Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110, Kuwait
Received 2 December 2002; returned 24 December 2002; revised 2 February 2003; accepted 3 February 2003
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Abstract |
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Materials and methods: Antibiotic resistance was detected by disc diffusion. The Etest was used to determine mupirocin MIC. The presence of mupA was detected by PCR using specific primers. Curing, transfer experiments, pulsed-field gel electrophoresis (PFGE) and DNA hybridization were used to study the genetic location of mupA.
Results: The isolates had mupirocin MICs > 1024 mg/L and were resistant to methicillin, gentamicin, kanamycin, streptomycin, erythromycin, tetracycline, ciprofloxacin, cadmium acetate, propamidine isethionate and ethidium bromide. They carried two plasmids of 26 and 2.8 kb. Curing and transfer experiments demonstrated that the 26 kb plasmid encoded resistance to cadmium acetate, propamidine isethionate and ethidium bromide. Loss of mupirocin resistance corresponded to the loss of a 40 kb DNA fragment from a 175 kb SmaI chromosomal fragment. The mupA gene was detected only in the genomic DNA of the mupirocin-resistant strains and in their derivatives cured of the 26 kb plasmid. A labelled mupA probe hybridized to the 175 kb SmaI fragment only in the mupirocin-resistant isolates.
Conclusion: The absence of mupA on any of the plasmids and its detection only in the chromosomal DNA of the parents and in their derivatives cured of the 26 kb plasmid strongly supports a chromosomal location for mupA in these isolates.
Keywords: mupA, chromosomal resistance, Staphylococcus aureus
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Introduction |
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High-level, mupirocin-resistant, methicillin-resistant S. aureus (MRSA) were first isolated from patients in the Burns Unit of Ibn Sina Hospital, in Kuwait in November 1996.7 This resistance was mediated by mupA located on a 38 kb conjugative plasmid but was not detected on the genomic DNA of low-level mupirocin-resistant MRSA isolates from the same hospital.10 In a continuing effort to monitor MRSA isolates from the Burns Unit for mupirocin resistance, we identified high-level mupirocin-resistant MRSA isolates that lacked the 38 kb conjugative mupA plasmid. These isolates were studied for the genetic location of their high-level mupirocin resistance determinants.
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Materials and methods |
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Fourteen MRSA isolates expressing high-level mupirocin resistance were isolated from patients in the Burns Unit, Ibn Sina Hospital in Kuwait, between January and February 2000. Strains WBG18765 and WBG5415 were used as recipients in mixed-culture transfer (MCT) and conjugation experiments, respectively. Strains WBG541 and WBG1876 were provided by Professor W. B. Grubb (Curtin University of Technology, Perth, Australia).
Susceptibility to antimicrobial agents
Susceptibility to antimicrobial agents was tested by disc diffusion on MuellerHinton agar as described previously.7 The MICs of mupirocin were determined using Etest strips (AB Biodisk, Solna, Sweden) according to the manufacturers instructions. S. aureus strain ATCC 25923 was used as a control strain. Mupirocin powder was a gift from SmithKline Beecham, Worthing, UK.
Plasmid isolation and analysis
Plasmid isolation and curing of resistance determinants were carried out as described previously.7 Plasmid transfer was investigated in conjugation and in MCT experiments as described previously using strains WBG541 and WBG1876 as respective recipient strains.10
Pulsed-field gel electrophoresis (PFGE)
PFGE of SmaI-digested genomic DNA was carried out as described previously.7
Amplification of mupA
PCR was carried out using previously published primers and conditions.8,10 Mup1 (5'-CCCATGGCTTACCAGTTGA-3') and Mup2 (5'-CCATGGAGCACTATCCGAA-3') were used to generate a 1.5 kb product. The primers were purchased from Gibco-BRL Custom primers (Life Technologies, UK). Genomic DNA isolated from S. aureus strain ATCC 25923 was used as a negative control, whereas DNA from the mupirocin resistance plasmid pXU124 was used as a positive control.
Southern blot analysis
The 1.5 kb PCR product from isolate 1061 was purified by elution from low melting point agarose following treatment with agarase (Roche Applied Science, Mannheim, Germany) according to the manufacturers instructions and labelled by the random priming method with digoxigenin-11-dUTP using the DIG High Prime DNA labelling and detection kit (Roche Applied Science), to produce a mupA gene probe. PFGE-separated SmaI-digested genomic DNA was transferred to a nylon membrane (Roche Applied Science) by Southern blotting. DNA hybridization, post-hybridization washes and detection were carried out using the High Prime DNA labelling and detection kit following instructions provided by the kit manufacturer (Roche Applied Sciences). Autoradiographs were obtained with Kodak X-OMAT AR film.
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Results |
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Loss of resistance determinants following growth at 43.5°C
The results of curing experiments are summarized in Table . Loss of resistance to cadmium acetate, propamidine isethionate and ethidium bromide was accompanied by the loss of the 26 kb plasmid. High-level mupirocin resistance was lost in up to 2% of the colonies screened, but this was not accompanied by plasmid loss. One colony, designated 1061B, lost resistance to cadmium acetate, propamidine isethionate, ethidium bromide and mupirocin, but still lost only the 26 kb plasmid (Table
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Transfer of resistance determinants
None of the three isolates transferred mupirocin resistance in conjugation and MCT experiments. However, they all co-transferred resistance to cadmium acetate, propamidine isethionate and ethidium bromide, together with the 26 kb plasmid in MCT experiments with selection for any of the three compounds. Since high-level mupirocin resistance was lost on curing and since it has been reported previously on non-conjugative plasmids,3,5,6 it could be located on the 26 kb plasmid. Failure to cotransfer it with resistance to cadmium acetate, propamidine isethionate and ethidium bromide could be due to its transposition or deletion during transfer. To establish whether this was the case, the 26 kb plasmids in the parent MRSA isolates and in the MCT transcipients were compared by EcoRI restriction analysis. There were no differences in size or restriction patterns of the plasmids from both backgrounds (data not shown).
Amplification and location of mupA
All of the 14 parental isolates and the mupirocin-resistant derivatives of 1061, 1063 and 1065 cured of the 26 kb plasmid contained mupA by PCR, but mupA was not detected in any of the mupirocin-susceptible derivatives (Table ). When isolates 1061, 1063 and 1065 and their mupirocin-susceptible derivatives were compared by PFGE, a novel 135 kb SmaI fragment in the mupirocin-susceptible strains replaced a 175 kb fragment present in the parental isolates (Figure 1a). Hence, the loss of mupirocin resistance corresponded to the loss of a 40 kb DNA fragment from the parental isolate.
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Discussion |
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It is well established that mupA encodes plasmid-borne high-level mupirocin resistance1,2 but it has also been reported on the chromosome of low-level mupirocin-resistant S. aureus.8,9 This is the first report of its location on the chromosomal DNA of high-level mupirocin-resistant MRSA isolates. The size of DNA deleted in the mupirocin-susceptible derivatives (40 kb) is similar to the size of the conjugative mupA plasmid (38 kb) in MRSA isolated previously in the Burns Unit.7,10 Further work is needed to determine whether the chromosomal mupA in these isolates resulted from the integration of a mupirocin resistance plasmid into the S. aureus chromosome.
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Acknowledgements |
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Footnotes |
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References |
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2 . Hogson, J. E., Curnock, S. P., Dyke, K. G. H., Morris, D., Sylvester, D. R. & Gross, M. S. (1994). Molecular characterization of the gene encoding high-level mupirocin resistance in Staphylococcus aureus J2870. Antimicrobial Agents and Chemotherapy 38, 12058.[Abstract]
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7 . Udo, E. E., Farook, V. S., Mokadas, E. M., Jacob, L. E. & Sanyal, S. C. (1999). Molecular fingerprinting of mupirocin-resistant Staphylococcus aureus from a Burn unit. International Journal of Infectious Disease 3, 827.[CrossRef]
8 . Ramsey, M. A., Bradley, S. F., Kauffman, C. A. & Morton, T. A. (1996). Identification of chromosomal location of mupA gene, encoding low-level mupirocin resistance in staphylococcal isolates. Antimicrobial Agents and Chemotherapy 40, 28203.[Abstract]
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Fujimura, S., Watanabe, A. & Beighton, D. (2001). Characterization of the mupA gene in strains of methicillin-resistant Staphylococcus aureus with low level of resistance to mupirocin. Antimicrobial Agents and Chemotherapy 45, 6412.
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Udo, E. E., Jacob, L. E. & Mathew, B. (2001). Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance. Journal of Medical Microbiology 50, 90915.