 (IncP-9); R46 (IncN); Sa (IncW); and RP8, R40c, and R91 (unclassified). The disinfectants chosen included six found by Delmotte et al. (6) to differentiate clinical isolates of P. aeruginosa. With one exception, the inhibitors failed to discriminate among the P. aeruginosa strains. Table 1 lists those compounds that either failed to distinguish between R- and R+ bacteria or that were not inhibitory at the concentrations tested. Resistance to hexachlorophene. Two plasmids, pMG1 and pMG2, both belonging to Inc P-2, provided resistance to hexachlorophene. RPU21 and derivatives carrying other plasmids had a minimal inhibitory concentration of 0.1 mM hexachlorophene. PU21 carrying pMG1 or pMG2 demonstrated a fivefold increase in hexachlorophene resistance. Kinetics of hexachlorophene transfer. Figure 1 shows the kinetics of transfer of hexachlorophene resistance in a mating between PU1(pMG1) and PU21. Transfer of resistance to hexachlorophene and to gentamicin was delayed as compared with transfer of sulfonamide resistance, but transconjugants selected with sulfadiazine at 70 min or with gentamicin or
HEXACHLOROPHENE RESISTANCE
VOL. 13, 1978
hexachlorophene at later times were uniformly resistant to the other agents, indicating that all three markers were transferred together and that there was a phenotypic lag in the expression of hexachlorophene resistance. Resistance ofother plasmids to hexachlorophene. PU21 and PA0303 derivatives carrying the following additional plasmids were tested for hexachlorophene susceptibility: RP4-pMG2, R527, and R1033 (IncP-1); pMG6, kR61, and Rmsl59 (IncP-2); pMG7 (IncP-3); R679, R1162, and R5265 (IncP-4); Rmsl63 (IncP-5); Rmsl49 (IncP-6); Rmsl48 (IncP-7); and RP1-1, pVS1, FP5, and R716 (unclassified). None gave enhanced hexachlorophene resistance.
25 0
20
aeruginosa Compound Noninhibitorya Chloramine-T ......... Chlorobutanol ......... o-Chlorophenol ........ Triclocarban ........... Tetrachlorophene ....
Nondiscriminatoryb Benzalkonium chloride Cetrimide ............. Chlorhexidine .......... 4-Chloro-m-cresol ...... Dichlorophene ......... 2,4,5-Trichlorophenol ...
*
SulfodiazineR
A
GentomicinR
o Hexachlorophene
15
-
Izi
0o
0
20
40
60
80
MAT/ING TIME (minutes)
FIG. 1. Kinetics of transfer of sulfadiazine (0), gentamicin (A), and hexachlorophene (EJ) resistance in a mating between PUI(pMG1) and PU21.
Concn 10 mM 10 mM 3 x 10-3 dilution 10 mM 1 mM 4 x 10-4 dilution 10 mM 0.2 mM
10 mM 1 mM 0.5 mM
10-1 dilution Compounds that failed to inhibit R- or R+ P. PU21 at the indicated concentrations. aeruginosa " Compounds that inhibited R- and R+ PU21 equally. Concentration figures indicate miniimal inhibitory concentration.
Wescodyne ............
-
~4
DISCUSSION K Hexachlorophene [2,2'-methylene bis-(3,4,6trichlorophenol)] is a bactericidal agent with multiple effects on gram-positive and gram-negative bacteria (10). Studies by Comer, Gerhardt, and co-workers suggest that its primary site of (In action is inhibition of respiration by the mem- I4* brane-bound portion of the electron transport chain (4, 9, 14, 16). How R plasmids interfere with hexachlorophene action is not known, although they could affect either permeability to the inhibitor or an alteration at its site of action. No growth has been observed with PU21, PU21(pMG1), or PU21(pMG2) with hexachlorophene as a sole carbon source, nor do these plasmids allow growth on diphenylmethane, which is metabolized by some soil Pseudomonas species (8). The designation Hex is proposed to TABLE 1. Disinfectants tested on R+ and R- P.
635
describe this phenotype (15). In this survey Hex was found on only 2 of 38 Pseudomonas plasmids tested. Delmotte et al. (6) found considerable variation in the susceptibility of clinical isolates of P. aeruginosa to hexachlorophene. P. aeruginosa is known to be one of the gram-negative bacteria that emerges following hexachlorophene treatment of skin (7) and can also contaminate stocks of hexachlorophene-containing soaps and detergents (1). Whether plasmids play a role in the resistance of natural isolates of P. aeruginosa to hexachlorophene and other antiseptics appears worthy of further study.
a
ACKNOWLEDGMENT This work was supported by a grant from the National Science Foundation (PCM75-03932).
636
SUTTON AND JACOBY LITERATURE CITED
1. Anderson, K. 1962. The contamination of hexachloro-
2.
3. 4.
5.
6.
7. 8.
phene soap with Pseudomonas pyocyanea. Med. J. Aust. 49:463. Bergan, T. 1975. Epidemiological typing of Pseudomonas aeruginosa, p. 189-235. In M. R. W. Brown (ed.), Resistance of Pseudomonas aeruginosa. John Wiley & Sons, London. Clark, D. L., A. A. Weiss, and S. Silver. 1977. Mercury and organomercurial resistances determined by plasmids in Pseudomonas. J. Bacteriol. 132:186-196. Corner, T. R., H. L. Joswick, J. N. Silvernale, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: lysis and fixation of bacterial proptoplasts. J. Bacteriol. 108:501-507. Delmotte, A., J. Beumer, E. Cotton, N. DekeyserDelmotte, M. Millet, K. G. Vanden Abbeele, W. Von Grunigen, and E. Yourassowsky. 1971. Etude sur la sensibilit6 du bacille pyocyanique (Pseudomonas aeruginosa) aux antiseptiques et aux antibiotiques. I. Variations de la sensibilite du bacille pyocyanique aux antiseptiques et antibiotiques au cours du temps et reproductibilite des resultats. Therapie 26:629-644. Delmotte, A., J. Beumer, E. Cotton, N. DekeyserDelmotte, M. Millet, K. G. Vanden Abbeele, W. Von Grunigen, and E. Yourassowsky. 1971. Etude sur la sensibilite du bacille pyocyanique (Pseudomonas aeruginosa) aux antiseptiques et aux antibiotiques. II. De la notion statistique de la sensibilite du bacille pyocyanique aux antiseptiques a la conception de 1'antiseptogramme. Therapie 26:645-651. Evans, Z. A., R. C. Rendtorff, H. Robinson, and E. W. Rosenberg. 1973. Ecological influence of hexachlorophene on skin bacteria. J. Invest. Dermatol. 60:207-214. Francis, A. J., R. J. Spanggord, G. I. Ouchi, R. Bram-
ANTIMICROB. AGENTS CHEMOTHER. hall, and N. Bohonos. 1976. Metabolism of DDT analogues by a Pseudomonas sp. Appl. Environ. Microbiol. 32:213-216. 9. Frederick, J. J., T. R. Corner, and P. Gerhardt. 1974. Antimicrobial actions of hexachlorophene: inhibition of respiration in Bacillus megaterium. Antimicrob. Agents Chemother. 6:712-721. 10. Gump, W. S., and G. R. Walter. 1968. The bis-phenols, p. 257-277. In C. A. Lawrence and S. S. Block (ed.), Disinfection, sterilization, and preservation. Lea & Febiger, Philadelphia. 11. Jacoby, G. A. 1974. Properties of R plasmids determining gentamicin resistance by acetylation in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6:239-252. 12. Jacoby, G. A. 1977. Classification of plasmids in Pseudomonas aeruginosa, p. 119-126. In D. Schlessinger (ed.), Microbiology-1977. American Society for Microbiology, Washington, D.C. 13. Jacoby, G. A., and J. A. Shapiro. 1977. Plasmids studied in Pseudomonas aeruginosa and other pseudomonads, p. 639-656. In A. I. Bukhari, J. A. Shapiro, and S. Adhya (ed.), DNA insertion elements, plasmids, and episomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 14. Joswick, H. L., T. R. Corner, J. N. Silvernale, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: release of cytoplasmic materials. J. Bacteriol. 108:492-500. 15. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III, N. Datta, and S. Falkow. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. 16. Silvernale, J. N., H. L. Joswick, T. R. Corner, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: cytological manifestations. J. Bacteriol. 108:482-491.
