Antonie van Leeuwenhoek 61: 333-337, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Plasmid-determined resistance to arsenic and antimony in

Pseudomonas aeruginosa Carlos Cervantes & Jaime Ch~ivez lnstituto de lnvestigaciones Quimico-Biol6gicas, Universidad Michoacana, Morelia, Mich., M~xico Received 26 September 1991; accepted in revised form 13 February 1992

Key words: arsenate resistance, arsenite resistance, plasmid, Pseudomonas Abstract

Resistance to arsenic salts in a Pseudomonas aeruginosa clinical isolate was shown to be determined by a 100kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protected P. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.

Introduction

Plasmids conferring resistance to heavy metals are frequently found in a diversity of bacterial species of different origins (Silver and Misra, 1988; Misra et al., 1989). Plasmid-mediated resistance to arsenic ions is a common trait in many R-factors from both gram negative and gram positive bacteria (Novick and Roth, 1968; Hedges and Baumberg, 1973; Efstathiou and McKay, 1977; Nakahara et al., 1977; Smith, 1978; Summers et al., 1978; Gotz et al., 1983; Silver and Nakahara, 1983; CervantesVega et al., 1986). The resistance spectrum of the ars operon in Escherichia coli and Staphylococcus aureus includes arsenate, arsenite and antimonite, which are all inducers of the operon (Silver et al., 1981). The mechanism of resistance to arsenic proved to be an accelerated efflux of the toxic ions (Silver and Keach, 1982; Mobley and Rosen, 1982; Rosen and Borbolla, 1984). Nucleotide sequence analysis of the cloned ars operon from E. coli revealed that arsenate and arsenite efflux is carried out by a membrane translocation channel (ArsB) energized by a membrane ATPase (ArsA) (Chen et

al., 1986; Rosen et al., 1988; Silver et al., 1989; Tisa and Rosen, 1990). In this initial report we describe the properties of the arsenic and antimony resistances from Pseudomonas aeruginosa plasmid pUM310.

Materials and methods

Strains The origin of the Pseudomonas aeruginosa clinical strains has been described (Cervantes-Vega et al., 1986). P. aeruginosa PU21 (FP-, ilv, leu, Str r, Rif r) was a gift from G. Jacoby (Massachusetts General Hospital, Boston, MA). P. aeruginosa strains bearing plasmids R3108 (420 kb) or FP2 (90 kb), used as molecular size standards, were kindly provided by B. Holloway (Monash University, Clayton, Victoria, Australia). Conjugation Log phase cultures of the clinical isolates grown at 37~ were mixed 1:1 (vol/vol) with overnight cultures of PU21 grown at 43~ (Holloway, 1965).

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with varying concentrations of the toxic ions were incubated for 18h and turbidity was determined. Na2HAsO4, NaAsO2 and SbCI3 (Merck Co.) were used as sources for the toxic ions. Induced cultures were treated for l h with subinhibitory amounts of the inducing ion and then challenged with an inhibitory concentration of the tested ion. Uninduced cultures were similarly challenged but not pretreated. Turbidity was measured at intervals.

Results and discussion

Fig. 1. Agarose gel electrophoresis of plasmids isolated from Pseudomonas aeruginosa strains. A, mixture of molecularsize standard plasmidsR3108 (420kb) and FP2 (90 kb); B, plasmidless PU21; C, clinical isolate PSS08; D, transconjugant PU21 (pUM310). Numbersindicatemolecularsize in kilobases. CHR marks the position of chromosomalDNA. Plasmidswere separated in 0.7% agarose at 120V for 3 h, stained with ethidium bromide and photographed under UV illumination.

After static incubation at 43~ for 24 h, transconjugants were selected on nutrient agar plates containing 500/xg/ml rifampicin and 20 mg/ml sodium arsenate. Plasmid analysis Plasmid D N A was isolated by the alkaline lysis procedure of Casse et al. (1979). Plasmids were separated by gel electrophoresis in 0.7% agarose in Tris-borate buffer (Meyers et al., 1976) at 120 V for 3 h. Bands were stained with ethidium bromide and photographed under UV illumination. Growth experiments Assays were routinely initiated by diluting 1:50 cultures grown overnight at 37 ~C with aeration in nutrient broth (Bioxon, M6xico). Turbidity was measured with a Bausch & Lomb spectrophotometer at 590 nm. For susceptibility testing, cultures

In a collection of P. aeruginosa clinical isolates we found several arsenic resistant strains able to transfer the resistance phenotype to P. aeruginosa strain PU21 (Cervantes-Vega et al., 1986). Clinical strain PSS08 was chosen for further study. By agarose gel electrophoresis, PSS08 showed two plasmid bands of 45kb and 100kb (Fig. 1). Arsenic-resistant transconjugants contained only the 100 kb plasmid, designated pUM310, which was absent in the recipient strain PU21 (Fig. 1). Thus, arsenic resistance was assigned to plasmid pUM310. It can be seen from Fig. 2 that PU21 (pUM310) strain showed resistance to arsenate, arsenite and antimony ions as compared to the plasmid-less sensitive strain PU21 that was inhibited at lower ion concentrations. PU21 (pUM310) was however inhibited by 5 mg/ml arsenite (Fig. 2B), 200ug/ml antimony (Fig. 2C) and by 40 mg/ml arsenate (data not shown). The resistance spectrum was similar to those found in E. coli and S. aureus (Silver et al., 1981). When comparing the levels of ion sensitivity in P. aeruginosa strains (Fig. 2) and those reported for E. coli and S. aureus (Silver et al., 1981) some differences were observed: S. aureus strains were much more sensitive to arsenate, arsenite and antimony than both E. coli and P. aeruginosa. On the other hand, E. coli cells were more sensitive to arsenate and antimony, but similarly sensitive to arsenite, than P. aeruginosa. Therefore, P. aeruginosa seemed to be generally more resistant to arsenic and antimony than E. coli and S. aureus. Resistance to arsenate and arsenite in PU21 (pUM310) was induced by previous exposure to subinhibitory concentrations of either of the two

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Fig. 2. Susceptibility to arsenic and antimony in P. aeruginosa PU21 (O) and PU21 (pUM310) (O). Cells were grown at, 7 C with aeration for 18 h in nutrient broth with the indicated concentrations of the salts and turbidity was measured. A. sodium arsenate: B, sodium arsenite; C, antimony chloride.

anions (Fig. 3). Induction of arsenate resistance by arsenite and of arsenite resistance by arsenate was equally efficient as compared to the induction with

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the same ion (Fig. 3). In E. coli, the ars operon consists of two distinct regions, one encoding arsenate resistance and another encoding arsenite and

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Fig. 3. Induction of arsenate and arsenite resistances in P. aeruginosa PU21 (pUM310). Control with no additions (O): uninduced cultures with 40 mg/ml sodium arsenate (A) or 0.6 mg/ml sodium arsenite (B) added at time zero (O): induced cultures pretreated for 1 h with either 4 mg/ml sodium arsenate ( I ) or 0.06 mg/ml sodium arsenite (El) followed by the addition of 40 mg/ml sodium arsenate (A) or 0.6 mg/ml sodium arsenite (B).

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Fig. 4. Effectof phosphate on arsenate and arsenite toxicityin P. aeruginosa PU21(pUM310).Controlwith no additions (O); cultures with 40mg/ml sodium arsenate (A) or 0.6mg/ml sodium arsenite (B) with (11)or without (0) 10mg/mlsodiumphosphate.

antimony resistances (Chen et al., 1985). Resistance to both arsenate and arsenite was clearly induced by antimony (data not shown) but we were unable to show induction of antimony resistance by any ion because growth inhibition of P. aeruginosa cultures by antimony was not complete unless very high antimony concentrations were used. Silver et al. (1981) reported similar problems trying to induce antimony resistance in S. aureus but finally demonstrated induction using a minimal medium. By its chemical similarity with phosphate, arsenate is usually taken up by the bacterial phosphate transport systems (Willsky and Malamy, 1980), phosphate being a competitive inhibitor of arsenate uptake (Silver, 1978). Accordingly, we found that phosphate reversed the toxic effects of arsenate (at 40 mg/ml) against PU21 (pUM310), even at a 3-times lower phosphate concentration (i.e. 215 mM arsenate and 70 mM phosphate; Fig. 4A). However, phosphate was unable to protect PU21 (pUM310) from arsenite (at 0.6mg/ml) even at a

15-times higher phosphate concentration (4.6 mM arsenite and 70mM phosphate; Fig. 4B). Similar results were obtained with plasmid-less sensitive PU21 strain (data not shown). These data corroborate the previous finding that arsenite is not taken up by the phosphate (arsenate) transport pathway (Silver et al., 1981). Our results show that the properties of plasmidmediated resistance to arsenic and antimony ions in P. aeruginosa are comparable to those reported in E. coli and S. aureus (Silver et al., 1981), thus suggesting that a similar mechanism of resistance, presumably involving arsenic ions efflux, is probably functioning in P. aeruginosa plasmid pUM310.

Acknowledgements We thank S. Silver for helpful comments. This work was partially supported by Coordinaci6n de la Investigaci6n Cientffica, UMSNH.

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