Association of Metal Tolerance with Multiple Antibiotic Resistance of ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1984, p. 1238-1242

Vol. 47, No. 6

0099-2240/84/061238-05$02.00/0 Copyright © 1984, American Society for Microbiology

Association of Metal Tolerance with Multiple Antibiotic Resistance of Bacteria Isolated from Drinking Watert JON J. CALOMIRIS, JOHN L. ARMSTRONG, AND RAMON J. SEIDLER* Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804 Received 9 September 1983/Accepted 30 January 1984

Bacterial isolates from the drinking water system of an Oregon coastal community were examined to the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels of Cu2", Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance. assess

Through research in our laboratory, we have been able to demonstrate that significant increases of multiple-antibioticresistant (MAR) bacteria occur in various drinking water systems (5). Multiple antibiotic resistance was expressed by 18.6% of the standard plate count (SPC) bacteria isolated from the intake for untreated water and 67.8% of the drinking water SPC bacteria isolated from the distribution system. The enhancement of MAR bacteria observed in municipal water systems may be of health significance since human infections caused by such organisms could be difficult to treat with drugs. Grabow et al. (8) have called for the need to review water quality standards since bacterial types usually considered harmless could pose a health threat should they possess R factors that confer multiple drug resistance. Microorganisms resistant to both antibiotics and metals have been isolated from nosocomial and burn wound infections, infections treated with metal-based antimicrobial agents (12), and various metal-contaminated environments such as estuaries (1, 24), soils (10, 11), and sewage (25). Nakahara et al. (13, 14, 15) have suggested that the combined expressions of antibiotic resistance and metal tolerance may not be a fortuitous phenomenon but rather is caused by selection resulting from metals present in an environment. In the present study, SPC bacteria were tested for their tolerances to those metals associated with water treatment and distribution. It was found that a high percentage of the SPC bacteria were both metal tolerant and antibiotic resistant. MATERIALS AND METHODS The drinking water system serving an Oregon resort coastal community of 6,000 residents and up to 7,000 visitors annually was selected for this study. The raw water sources consist of two virgin mountain streams which receive a chlorine percolation treatment and are then distributed ca. 16.0 km throughout a sprawling community through concrete and galvanized iron pipes. Many residences are served on site by copper pipes. Samplings were performed on 29

March, 27 May, 8 July, and 14 July 1980 at the raw water source and private residences ca. 2.5 and 15.5 km from the chlorination station. By standard methods (3), water samples were collected in sterile plastic carboys, transported to the laboratory, and membrane filtered with GN-6 Gelman filters with a 0.45-,um pore size. Filters were placed in petri plates containing M-SPC agar, an SPC medium (23), and incubated for 48 h at 35°C. Plates were then examined at x 15 magnification, and randomly picked colonies were streaked onto tryptic soy agar (Difco Laboratories, Detroit, Mich.) supplemented with 0.3% yeast extract (Difco). Isolates were identified to the genus level by the method of LeChavallier et al.

(9).

By a replica plate method that uses colonies on a master plate as inocula, the resistance of each isolate to five antibiotics (Sigma Chemical Co., St. Louis, Mo.) was determined by growth on Mueller-Hinton medium (Difco) as previously described (4, 5). All isolates were also tested for tolerance to six metals by a replica plate method with 24-h broth (Mueller-Hinton broth; Difco) cultures adjusted to about 105 cells per ml as the inocula (26). A multipoint inoculator with stainless steel prongs was used to deliver ca. 10-LI drops from the broth culture tubes onto plates containing Mueller-Hinton medium with and without supplemented metals. The experimental plates were prepared by supplementing Mueller-Hinton medium with metal salts for final cationic concentrations of (micrograms per milliliter): 200, 400, 800, 1,600, and 3,200 for Al3+, Cu2+, Sn2+, and Zn2+; 800, 1,600, 2,400, and 3,200 for Pb2+; and 25, 50, 100, 200, and 400 for Cd2+. The compounds used were A12(SO4)3, CuCl2 * 2H20, Pb(NO3)2, and ZnC12 (J. T. Baker Chemical Co., Phillipsburg, N.J.); SnC12 (Sigma); and CdCl2 * 2.5 H20 (Mallinckrodt, Inc., St. Louis, Mo.). The salts-agar media were adjusted to a final pH of 7.0 by using NaOH. After inoculation, the agar plates were incubated for 18 h at 350C and then examined to determine the MIC for growth. Unlike antibiotic resistance, there are no universally acceptable metal ion concentrations which are used to designate microbial metal tolerance. In our preliminary studies, medium composition was found to greatly influence the sensitivities of the microbes to the test metals. On minimal agar medium or in minimal broth, the sensitivities of the tested isolates to metals increased 20-fold or more. However, minimal media would not support the growth of many

* Corresponding author. t Technical Paper no. 7055, Oregon Agricultural Experiment Station, Corvallis, OR 97331-3804. 1238

RESISTANT BACTERIA FROM DRINKING WATER

VOL. 47. 1984 LUJ

FIJ 0

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0 I-

50

0.1 N

N

N

N

0-

LUJ

LU

0r

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0

1

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TOLERANCE TO METALS FIG. 1. Tolerance of isolates to metals. Frequencies of raw water (-- -) and distribution water ( ) isolates tolerant to none, one, two, or three of the metals Cu2+, Pb2, and Zn2+.

isolates. Mueller-Hinton agar was finally selected because strains grew well, and this medium was also used in the antibiotic sensitivity testing. Metal tolerance levels were chosen after an extensive analysis of MIC measurements on hundreds of raw water and distribution water isolates exposed to six concentrations of each metal tested. Histograms representing the fraction of isolates growing at each metal concentration were prepared. By stringent criteria, the MIC was designated to be a twofold dilution of the metal ion concentration which inhibited the growth of most, if not all, of the tested cultures on Mueller-Hinton medium. MIC values indicative of metal tolerance were (micrograms per milliliter): 200 for Cd>, 1,600 for Zn2+, 3,200 for Cu> and Pb-, and greater than 3,200 for Al3 and Sn2> Strains of Esuherichia coli (ATCC 25922), Pseiudomonas aerliginosa (ATCC 27853), and Staphylococcus aiureius (ATCC 25923) which are commonly used in antibiotic sensitivity testing were used as controls. One Bacillius and two AcInetobac ter isolates were selected as controls for the metal tolerance testing since against them the various metals have broad MIC ranges. Statistical analyses of the data were performed by a comparison of proportions by the Z test, with confidence levels of 5% being considered significant. P values were calculated by comparing occurrences of metal tolerance and antibiotic resistances among all raw water and all distribution water isolates. RESULTS Of the 393 isolates tested in this study, approximately twothirds of the 216 distribution water isolates and two-thirds of the 177 raw water isolates expressed tolerance to one or more of the tested materials. However, a significant proportion (, with 24.4% tolerant to two and 20.9% tolerant to all three metals (Fig. 1). Of the antibiotic-sensitive isolates, only 4.3%G were tolerant to two and none were tolerant to all three metals. Of the raw water isolates, only 2.8% of the MAR and 1.5% of the antibiotic-sensitive isolates were multiply metal tolerant to Cu2 ', Pb-2, and Zn2>. Furthermore, positive associations (P < 0.0001) of multiple antibiotic resistance with tolerance to Cu2> Pb2+, and Zn2+ were found for distribution water isolates, whereas both MAR and antibiotic-sensitive isolates from raw waters displayed similar low levels of tolerance to these metals (Fig. 2). Tolerance to both Al3 + and Sn- + was expressed by

1239

significantly greater (P < 0.0001) proportions of raw water isolates (65.0%) than distribution water isolates (20.6%). Although similar proportions of MAR isolates (65.1%) and non-MAR isolates (65.0%) from raw waters were tolerant to both metals, a significantly greater (P < 0.0001) percentage of antibiotic-sensitive isolates (43.8%) than MAR isolates (14.1%) from distribution waters were tolerant to Al3 and Sn>+ Antibiotic and metal resistance phenotypes were associated with nearly all the genera of the bacterial isolates (Table 1). Of the genera represented by isolates from both raw and distribution waters, greater numbers of the PseiudomonasAlcaligenes group, Acinetobacter, Enterobacter, and Moraxella isolates from distribution waters exhibited resistances to more compounds than isolates of corresponding genera from raw water. Resistance to kanamycin and tolerances to Cu2 , Pb>+, and Zn2 were expressed by larger proportions of distribution water isolates than raw water isolates. For example, the percentages of resistant raw water isolates and distribution water isolates were, respectively: with Enterobacter, 6.7 and 62.5 for kanamycin, 0 and 62.5 for Cu>, 13.3 and 56.3 for Pb-t, and 0 and 56.3 for Zn2> with Acinetobacter, 30.8 and 60.0 for kanamycin, 0 and 30.0 for streptomycin, 30.8 and 70.0 for tetracycline, 0 and 60.0 for Cu>, 0 and 90.0 for Pb2, and 7.7 and 50.0 for Zn>+. The relation of metal tolerance to multiple antibiotic resistance varied when isolates of different distribution water sites were compared (Table 2). Isolates from sample site A, ca. 2.5 km from the chlorination station, exhibited resistance patterns more similar to those of the raw water isolates, where large proportions of both MAR and antibiotic-sensitive isolates were similarly sensitive to Pb>' and Zn>2 while tolerant to Al3 t and Sn 2 '. In contrast, the isolates from sites B and C, ca. 15.5 km from the chlorination source, displayed positive associations (P < 0.04) of tolerance to Cu2+, Pb2+, and Zn>2 with multiple antibiotic resistance. DISCUSSION Dynamic changes in the raw water microbial population upon entering the distribution system were reflected by a ca. 20,000-fold decrease in the numbers of SPC bacteria per milliliter as well as by changes in the predominant genera. Also observed was an increase in the percentage of MAR bacteria from 20.4% of the raw water isolates to 36.7% of the distribution water isolates. The observed occurrences appar-

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TOLERANCE TO METALS FIG. 2. Tolerance to metals and sensitivity to antibiotics. Frequencies of MAR (O) and antibiotic-sensitive (0) isolates from raw waters (-- -) and distribution waters ( ) tolerant to none, one, two, or three of the metals Cu-+, Pb2, and Zn-.

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APPL. ENVIRON. MICROBIOL.

CALOMIRIS, ARMSTRONG, AND SEIDLER

TABLE 1. Resistance to antibiotics and tolerance to metals expressed by at least 50% of the raw water isolates or distribution water isolates or both of each genus or group Genus

Hafniae Escherichia Klebsiella Serratia Micrococcus Staphylococcus Arthrobacter Acinetobacter Enterobacter PseudomonasAlcaligenes group Aeromonas

Moraxella Citrobacter

of Source (no. isolates tested)

Raw (28) Raw (9) Raw (23) Raw (9) Distribution Distribution Distribution Raw (13) Distribution Raw (15) Distribution Raw (14)

were resistant or tolerant" 50% of thePb>+ isolates ofZn2each genus Al'+ Antibiotics Cd'+ CM to which KM at leastCu> SM TC and metals Sn>2

+

+ + + +

(32) (30) (10) (10)

+

+

+ + + +

+ + + +

+ + + +

+

+

+

+

+ +

+

+

+

+

+

+

+

+

+ + + +

+

(16)

Distribution (48) Raw (18) Distribution (6) Raw (13) Distribution (25) Raw (14)

+ +

+ +

+ +

+ +

+

+

+ +

+

+

+ +

+ + +

+ +

+

+

+

+ +

Distribution (16)

"SM, Streptomycin; TC, tetracycline; CM, chloramphenicol; KM, kanamycin. +, Metal tolerant or antibiotic resistant.

ently reflected water treatment processes and possibly the added influences from subsequent water distribution. Although results of research in our laboratory suggest an association of chlorine disinfection with selection for antibiotic resistance (4), additional factors such as metal exposures from distribution water pipe materials could indirectly explain the significant increase of MAR bacteria in distribution networks. For this reason, bacterial isolates were studied to determine the manner by which metal tolerance was associated with multiple antibiotic resistance. Positive correlationis were observed between tolerance to Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance. Because tolerance to these metals was expressed by greater proportions of drinking water isolates than raw water isolates, microbial selection phenomena for metal tolerance must have occurred during treatment or within the distribution system. Since multiple tolerance to Cu2+, Pb2+, and Zn2+ is significantly associated with distribution water isolates that are MAR but not those that are antibiotic sensitive, it appears that simultaneous selection for metal- and drugresistant bacteria may occur within the drinking water system. The relation of metal tolerance to antibiotic resistance is demonstrated further by the phenotypic trends of the iso-

lates of various genera. In comparing the isolates of genera found in both raw and distribution waters, greater percentages of isolates from drinking waters were resistant to antibiotics and metals than the raw water isolates of corresponding genera. This finding was illustrated when isolates of the genera Acinetobacter and Enterobacter were compared, whereby greater proportions of the distribution water isolates than raw water isolates were resistant to kanamycin and tolerant to Cu2+, Pb2+, and Zn2+. Previous studies have demonstrated the role of plasmids in conferring resistance to both antibiotics and metals. McHugh et al. (12) have shown plasmids conferring antibiotic and metal resistance to be present in Salmonella typhimurium isolates from human burn wounds treated with silver nitrate solution. Others have demonstrated genetic linkages (presumably by plasmids) between antibiotic resistance in Enterobacter aerogenes and tolerance to Cd2+ and Zn2+ (17), whereas Timoney et al. have demonstrated linkage between Hg2+, Cd2+, Zn2+ and ampicillin resistance (24). Novick et al. (16) have demonstrated penicillinase plasmids of S. aureus to be responsible for resistance to erythromycin and various inorganic ions, including Cd2+, Pb2+, Hg2+, and Zn2+. However, these researchers have emphasized that complicated sets of relationships exist between the host cell

TABLE 2. Incidence of metal tolerance of MAR and antibiotic-sensitive isolates obtained from four sampling sites Sample site

Distance*(km)from

chlorination station

Raw

Distribution A

2.5

Distribution B

15.5

Distribution C

15.5

c% of isolates tolerant to:

Antibiotic

phenotype (no. of isolates)

Cu>+

MAR (109) Sensitive (68) MAR (23) Sensitive (19) MAR (28) Sensitive (12) MAR (65) Sensitive (10)

4.6 1.5 13.0 0 57.1 16.7 55.4 20.0

Pb>+ 23.0 29.4 4.3 10.5 71.4 16.7 78.5 20.0

Zn2'

Al"

6.4 1.5 8.7 5.3 67.9 16.7 53.8 10.0

67.0 68.0 34.8 68.4 35.7 33.3 40.0 30.0

2+

Sn> 93.6 94.1 60.9 89.5 60.7 58.3 55.4 10.0

2+

Cd> 25.6 29.4 30.4 52.6 10.7 8.3 38.5 0

RESISTANT BACTERIA FROM DRINKING WATER

Vol. 47, 1984

and the plasmid with respect to resistance to metals. For example, some S. aureius strains possessing plasmids conferring Cd-+ resistance were shown to mutate and become Cd> sensitive with the mutation not being plasmid linked. Also, S. aiurelis isolates not containing plasmids were shown to mutate and become Cd-+ resistant. Standard, previously successful techniques for demonstrating plasmid transfer from other environmental cultures (22) failed in the present study in demonstrating mobilization of antibiotic or metal resistance from distribution isolates into proven E. (-oli or Klebsiella spp. recipients. Thus, to account for the frequent incidence of metal tolerance and multiple antibiotic resistance among the distribution water isolates in our study, the interactions of selection mechanisms involving both chromosomal and perhaps plasmid genetic elements need to be considered. Microbial populations in source water are exposed to metals during transport through and colonization of metal pipes in the distribution system. Since the treatment process of the source raw water of this study is only chlorination, most metal exposures can be attributed to the pipe environment. Several studies have illustrated that water distribution pipes are colonized by diverse bacterial populations (2, 19, 20). Olson and colleagues (19, 20) have shown through the use of X-ray energy-dispersive microanalysis that inner

surfaces of galvanized iron distribution pipes also contain a complex array of mineral deposits. Minerals found deposited on the amorphous pipe surfaces as well as on the surface of colonizing bacteria such as Gallionella spp. included phosphorous, calcium, iron, aluminum, silicon, and zinc. Exposures of microbial populations to pipe surfaces and corrosion materials that deposit and accumulate in distribution systems, especially at dead ends, could eliminate some microbial groups while allowing metal-tolerant organisms to survive, colonize pipe surfaces, and ultimately be shed into the drinking water. This was noted in the present study by the differences in metal tolerance among isolates from various water distribution sites. Tolerance to Cu>+, Pb>, and Zn>+ and positive associations with multiple antibiotic resistance were expressed by most of the isolates from the sites near the end of the distribution system. However, most of the isolates from the site near the water source, just as the raw water isolates, were neither tolerant to these metals nor MAR. The health significance of MAR and metal-tolerant SPC bacteria may be influenced by various factors such as exposure conditions and host susceptibility. However, epidemiological studies have shown nosocomial infection outbreaks are often associated with bacteria possessing R factors (6, 18, 21). Gardner and Smith. (7) have emphasized that efforts to control R-mediated hospital outbreaks should be directed against environmental selection from overuse of antibiotics and conditions that allow colonization by resistant organisms. With drinking water, health quality standards with regard to the presence of MAR SPC bacteria have not been established. However, proper maintenance procedures should and can be practiced to minimize the occurrence of high concentrations of SPC bacteria in certain areas of some distribution network systems (2, 23). ACKNOWLEDGMENTS

This study was funded by the Environmental Protection Agency. Drinking Water Research Division, Cooperative Agreement CR

807123-01-0. The technical skills of Debbie S. Shigeno are acknowledged.

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1342. 17. Pickett, A. W., and A. C. R. Dean. 1976. Antibiotic resistance of cadmium- and zinc-tolerant strains of Klebsiella (Aerobacter) aerogenes growing in glucose-limited chemostat. FEMS Microbios Lett. 1:165-167. 18. Richmond, M. H. 1972. Some environmental consequences of the use of antibiotics: or "'what goes up must come down." J. Appl. Bacteriol. 35:155-176. 19. Ridgway, H. F., and B. H. Olson. 1981. Scanning electron microscope evidence for bacterial colonization of a drinkingwater distribution system. AppI. Environ. Microbiol. 41:274287. 20. Ridgway, H. F., E. G. Means, and B. H. Olson. 1981. Iron bacteria in drinking-water distribution systems: elemental anal-

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25. Varma, M. M., W. A. Thomas, and C. Prasad. 1976. Resistance to inorganic salts and antibiotics among sewage-borne Enterobacteriaceae and Achromobacteriaceae. J. Appl. Bacteriol. 41:347-349. 26. Washington, J. A., II, and A. L. Barry. 1974. Dilution test procedures, p. 410-417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.