APPLIED AND ENVIRONMENTAL MICROBIOLoGY, Apr. 1977, p. 975-976 Copyright (© 1977 American Society for Microbiology
Vol. 33, No. 4 Printed in U.S.A.
Linkage of Mercury, Cadmium, and Arsenate and Drug Resistance in Clinical Isolates of Pseudomonas aeruginosa HIDEOMI NAKAHARA,* TOMOAKI ISHIKAWA, YASUNAGA SARAI, ISAMU KONDO, HIROYUKI KOZUKUE, AND SIMON SILVER Departments of Hygiene and Microbiology, The Jikei University School of Medicine, Tokyo 105, Japan,* and Department of Biology, Washington University, St. Louis, Missouri 63130 Received for publication 29 September 1976
Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.
It is well known that many bacterial isolates are resistant to heavy metal ions (2, 6, 9-11). For example, besides penicillin resistance, the penicillinase plasmid of Staphylococcus aureus carries genes determining resistance to several metallic ions such as Hg, Cd, As, Pb, and Zn (7). We also observed that resistances to Hg and Cd mediated by the penicillinase plasmid were controlled by quite different mechanisms (3). Furthermore, R-factor-mediated resistance to Hg, Co, and Ni has also been observed in Escherichia coli (8). It is of interest that resistance to these metals is mediated by the same plasmid that determines resistance to drugs. Most of these metals have recently been listed as established or possible causes of environmental pollution. In addition, methyl mercury is known to cause Minamata disease, and cadmium is the causative agent of Itai-Itai disease in Japan. Studies of the epidemiology, genetics, and biochemistry of drug-resistant bacteria indicate that the origin, selection, spread, and prevalence of drug-resistant microorganisms resulted from use of drugs. In contrast, several investigations suggest a correlation between resistance to metals and drugs and exposure of bacteria to a hospital environment (1, 5). However, the nature of the factors contributing to these metal-resistant organisms has not yet been explained by epidemiological and genetic investigations. These metal-resistant isolates do not appear to originate by chance. We presumed that one of the factors selecting for metal-resistant organisms may be environmental contamination by these metals. Our previous studies of the metal resistance of 415 isolates of S. aureus derived from clinical lesions (4) indicated that the frequency of metal resistance in isolates of S. aureus was higher than that of drug resistance. Most of these metal-resistant isolates were multiply metal resistant and also multiply drug resistant. Fur-
thermore, 8.0% of the cultures tested were metal resistant but drug sensitive. In the current investigation, we assayed both metal and drug resistances in 787 P. aeruginosa. isolates from clinical lesions and attempted to demonstrate a relation between the two phenomena. The four metals tested (Hg, Cd, As, and Pb) were provided as HgCl2, CdCl2, Na2HAsO4, and Pb(CH,COO)2, respectively. Effective concentrations of the metals were determined experimentally, whereas the concentrations of the test drugs were selected arbitrarily. Those cultures not inhibited by 100 ,.g of streptomycin, tetracycline, chrolamphenicol, or kanamycin per ml or by 25 ,g of gentamicin or dibekacin per ml were regarded as resistant to each of the antibiotics. Figure 1 shows a clear-cut bimodal distribution of susceptibility to three of the metals, but only a single peak of resistance to Pb. It can be seen that resistance was demonstrable in media containing the following concentrations of metals (in micrograms per milliliter): HgCl2, 10; CdCl2, 400; and Na2HAsO4, 400. The frequencies of resistance to these concentrations of Hg, Cd, and As were 75.1, 96.5, and 98.8%, respectively (Table 1). A greater fraction ofthe isolates were resistant to each metal than to each antibiotic. Cultures with resistance to all three metals (triple) were isolated most frequently, followed by those resistant to two metals (double) and then those resistant to one (single) (Table 2). Among the doubles, those resistant to Cd and As were isolated most frequently. In sum, a total of 99.8% of the cultures tested comprised metal-resistant isolates, and 99.5% of these were multiply metal resistant. In addition, 53.2% of these multiply metal resistant isolates were also multiply drug resistant. The number that were metal sensitive and drug resistant was small, comprising only 0.2% of the isolates. This contrasts with 19.4% of the isolates that 975
976
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 2. Patterns of resistance of P. aeruginosa isolates to heavy metal ions 0
13 .1
Type of resistance
1.6
6.25
25
100
400
1600
6400
Minimal inhibitory concentration (ogIml)
FIG. 1. Minimal inhibitory concentration distribution of resistance to Hg, Cd, As, and Pb among 787 P. aeruginosa isolates. Minimal inhibitory concentration, as determined by the agar dilution method, is defined as the lowest concentration at which no visible growth of bacteria is detected. From these patterns, those cultures not inhibited by 10 pg of HgCl2 per ml or by 400 Mg of either CdCl2 or Na2HAsO4 per ml were regarded as resistant to the respective metal. The distribution pattern of Pb resistance revealed a single peak. TABLE 1. Frequency of isolation of metal- and drugresistant isolates of P. aeruginosa Determination
Metal Hg Cd As Drug" SM TC CP KM GM DKB
No. of isolates
% of isolates
591 759 779
75.1 96.5 98.9
405 125 324 404 108
51.5 15.9 41.2 51.3 13.7
113
14.4
SM, Streptomycin; TC, tetracycline; CP, chloramphenicol; KM, kanamycin; GM, gentamicin; DKB, dibekacin.
were metal resistant but drug sensitive. A similar result was obtained in S. aureus (4). In conclusion, the results of this study may be summarized as follows: (i) the frequency of metal resistance was higher than that of antibiotic resistance; (ii) most of these metal-resistant isolates were multiply metal resistant and also multiply drug resistant; and (iii) there were many isolates that were metal resistant but drug sensitive. These results should be noted in view of the
Triple Hg, Cd, As Double Hg, Cd Hg, As Cd, As Single Cd As Sensitive
No. of isolates M
Isolation frequency among tested isolates (%)
559 (71.0)
71.0
2 (0.3) 196 (24.9) 21 (2.7)
27.9
2 (0.25) 2 (0.25) 5 (0.6)
0.5
0.6
fact that these metals are a cause of environmental pollution. We are grateful to R. R. Colwell, S. Mitsuhashi, and M. Inoue for helpful and constructive discussions. We thank K. Kurosaka, K. Machida, T. Sawahata, K. Itoh, and Y. Tsuruta for gifts for clinical isolates. This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education (no. 177156). LITERATURE CITED 1. Barbara, M. H. 1970. Distribution of mercury resistance among Staphylococcus aureus isolated from a hospital community. J. Hyg. 68:111-119. 2. Hamdy, M. K., and 0. R. Noyes. 1975. Formation of methyl mercury by bacteria. Appl. Microbiol. 30:424432. 3. Kondo, I., T. Ishikawa, and H. Nakahara. 1974. Mercury and cadmium resistances mediated by the penicillinase plasmid in Staphylococcus aureus. J. Bacteriol. 117:1-7. 4. Kondo, I., H. Nakahara, and T. Ishikawa. 1975. The possibility of a specific plasmid mediating metal resistances, p. 145-152. In S. Mitsuhashi and H. Hashimoto (ed.), Microbiol drug resistance. University of Tokyo Press, Tokyo. 5. Moore, B. 1960. A new screen test and selective medium for the rapid detection of epidemic strains of Staph. aureus. Lancet ii:453-458. 6. Nelson, J. D., Jr., W. Blair, F. E. Brinckman, R. R. Colwell, and W. P. Iverson. 1973. Biodegradation of phenylmercuric acetate by mercury-resistant bacteria. Appl. Microbiol. 26:321-326. 7. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342. 8. Smith, D. H. 1967. R factors mediate resistance to mercury, nickel and cobalt. Science 156:1114-1116. 9. Summers, A. O., and S. Silver. 1972. Mercury resistance in plasmid-bearing strains ofEscherichia coli. J. Bacteriol. 112:1228-1236. 10. Tonomura, K., K. Maeda, F. Futai, T. Nakagami, and M. Yamada. 1968. Stimulative vaporization of phenylmercuric acetate by mercury-resistant bacteria. Nature (London) 217:644-646. 11. Walker, J. D., and R. R. Colwell. 1974. Mercury-resistant bacteria and petroleum degradation. Appl. Microbiol. 27:285-287.
Vol. 33, No. 4 Printed in U.S.A.
Linkage of Mercury, Cadmium, and Arsenate and Drug Resistance in Clinical Isolates of Pseudomonas aeruginosa HIDEOMI NAKAHARA,* TOMOAKI ISHIKAWA, YASUNAGA SARAI, ISAMU KONDO, HIROYUKI KOZUKUE, AND SIMON SILVER Departments of Hygiene and Microbiology, The Jikei University School of Medicine, Tokyo 105, Japan,* and Department of Biology, Washington University, St. Louis, Missouri 63130 Received for publication 29 September 1976
Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.
It is well known that many bacterial isolates are resistant to heavy metal ions (2, 6, 9-11). For example, besides penicillin resistance, the penicillinase plasmid of Staphylococcus aureus carries genes determining resistance to several metallic ions such as Hg, Cd, As, Pb, and Zn (7). We also observed that resistances to Hg and Cd mediated by the penicillinase plasmid were controlled by quite different mechanisms (3). Furthermore, R-factor-mediated resistance to Hg, Co, and Ni has also been observed in Escherichia coli (8). It is of interest that resistance to these metals is mediated by the same plasmid that determines resistance to drugs. Most of these metals have recently been listed as established or possible causes of environmental pollution. In addition, methyl mercury is known to cause Minamata disease, and cadmium is the causative agent of Itai-Itai disease in Japan. Studies of the epidemiology, genetics, and biochemistry of drug-resistant bacteria indicate that the origin, selection, spread, and prevalence of drug-resistant microorganisms resulted from use of drugs. In contrast, several investigations suggest a correlation between resistance to metals and drugs and exposure of bacteria to a hospital environment (1, 5). However, the nature of the factors contributing to these metal-resistant organisms has not yet been explained by epidemiological and genetic investigations. These metal-resistant isolates do not appear to originate by chance. We presumed that one of the factors selecting for metal-resistant organisms may be environmental contamination by these metals. Our previous studies of the metal resistance of 415 isolates of S. aureus derived from clinical lesions (4) indicated that the frequency of metal resistance in isolates of S. aureus was higher than that of drug resistance. Most of these metal-resistant isolates were multiply metal resistant and also multiply drug resistant. Fur-
thermore, 8.0% of the cultures tested were metal resistant but drug sensitive. In the current investigation, we assayed both metal and drug resistances in 787 P. aeruginosa. isolates from clinical lesions and attempted to demonstrate a relation between the two phenomena. The four metals tested (Hg, Cd, As, and Pb) were provided as HgCl2, CdCl2, Na2HAsO4, and Pb(CH,COO)2, respectively. Effective concentrations of the metals were determined experimentally, whereas the concentrations of the test drugs were selected arbitrarily. Those cultures not inhibited by 100 ,.g of streptomycin, tetracycline, chrolamphenicol, or kanamycin per ml or by 25 ,g of gentamicin or dibekacin per ml were regarded as resistant to each of the antibiotics. Figure 1 shows a clear-cut bimodal distribution of susceptibility to three of the metals, but only a single peak of resistance to Pb. It can be seen that resistance was demonstrable in media containing the following concentrations of metals (in micrograms per milliliter): HgCl2, 10; CdCl2, 400; and Na2HAsO4, 400. The frequencies of resistance to these concentrations of Hg, Cd, and As were 75.1, 96.5, and 98.8%, respectively (Table 1). A greater fraction ofthe isolates were resistant to each metal than to each antibiotic. Cultures with resistance to all three metals (triple) were isolated most frequently, followed by those resistant to two metals (double) and then those resistant to one (single) (Table 2). Among the doubles, those resistant to Cd and As were isolated most frequently. In sum, a total of 99.8% of the cultures tested comprised metal-resistant isolates, and 99.5% of these were multiply metal resistant. In addition, 53.2% of these multiply metal resistant isolates were also multiply drug resistant. The number that were metal sensitive and drug resistant was small, comprising only 0.2% of the isolates. This contrasts with 19.4% of the isolates that 975
976
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 2. Patterns of resistance of P. aeruginosa isolates to heavy metal ions 0
13 .1
Type of resistance
1.6
6.25
25
100
400
1600
6400
Minimal inhibitory concentration (ogIml)
FIG. 1. Minimal inhibitory concentration distribution of resistance to Hg, Cd, As, and Pb among 787 P. aeruginosa isolates. Minimal inhibitory concentration, as determined by the agar dilution method, is defined as the lowest concentration at which no visible growth of bacteria is detected. From these patterns, those cultures not inhibited by 10 pg of HgCl2 per ml or by 400 Mg of either CdCl2 or Na2HAsO4 per ml were regarded as resistant to the respective metal. The distribution pattern of Pb resistance revealed a single peak. TABLE 1. Frequency of isolation of metal- and drugresistant isolates of P. aeruginosa Determination
Metal Hg Cd As Drug" SM TC CP KM GM DKB
No. of isolates
% of isolates
591 759 779
75.1 96.5 98.9
405 125 324 404 108
51.5 15.9 41.2 51.3 13.7
113
14.4
SM, Streptomycin; TC, tetracycline; CP, chloramphenicol; KM, kanamycin; GM, gentamicin; DKB, dibekacin.
were metal resistant but drug sensitive. A similar result was obtained in S. aureus (4). In conclusion, the results of this study may be summarized as follows: (i) the frequency of metal resistance was higher than that of antibiotic resistance; (ii) most of these metal-resistant isolates were multiply metal resistant and also multiply drug resistant; and (iii) there were many isolates that were metal resistant but drug sensitive. These results should be noted in view of the
Triple Hg, Cd, As Double Hg, Cd Hg, As Cd, As Single Cd As Sensitive
No. of isolates M
Isolation frequency among tested isolates (%)
559 (71.0)
71.0
2 (0.3) 196 (24.9) 21 (2.7)
27.9
2 (0.25) 2 (0.25) 5 (0.6)
0.5
0.6
fact that these metals are a cause of environmental pollution. We are grateful to R. R. Colwell, S. Mitsuhashi, and M. Inoue for helpful and constructive discussions. We thank K. Kurosaka, K. Machida, T. Sawahata, K. Itoh, and Y. Tsuruta for gifts for clinical isolates. This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education (no. 177156). LITERATURE CITED 1. Barbara, M. H. 1970. Distribution of mercury resistance among Staphylococcus aureus isolated from a hospital community. J. Hyg. 68:111-119. 2. Hamdy, M. K., and 0. R. Noyes. 1975. Formation of methyl mercury by bacteria. Appl. Microbiol. 30:424432. 3. Kondo, I., T. Ishikawa, and H. Nakahara. 1974. Mercury and cadmium resistances mediated by the penicillinase plasmid in Staphylococcus aureus. J. Bacteriol. 117:1-7. 4. Kondo, I., H. Nakahara, and T. Ishikawa. 1975. The possibility of a specific plasmid mediating metal resistances, p. 145-152. In S. Mitsuhashi and H. Hashimoto (ed.), Microbiol drug resistance. University of Tokyo Press, Tokyo. 5. Moore, B. 1960. A new screen test and selective medium for the rapid detection of epidemic strains of Staph. aureus. Lancet ii:453-458. 6. Nelson, J. D., Jr., W. Blair, F. E. Brinckman, R. R. Colwell, and W. P. Iverson. 1973. Biodegradation of phenylmercuric acetate by mercury-resistant bacteria. Appl. Microbiol. 26:321-326. 7. Novick, R. P., and C. Roth. 1968. Plasmid-linked resistance to inorganic salts in Staphylococcus aureus. J. Bacteriol. 95:1335-1342. 8. Smith, D. H. 1967. R factors mediate resistance to mercury, nickel and cobalt. Science 156:1114-1116. 9. Summers, A. O., and S. Silver. 1972. Mercury resistance in plasmid-bearing strains ofEscherichia coli. J. Bacteriol. 112:1228-1236. 10. Tonomura, K., K. Maeda, F. Futai, T. Nakagami, and M. Yamada. 1968. Stimulative vaporization of phenylmercuric acetate by mercury-resistant bacteria. Nature (London) 217:644-646. 11. Walker, J. D., and R. R. Colwell. 1974. Mercury-resistant bacteria and petroleum degradation. Appl. Microbiol. 27:285-287.
