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A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923
FEMS Microbiology Letters 189 (2000) 79^80
www.fems-microbiology.org
A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923 Edet E. Udo *, Latha E. Jacob, Bindu Mathew Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait Received 20 March 2000; received in revised form 1 June 2000; accepted 2 June 2000
Abstract A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cadmium resistance; cadA; ATCC25923; S. aureus
Cadmium resistance is widespread in many bacterial species. In Staphylococcus aureus cadmium resistance is mediated by di¡erent genes. Two of these genes, cadA and cadB have been studied extensively [1^3]. The cadA gene confers high level resistance by an energy-dependent e¥ux mechanism [2,3], is usually located on large plasmids such as pI258 [1,2] that also encode penicillinase production and resistance to other heavy metal ions [1,4], aminoglycosides [4] and nucleic acid binding compounds [5]. The cadB gene confers low level cadmium resistance and has been demonstrated on a large plasmid, pII147 [1] and on small multicopy plasmids [6]. A chromosomal cadmium resistance determinant that is di¡erent from cadA and cadB has also been reported [7]. This paper reports a plasmid encoded high level cadmium resistance in S. aureus strain ATCC25923. S. aureus strain ATCC25923 was isolated from a clinical sample in 1945 [8]. It is recommended for use as a reference strain for disk susceptibility testing of S. aureus [9,10] and is widely used in clinical microbiology laboratories as a control strain for the disk susceptibility testing of S. aureus. In addition to antibiotic resistance testing, we routinely test S. aureus isolates for resistance to heavy metal ions such as cadmium, arsenate, arsenite, mercuric chloride, phenylmercuric acetate, propamidine isothionate and ethidium bromide. While using S. aureus strain
* Corresponding author. Fax: +965 (533) 2719 ; E-mail : [email protected]
ATCC25923 as a control with disk di¡usion tests, it was observed to be resistant to cadmium. The result was con¢rmed by testing S. aureus ATCC25923 from three di¡erent sources (Mast Diagnostics, Oxoid Laboratories, the CDC, Atlanta, USA), for susceptibility to cadmium and other compounds. The strains from all three sources were resistant to cadmium but susceptible to arsenate, arsenite, mercuric chloride, phenylmercuric acetate, propamidine isothionate and ethidium bromide. It did not produce penicillinase when tested with nitroce¢n (Oxoid, Basingstoke, England) and was susceptible to methicillin, penicillin G, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, chloramphenicol, tetracycline, trimethoprim, cipro£oxacin, mupirocin, rifampicin, fusidic acid, vancomycin and teicoplanin. The MIC of cadmium sulfate determined by the macro broth dilution method with cadmium concentrations ranging from 0.5 to 512 mg l31 was 256 mg l31 for S. aureus strains ATCC25923 and WBG516 (carrying pI258) and 4 mg l31 for RN450 (a plasmid free isolate that is susceptible to heavy metals and antibiotics [4]). Plasmid analysis [4] revealed that S. aureus strain ATCC25923 contained a 25.9-kb plasmid. In curing experiments, four of 96 colonies lost cadmium resistance together with the 25.9-kb plasmid. In order to con¢rm that the 25.9-kb plasmid encoded cadmium resistance, ATCC25923 was used as a donor in mixed culture transfer experiments with an S. aureus recipient strain WBG1876 as described previously [4]. ATCC25923 transferred cadmium resistance to WBG1876 at an average frequency of
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 5 6 - 1
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E.E. Udo et al. / FEMS Microbiology Letters 189 (2000) 79^80
testing it against a known incompatibility group 1 plasmid, pWBG115, as described previously [5]. The results demonstrated that pXU5 belongs to the same S. aureus incompatibility group as pI258. These results have demonstrated that plasmid pXU5, present in S. aureus strain ATCC25923, contained sequences homologous to the cadA and encoded high level cadmium resistance but not resistance to mercuric chloride, arsenate, arsenite, antibiotics or penicillinase production. The presence of plasmid pXU5 in S. aureus strain ATCC25923 isolated in 1945 suggests that cadmium resistance plasmids existed in S. aureus before the appearance of penicillin resistance probably due to the presence of toxic heavy metals in the environment before the introduction and clinical use of penicillins. Acknowledgements This study was supported by grant MI091 from Kuwait University Research Administration.
Fig. 1. Hybridisation of pXU5 with a cadA probe. A: Lane 1, HindIII digests of lambda DNA. Sizes are in kb. Lane 2, EcoRI digest of pI258, sizes in kb from the top are 12.2, 7.2, 6.6, 2.2. Lane 3, EcoRI digest of pXU5. Sizes in kb from the top are 6.9, 4.8, 4.2, 3.4, 2.7, 2.3, 1.6. B: Autoradiograph of plasmids in panel A after hybridisation with a DIG-II-dUTP labelled cadA probe. The probe hybridised to the 2.3-kb EcoRI fragment of pXU5 and a large EcoRI fragment of pI258.
6.2U1036 transcipients per donor and representative transcipients analysed for plasmids contained a 25.9-kb plasmid, pXU5. The presence of the cadA gene on pXU5 was investigated in DNA hybridisation experiments. The 3.7-kb XbaI^BgllI cadA probe was recovered from pI258 [2] in 1% low melting point agarose after agarase treatment as instructed by the manufacturer (Boehringer Mannheim), labelled by the random primed method using DIG-IIdUTP (Boehringer Mannheim) and used to probe EcoRI digested plasmid pXU5. DNA homology was detected with a chemiluminescent substrate CSPD0 (Boehringer Mannheim) according to the manufacturer's protocol. As shown in Fig. 1B, the cadA probe hybridised to a large EcoRI fragment of pI258 and to the 2.3-kb fragment of pXU5 indicating that it contains DNA homologous to cadA. The incompatibility group of pXU5 was determined by
References [1] Shalita, Z., Murphy, E. and Novick, R.P. (1980) Penicillinase plasmids of Staphylococcus aureus: structural and evolutionary relationships. Plasmid 3, 291^311. [2] Nucifora, G., Chu, L., Misra, T.K. and Silver, S. (1989) Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA results from a cadmium-e¥ux ATPase. Proc. Natl. Acad. Sci. USA 86, 3544^3548. [3] Silver, S. and Phung, L.T. (1996) Bacterial heavy metal resistance: new surprises. Annu. Rev. Microbiol. 50, 753^789. [4] Udo, E.E. and Grubb, W.B. (1991) Transfer of resistance determinants from a multi-resistant Staphylococcus aureus isolate. J. Med. Microbiol. 35, 72^79. [5] Townsend, D.E., Ashdown, N. and Grubb, W.B. (1985) Evolution of Australian isolates of methicillin-resistant Staphylococcus aureus: a problem of incompatibility? J. Med. Microbiol. 20, 49^61. [6] El-Solh, N. and Ehrlich, S.D. (1982) A small cadmium resistance plasmid isolated from Staphylococcus aureus. Plasmid 7, 77^84. [7] Witte, W., Green, L., Misra, T.K. and Silver, S. (1986) Resistance to mercury and to cadmium in chromosomally resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 29, 663^669. [8] Staphylococcus aureus ATCC25923. Product Information Sheet. American Type Culture Collection, Manassas, VA, USA. [9] WHO Expert Committee on Biological Standardization, 28 Report (1977) WHO Technical Report Series 610, pp. 122^123. [10] National Committee of Clinical Laboratory Standards (1999) Performance Standards for Antimicrobial Disk Susceptibility Testing. National Committee for Clinical Laboratory Standards, Wayne, PA.
FEMSLE 9483 14-7-00
www.fems-microbiology.org
A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923 Edet E. Udo *, Latha E. Jacob, Bindu Mathew Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait Received 20 March 2000; received in revised form 1 June 2000; accepted 2 June 2000
Abstract A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cadmium resistance; cadA; ATCC25923; S. aureus
Cadmium resistance is widespread in many bacterial species. In Staphylococcus aureus cadmium resistance is mediated by di¡erent genes. Two of these genes, cadA and cadB have been studied extensively [1^3]. The cadA gene confers high level resistance by an energy-dependent e¥ux mechanism [2,3], is usually located on large plasmids such as pI258 [1,2] that also encode penicillinase production and resistance to other heavy metal ions [1,4], aminoglycosides [4] and nucleic acid binding compounds [5]. The cadB gene confers low level cadmium resistance and has been demonstrated on a large plasmid, pII147 [1] and on small multicopy plasmids [6]. A chromosomal cadmium resistance determinant that is di¡erent from cadA and cadB has also been reported [7]. This paper reports a plasmid encoded high level cadmium resistance in S. aureus strain ATCC25923. S. aureus strain ATCC25923 was isolated from a clinical sample in 1945 [8]. It is recommended for use as a reference strain for disk susceptibility testing of S. aureus [9,10] and is widely used in clinical microbiology laboratories as a control strain for the disk susceptibility testing of S. aureus. In addition to antibiotic resistance testing, we routinely test S. aureus isolates for resistance to heavy metal ions such as cadmium, arsenate, arsenite, mercuric chloride, phenylmercuric acetate, propamidine isothionate and ethidium bromide. While using S. aureus strain
* Corresponding author. Fax: +965 (533) 2719 ; E-mail : [email protected]
ATCC25923 as a control with disk di¡usion tests, it was observed to be resistant to cadmium. The result was con¢rmed by testing S. aureus ATCC25923 from three di¡erent sources (Mast Diagnostics, Oxoid Laboratories, the CDC, Atlanta, USA), for susceptibility to cadmium and other compounds. The strains from all three sources were resistant to cadmium but susceptible to arsenate, arsenite, mercuric chloride, phenylmercuric acetate, propamidine isothionate and ethidium bromide. It did not produce penicillinase when tested with nitroce¢n (Oxoid, Basingstoke, England) and was susceptible to methicillin, penicillin G, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, chloramphenicol, tetracycline, trimethoprim, cipro£oxacin, mupirocin, rifampicin, fusidic acid, vancomycin and teicoplanin. The MIC of cadmium sulfate determined by the macro broth dilution method with cadmium concentrations ranging from 0.5 to 512 mg l31 was 256 mg l31 for S. aureus strains ATCC25923 and WBG516 (carrying pI258) and 4 mg l31 for RN450 (a plasmid free isolate that is susceptible to heavy metals and antibiotics [4]). Plasmid analysis [4] revealed that S. aureus strain ATCC25923 contained a 25.9-kb plasmid. In curing experiments, four of 96 colonies lost cadmium resistance together with the 25.9-kb plasmid. In order to con¢rm that the 25.9-kb plasmid encoded cadmium resistance, ATCC25923 was used as a donor in mixed culture transfer experiments with an S. aureus recipient strain WBG1876 as described previously [4]. ATCC25923 transferred cadmium resistance to WBG1876 at an average frequency of
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 5 6 - 1
FEMSLE 9483 14-7-00
80
E.E. Udo et al. / FEMS Microbiology Letters 189 (2000) 79^80
testing it against a known incompatibility group 1 plasmid, pWBG115, as described previously [5]. The results demonstrated that pXU5 belongs to the same S. aureus incompatibility group as pI258. These results have demonstrated that plasmid pXU5, present in S. aureus strain ATCC25923, contained sequences homologous to the cadA and encoded high level cadmium resistance but not resistance to mercuric chloride, arsenate, arsenite, antibiotics or penicillinase production. The presence of plasmid pXU5 in S. aureus strain ATCC25923 isolated in 1945 suggests that cadmium resistance plasmids existed in S. aureus before the appearance of penicillin resistance probably due to the presence of toxic heavy metals in the environment before the introduction and clinical use of penicillins. Acknowledgements This study was supported by grant MI091 from Kuwait University Research Administration.
Fig. 1. Hybridisation of pXU5 with a cadA probe. A: Lane 1, HindIII digests of lambda DNA. Sizes are in kb. Lane 2, EcoRI digest of pI258, sizes in kb from the top are 12.2, 7.2, 6.6, 2.2. Lane 3, EcoRI digest of pXU5. Sizes in kb from the top are 6.9, 4.8, 4.2, 3.4, 2.7, 2.3, 1.6. B: Autoradiograph of plasmids in panel A after hybridisation with a DIG-II-dUTP labelled cadA probe. The probe hybridised to the 2.3-kb EcoRI fragment of pXU5 and a large EcoRI fragment of pI258.
6.2U1036 transcipients per donor and representative transcipients analysed for plasmids contained a 25.9-kb plasmid, pXU5. The presence of the cadA gene on pXU5 was investigated in DNA hybridisation experiments. The 3.7-kb XbaI^BgllI cadA probe was recovered from pI258 [2] in 1% low melting point agarose after agarase treatment as instructed by the manufacturer (Boehringer Mannheim), labelled by the random primed method using DIG-IIdUTP (Boehringer Mannheim) and used to probe EcoRI digested plasmid pXU5. DNA homology was detected with a chemiluminescent substrate CSPD0 (Boehringer Mannheim) according to the manufacturer's protocol. As shown in Fig. 1B, the cadA probe hybridised to a large EcoRI fragment of pI258 and to the 2.3-kb fragment of pXU5 indicating that it contains DNA homologous to cadA. The incompatibility group of pXU5 was determined by
References [1] Shalita, Z., Murphy, E. and Novick, R.P. (1980) Penicillinase plasmids of Staphylococcus aureus: structural and evolutionary relationships. Plasmid 3, 291^311. [2] Nucifora, G., Chu, L., Misra, T.K. and Silver, S. (1989) Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA results from a cadmium-e¥ux ATPase. Proc. Natl. Acad. Sci. USA 86, 3544^3548. [3] Silver, S. and Phung, L.T. (1996) Bacterial heavy metal resistance: new surprises. Annu. Rev. Microbiol. 50, 753^789. [4] Udo, E.E. and Grubb, W.B. (1991) Transfer of resistance determinants from a multi-resistant Staphylococcus aureus isolate. J. Med. Microbiol. 35, 72^79. [5] Townsend, D.E., Ashdown, N. and Grubb, W.B. (1985) Evolution of Australian isolates of methicillin-resistant Staphylococcus aureus: a problem of incompatibility? J. Med. Microbiol. 20, 49^61. [6] El-Solh, N. and Ehrlich, S.D. (1982) A small cadmium resistance plasmid isolated from Staphylococcus aureus. Plasmid 7, 77^84. [7] Witte, W., Green, L., Misra, T.K. and Silver, S. (1986) Resistance to mercury and to cadmium in chromosomally resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 29, 663^669. [8] Staphylococcus aureus ATCC25923. Product Information Sheet. American Type Culture Collection, Manassas, VA, USA. [9] WHO Expert Committee on Biological Standardization, 28 Report (1977) WHO Technical Report Series 610, pp. 122^123. [10] National Committee of Clinical Laboratory Standards (1999) Performance Standards for Antimicrobial Disk Susceptibility Testing. National Committee for Clinical Laboratory Standards, Wayne, PA.
FEMSLE 9483 14-7-00