Plasmid-Controlled Resistance to Copper in Escherichia coli

Vol. 154, No. 3

JOURNAL OF BACTERIOLOGY, June 1983, p. 1263-1268

0021-9193/83/061263-06$02.00/0 Copyright C 1983, American Society for Microbiology

Plasmid-Controlled Resistance to Copper in Escherichia coli TIMOTHY J. TETAZ AND RICHARD K. J. LUKE* School of Agriculture, La Trobe University, Bundoora, Victoria, 3083, Australia Received 8 November 1982/Accepted 12 March 1983

The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004. Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K. Sensitive strains of E. coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.

Copper sulfate has been widely used in pig feeds since Barber et al. (3) first described its growth-promoting capacity. Fuller et al. (12) and Smith and Jones (21) attempted to isolate copper-resistant (Cur) enteric bacteria from the feces and gut contents of pigs which had been fed a copper-supplemented diet, but they were unsuccessful. The observation that the addition of copper sulfate to common bacteriological media can lead to a significant decrease in pH led us to employ a modified procedure during an attempt to isolate Cur strains of Escherichia coli from the effluent of a piggery where pigs were fed a copper-supplemented diet. This facilitated the detection of Cur E. coli strains of porcine origin. Copper resistance of clinical isolates of E. coli (10) and of coliforms isolated from sewage (17, 24) has been reported. However, the genetic basis for the copper resistance of these isolates was not investigated. We found that the copper resistance of one of our porcine E. coli isolates is controlled by a conjugative plasmid, which we have designated pRJ1004. This paper describes some of the genetic and physical characteristics of this plasmid. MATERIALS AND METHODS Bacterial strains and plasmids. E. coli strains used are described in Table 1. Cur isolates (RJ92 through RJ95) were obtained by plating suitably diluted samples (0.1 ml) of piggery effluent onto nutrient agar (NA) containing 10 mM CUSO4. Plates were incubated overnight at 37°C, and representative colonies were picked off the primary isolation plates and restreaked on the same medium. Strains were identified according to the procedures of Cowan and Steel (6) for the identification of gram-negative bacteria. Isolates RJ92 through RJ95 were identified as typical E. coli, with the exception of RJ94, which was identified as an indole-negative, non-lactose-fermenting E. coli. Standard R plasmids used in incompatibility tests were obtained from the collections of J. Pittard, Uni-

versity of Melbourne, Melbourne, Australia (R16, R40a, R386, Rldrd-16, R27, R144drd3, R648, R64drdll, R621a, R471a, RN3, RP1, R388, and R6K), and N. Datta, Hammersmith Hospital, London, England (R387 and Rtsl). Media. Nutrient broth (NB) consisted of nutrient broth no. 2 (25 g; Oxoid CM67) and yeast extract (3 g; Oxoid L20) made to 1 liter with distilled water. Ajax agar (15 g/liter) was added to prepare NA. Coppercontaining media were prepared by adding appropriate amounts of 1.0 M CuS04 * 5H20 to NA, adjusting the pH to 7.5 with NaOH, and autoclaving at 121°C for 15 min. When required, antibacterials were added to NA at the following concentrations (,ug/ml): tetracycline, 25; streptomycin, 15; chloramphenicol, 12; kanamycin, 20; nalidixic acid, 25; and rifampin, 150. Mating procedures. The method used to transfer resistance determinants, in 30-min or 24-h matings, was based on a procedure described by Smith (20). Briefly, broth cultures (5 to 10 ,ul) of a prospective donor strain and of an E. coli K-12 recipient strain (W3110-1 or J53-1) were inoculated into 10 ml of NB. After incubation at 37 or 25°C, the mixed culture was plated onto an appropriate selective medium. Transconjugants were purified by restreaking on the same selective medium. Plasmid transfer frequencies were determined by mixing equal volumes of donor and recipient cultures containing approximately 2 x 108 cells in the log phase of growth per ml. Mating mixtures were incubated without shaking at 37°C, usually for 30 min, and samples (0.1 ml) of suitable dilutions were plated on a selective medium. Incompatibility tests. The incompatibility group of pRJ1004 was determined by the method of Datta (7). Matings were performed as described above, and transfer frequencies were determined for each mating. In all matings, selection was made for the incoming plasmid only. Where no transfer of the donor plasmid occurred in 30 min, presumably because of entry exclusion controlled by the plasmid already present in the recipient, overnight matings were carried out. Twenty transconjugant clones from each mating were restreaked on the same selective medium, and a single colony from each restreaking was tested for phenotypic traits controlled by donor and recipient plasmids.

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Determination of resistance to antibacterial agents. Minimal inhibitory concentrations (MICs) of copper were determined by spotting overnight NB cultures of E. coli strains onto NA containing CuSO4 at concentrations ranging from 2 to 20 mM. MICs were recorded as the lowest concentration of CuS04 which prevented growth after overnight incubation at 37°C. Qualitative estimates of Cur were made by streaking isolates onto NA containing 7.5 mM CuS04. Those which were able to form colonies on this medium after overnight incubation at 37°C were designated Cur. Resistance to antibiotics was determined by placing Oxoid Multodisks on Mueller-Hinton agar (Oxoid CM337) plates that had each previously been spread with approximately 107 organisms. Resistance to heavy metals was determined by a disk assay method. Overnight NB cultures of the E. coli strains to be tested were diluted 1:102 in saline (0.9%o NaCl, wt/vol). Samples (0.1 ml) of the resulting suspensions were transferred to 3-ml overlays (Mueller-Hinton agar, diluted 1:3) at 56°C. Overlays, each containing approximately 5 x 105 organisms, were poured over the surface of agar plates (MuellerHinton agar, 20 ml of medium per plate) and allowed to set at 4°C for 10 min. Sterile (blank) antibiotic assay disks (Oxoid, 6.5 mm diameter) were aseptically placed in the center of each plate and impregnated with 10 ,ul of an inorganic salt solution. After overnight incubation at 37°C, the diameters of the zones of inhibition of bacterial growth were measured. Stock solutions of the following inorganic salts were used: 1.0 M Na2HAsO4; 0.3 M CdCl2; 0.5 M CoCl2; 1.0 M CuS04; 0.5 M (CH3COO)2Pb; 0.07 M HgCl2; 1.0 M AgNO3; and 0.5 M ZnSO4. Transormtion. Purified plasmid DNA used to transform E. coli K-12 recipient strain ED8739 was prepared by banding a sodium lauryl sarcosine (Sarkosyl) lysate of strain RJ97 in a CsCI-ethidium bromide isopycnic gradient. Recipient cells were grown overnight at 37C in NB and made competent for transformation by the method described by Mandel and Higa (18).

For transformation, CaCl2-treated cells (125 ,ul) mixed with purified plasmid DNA (in 0.01 M Tris, pH 8.0) in a total volume of 375 uJ, and the mixture was chilled in an ice bath. The mixture was maintained at 0°C for 15 min and then subjected to a heat pulse of 5 min at 37°C. The entire mixture was then added to 3 ml of NB and incubated with aeration for 60 min at 37°C before being plated onto NA containing 7.5 mM CuS04. Agprse gel electrophoresis. The procedures used for the isolation of plasmid DNA and for examination of this plasmid DNA by agarose gel electrophoresis were those described by Davey and Pittard (9). were

RESULTS Isolation of copper-resistant E. coli. Initial

studies (described below) defined the conditions required for the selection of Cur E. coli strains on CuS04-containing media. After application of diluted piggery effluent to NA plates containing 10 mM CuSO4 (pH adjusted), four Cur E. coli strains (RJ92 through RJ95) were isolated and retained for further study (Table 1). The sample of piggery effluent was obtained from a farm where approximately 180 ppm (,ug/g) of CuSO4 was routinely included in the pig feed. All the Cur E. coli isolates examined, including E. coli K-12 transconjugants harboring pRJ1004 (see below), produced similar brown colonies, 1 to 2 mm in diameter with dark brown centers, when streaked on NA containing CuSO4. Determination of CuSO4 MICs. The sensitivities to CuSO4 (MICs) of the E. coli strains shown in Table 1 were tested by an agar dilution method. The addition of CuSO4 led to a reduction in the pH of NA from its initial value of 7.49 to 5.63 with 4 mM CuSO4, 4.90 with 10 mM CuSO4, and 4.40 with 20 mM CuSO4. In experiments where the pH of NA was not adjusted

TABLE 1. E. coli strains used Strain

Relevant genotype/phenotypea

Source or reference

J53-1 J53-1 recA W3110-1

F- pro-22 met-63 gyrA F- pro-22 met-63 recA gyrA F- rpo F- hsdR hsdM thi-l thr-l leu-6 metB Tc Su pRJ1004 Cu Tc Cu Ap Km Tc Cu Km Tc F- pro-22 met-63 gyrA pRJ1004 F- rpo pRJ1004 F- pro-22 met-63 recA gyrA pRJ1004 F- hsdR hsdM thi-l thr-I leu-6 metB pRJ1004

Spontaneous Nalr mutant of J5-3 (1) recA mutant of J53-1 Spontaneous Rif' mutant of W3110 (1) Derivative of 803 (4, 25) This paper This paper This paper This paper Cur Tc' Sus transconjugant from RJ92 x J53-1 Cur transconjugant from RJ% x W31104 Cur transconjugant from RJ97 x J53-1 recA Cur transformant (DNA donor RJ97, recipient

ED8739 RJ92 RJ93 RJ94

RJ95 RJ96 RJ97 RJ98 RJ99

ED8739) F- rpo pRJ1004 Cur transconjugant from RJ99 x W3110-1 RJ101 F- pro-22 met-63 gyrA pRJ1004 Cur transconjugant from RJ100 x J53-1 a Genetic symbols are as described by Bachmann and Low (2). Symbols denoting plasmid traits (or suspected plasmid traits) are those recommended by Novick et al. (19), with the exception of Cu, which denotes cupric ion resistance. The phenotypic abbreviations Nall and Rifr indicate resistance to nalidixic acid and rifampin, respectively. RJ100

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after the addition of CuSO4, MICs of 4 and 8 mM CuSO4 were observed for copper-sensitive (Cus) E. coli K-12, and Cur E. coli strains, respectively. Adjustment of NA to pH 7.5 after the addition of CuSO4 was found to reduce the toxicity of CuSO4 for all E. coli strains (Cur and Cus) tested, but increases in MIC were more marked for Cur than for Cu5 strains. MICs of CuSO4 for E. coli K-12 strains were increased to 6 mM, whereas Cur isolates RJ92 through RJ95 and Cur E. coli K-12 strains harboring pRJ1004 (see below) were found to have MICs between 12 and 20 mM CuSO4. Thus, pH adjustment of CuSO4containing NA facilitated differentiation between Cur and Cus E. coli strains. MICs of CuSO4 varied considerably according to the composition of the medium into which CuSO4 was incorporated. When Isosensitest agar (Oxoid CM471) was substituted for NA (and pH adjusted), significantly higher MICs were obtained for both Cur and Cu5 E. coli strains (20 and 10 mM, respectively). Substitution of Oxoid L21 yeast extract for the less nutritionally-rich Oxoid L20 yeast extract in CuS04-containing NA also resulted in higher MICs of CuSO4 being obtained for all E. coli strains tested. In the experiments described in this paper, CuS04-containing NA was always prepared with Oxoid L20 yeast extract. Conjugative transfer of the determinant conferring the Cur phenotype. To determine whether the genetic determinants of copper resistance in isolates RJ92 through RJ95 might be carried on transmissible plasmids, these Cur strains were mated with the E. coli K-12 strain J53-1. After overnight incubation at 37 or 25°C, the mating mixtures were plated onto NA containing 7.5 mM CuSO4 and nalidixic acid. Transfer of a Cur determinant to J53-1 at 37 or 25°C was detected only in matings involving RJ92. In matings of a shorter duration (30 min) at 37°C, transfer of the Cur determinant from RJ92 to J53-1 occurred at a frequency of 4.0 x 10-6. Consistent cotransfer of the unselected markers Tcr and Sur from RJ92 was not observed. Tests of eight Cur transconjugants derived from a 30-min mating between RJ92 and J53-1 revealed that four were Tcr and none was Sur. Table 2 shows the frequencies at which the Cur determinant, originally present in RJ92, was transferred between various E. coli K-12 strains. The similar frequencies at which the Cur determinant was found to transfer from RJ97 into recipients J53-1 and J53-1 recA are consistent with the Cur determinant being carried on a plasmid (hereafter designated pRJ1004). Molecular weight of pRJ1004. Preliminary agarose gel electrophoresis experiments indicated that RJ92 contained four different-sized plas-

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mid species. The molecular weight of the largest (i.e., the slowest migrating) of these four plasmid species was estimated to be between 70,000 and 90,000. Electrophoresis of plasmid DNA isolated from the Cur transconjugant RJ97 indicated that RJ97 harbored only this largest of the four plasmid species present in RJ92. Plasmid DNA purified from RJ97 was examined by electron microscopy. Visual inspection of at least 200 open-circular DNA molecules on electron microscope grids did not reveal any that were significantly different in size from those which were measured. Contour length measurements were made of 16 open-circular DNA molecules and 37 linear molecules of the A c1857 S DNA (molecular weight, 32,500), which was used as the standard. These measurements enabled the molecular weight of pRJ1004 to be estimated at 78,000 (standard deviation, ±1,800). Transformation with pRJ1004 DNA. Plasmid DNA isolated from strain RJ97 was used to transform the recombination-deficient, modification-deficient E. coli K-12 recipient ED8739. The resulting Cur transformant RJ99 was found to transfer the Cur determinant to E. coli K-12 recipients W3110-1 and J53-1 at frequencies similar to those obtained when strains RJ96 and RJ97 were used as donors (Table 2). Agarose gel electrophoresis of plasmid DNA isolated from the Cur transformant RJ99 and from the two Cur transconjugants RJ100 and RJ101 indicated that these three strains each contained only one plasmid species, the molecular weight of which was approximately 78,000. On the basis of these results, it was concluded that pRJ1004 was a conjugative plasmid. Search for additional genetic markers on TABLE 2. Plasmid transfer frequencies for

pRJl004a Donor strain

Recipient strain

Frequency of transfer

9.0 x 10-6 7.7 x 10-5 1.1 X 1 -4 RJ97 2.6 x 10-6 RJ99 1.2 x 10-4 RJ99 a Genotypes and phenotypes of donor and recipient strains are shown in Table 1. Transfer frequencies are expressed as the number of Cur transconjugants per donor cell obtained in 30-min matings at 37°C. Spontaneous mutation of the donor strains to nalidixic acid or rifampin resistance was detected at an average frequency of 10' per donor cell during these matings. Despite numerous platings of overnight NB cultures of E. coli strains W3110-1, J53-1, and J53-1 recA onto NA containing 7.5 mM CUS04, no spontaneous Cur mutants of these recipient strains were detected.

RJ96 RJ97

W3110-1 J53-1 J53-1 recA W3110-1 J53-1

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TABLE 3. Incompatibility of standard R plasmids with pRJ1004 No. of IncompatiFrerecipient clones bility quency of Recipient straina group transfer" containing plasinids

2 x 10-5 W3110-1c 1 x 10-5 20/20 C W3110-1(R40a) 4 x 10-5 20/20 FII W3110-1(Rldrd-16)