1 National Creative Research Initiatives Center for ARS Network, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742; 2 Imagene Co., Ltd, Biotechnology Incubating Center, Seoul National University, Seoul 151-742; 3 Laboratory of Antimicrobial Resistant Pathogens, Department of Microbiology, National Institute of Health, Seoul 122-701, Korea
Received 16 September 2002; returned 4 December 2002; revised 26 December 2002; accepted 5 January 2003
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Abstract |
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Keywords: mupirocin, staphylococci, resistance mechanism
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Introduction |
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Mupirocin-resistant strains are divided into two groups: low- and high-level resistance (MICs 8256 and >256 mg/L, respectively).4 Low- and high-level resistance have been detected in both S. aureus and coagulase-negative staphylococci (CoNS).4 In most cases, low-level resistance to mupirocin is related to alterations in the host IRS.5,6 The clinical isolates resistant to a high level of mupirocin contain two distinct IRS enzymes: endogenous IRS plus an additional IRS encoded by the ileS-2 gene.7 This additional enzyme is usually encoded by transferable plasmids.811 The IRS encoded by the ileS-2 gene shares only 30% amino acid sequence similarity with the endogenous IRS of S. aureus.12
A survey of mupirocin susceptibility of Gram-positive pathogens isolated in Korea up to 1999 failed to detect mupirocin-resistant staphylococci.13 However, we considered it necessary to continue to monitor the emergence of the resistant strains, because the resistance rates of the reference antibiotics, such as methicillin and erythromycin, against Gram-positive bacteria are much higher than those of other countries,14 and mupirocin ointment has been used widely for the management of skin infections in Korea.
To determine the prevalence of mupirocin resistance in a Korean hospital, we investigated the rates of mupirocin resistance among the clinical isolates of staphylococci. The characteristics of the resistant strains were then analysed in terms of their resistance level and genotype.
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Materials and methods |
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A total of 523 clinical isolates of Gram-positive cocci, comprising 319 S. aureus and 204 CoNS, were collected from the Severance General Hospital, in Seoul, Korea, between January 2000 and January 2002. The strains were associated with bacteraemia, hospital-acquired pneumonia, or skin and soft tissue infections. Only one isolate per patient was used, in order to avoid strain duplication. In the antibiogram typing analysis according to NCCLS standards,15 these isolates showed different sensitivities to the tested drugs, including ciprofloxacin, kanamycin, tetracycline, vancomycin, erythromycin and methicillin, indicating that they are independent variants. The strains were stored in brainheart infusion broth plus 20% glycerol at 70°C until studied. Mupirocin was obtained from Hanmi Pharmaceutical Co., Ltd, Korea.
Determination of MICs
MICs were determined by a standardized agar dilution method with MuellerHinton agar.15 A microinoculator (Sakuma Co. Ltd, Tokyo, Japan) was used to inoculate the bacterial suspensions (104 cfu/spot). The stock solutions of test compounds were diluted in sterilized distilled water to give a serial, two-fold series, yielding final drug concentrations that ranged from 0.016 to 1024 mg/L. MIC determination was evaluated according to NCCLS standards.15 S. aureus ATCC 29213 was used as the control for the susceptibility test.
PCR amplification and sequence analysis of the ileS-2 gene
A G-spin genomic DNA extraction kit (INtRON Biotechnology, Korea) was used to isolate genomic DNA. To detect the ileS-2 gene, a 456 bp region in the ileS-2 gene was amplified by PCR, using the primers 5'-TATATTATGCGATGGAAGGTTGG-3' and 5'-AATAAAATCAGCTGGAAAGTGTTG-3'.16 PCR was performed with 30 cycles of denaturation at 95°C for 30 s, annealing at 45°C for 60 s and extension at 72°C for 60 s, by using 2U of Vent DNA polymerase (New England Biolabs, Beverly, MA, USA). The reaction products were analysed using 1.2% agarose gel electrophoresis. To confirm its identity, an entire sequence analysis of the ileS-2 gene was performed, in two randomly selected isolates of S. aureus highly resistant to mupirocin. The entire ileS-2 gene was amplified using two oligonucleotide primer pairs; one pair was Mup1 5'-CCCATGGCTTACCAGTTGA-3' and Mup2 5'-CCATGGAGCACTATCCGAA-3',17 and the other Mup3 5'-TTCGGATAG TGCTCCATG-3' and Mup4 5'-CCCCAGTTACACCGATAT-3'.18
Sequence analysis of the host ileS gene
Genomic DNA, isolated to detect the ileS-2 gene, was also used in this analysis. The primer pair SA ileS13D (5'-GATTTCCCAATGCGAGGTGGTTTACCAAACAAGGAACCGC-3') and SA ileS2833V (5'-CAACTTGTTGGCATCGTGGGATAGATGCGTCAATTCATC-3') was designed to amplify the entire coding sequence of the S. aureus ileS gene encoding the host IRS (GenBank accession no. X74219). The primer pair SE ileS10P (5'-GCCGAAAACTGATTTTCCTATGAGAGGTGGCTTACC-3') and SE ileS2436R (5'-CGTGCTTGTTCTAATGCACGGTTAACATCATCACG-3') was designed for the entire host IRS coding sequence of CoNS. (The sequence was submitted to GenBank, accession no. AF516209.) PCR was performed using 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 60 s and extension at 72°C for 90 s, by using 2U of Vent DNA polymerase.
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Results and discussion |
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The results are summarized in Table 1. Of the 319 clinical isolates of S. aureus, 237 (74.3%) were methicillin resistant, and of the 204 CoNS isolates, 163 (79.9%) were methicillin resistant. In S. aureus, high-level mupirocin resistance was detected in 16 (5%) of the isolates, of which 15 were methicillin resistant. Low-level mupirocin resistance was not detected.
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In the cases of both S. aureus and CoNS, the mupirocin resistance rates were higher in the methicillin-resistant than in the methicillin-susceptible isolates (P < 0.05). These results suggest that mupirocin resistance may be linked to an MRSA epidemic in the hospital. The connection between methicillin and mupirocin resistance has also been reported previously.3,8
ileS-2 gene in the high-level mupirocin-resistant strains
The ileS-2 gene was detected in all of the high-level resistant isolates (Table 2). Thus, the acquisition of an additional gene, ileS-2, appears to be the major mechanism for the high-level of mupirocin resistance in Korea. The ileS-2 gene was not detected in any of the low-level resistant strains. The entire sequence analysis of the ileS-2 gene, from the two isolates of the highly mupirocin-resistant S. aureus, showed that they are identical to those reported previously.12
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Point mutations in the host IRSs of the low-level resistant strains
A comparison of endogenous ileS sequences from highly mupirocin-resistant isolates with the published sequence (S. aureus ileS, GenBank accession no. X74219) revealed no point mutation. This confirmed that endogenous IRS is not responsible for high-level mupirocin resistance. The molecular reason for low-level resistance in CoNS was revealed when we isolated the structural gene encoding the host IRS from S. epidermidis and determined its DNA sequence. (The sequence was submitted to GenBank, accession no. AF516209.) A comparison of the ileS sequencessegregated from low-level mupirocin-resistant isolates of CoNSwith the wild-type sequence revealed substitutions at several sites. Many of the substitutions were silent and encoded the same amino acids (data not shown). Interestingly, all of the low-level mupirocin-resistant CoNS isolates contained a point mutation encoding the change Val-588 to Phe (Table 3). Val-588 is located seven amino acids upstream of the region encoding the evolutionarily conserved KMSKS motif. The same mutation was also reported in the host IRSs of low-level mupirocin-resistant S. aureus.5 The amino acid residues next to Val-588 in the staphylococcal IRSs are important for the interaction with mupirocin in other species.1,20 In fact, these residues were mutated in mupirocin-resistant IRSs in E. coli20 and Methanosarcina barkeri.21 In the crystal structure of S. aureus IRS with mupirocin, the amino group of Val-588 makes a specific hydrogen bond with the oxygen in the carbonyl ester group of mupirocin.22 Therefore, the amino acid change at Val-588 in CoNS is also believed to cause low-level resistance to mupirocin. In two strains, additional subsitutions were found at Arg-121 and Val-605 (Table 3); however, these residues are located far from the mupirocin binding region or active site, suggesting that they are not responsible for the resistance to mupirocin.
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Acknowledgements |
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Footnotes |
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References |
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2 . Sutherland, R., Boon, R. J., Griffin, K. E., Masters, P. J., Slocombe, B. & White, A. R. (1985). Antibacterial activity of mupirocin (pseudomonic acid), a new antibiotic for topical use. Antimicrobial Agents and Chemotherapy 27, 4958.[ISI][Medline]
3 . Schmitz, F. J. & Jones, M. E. (1997). Antibiotics for treatment of infections caused by MRSA and elimination of MRSA carriage. What are the choices? International Journal of Antimicrobial Agents 9, 119.[CrossRef][ISI]
4 . Cookson, B. D. (1998). The emergence of mupirocin resistance: a challenge to infection control and antibiotic prescribing practice. Journal of Antimicrobial Chemotherapy 41, 118.[Abstract]
5
.
Antonio, M., McFerran, N. & Pallen, M. J. (2002). Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 46, 43842.
6 . Farmer, T. H., Gilbart, J. & Elson, S. W. (1992). Biochemical basis of mupirocin resistance in strains of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 30, 58796.[Abstract]
7 . Morton, T. M., Johnston, J. L., Patterson, J. & Archer, G. L. (1995). Characterization of a conjugative staphylococcal mupirocin resistance plasmid. Antimicrobial Agents and Chemotherapy 39, 127280.[Abstract]
8
.
Leski, T. A., Gniadkowski, M., Skoczynska, A., Stefaniuk, E., Trzcinski, K. & Hryniewicz, W. (1999). Outbreak of mupirocin-resistant staphylococci in a hospital in Warsaw, Poland, due to plasmid transmission and clonal spread of several strains. Journal of Clinical Microbiology 37, 27818.
9
.
Ramsey, M. A., Bradley, S. F., Kauffman, C. A., Morton, T. M., Patterson, J. E. & Reagan, D. R. (1998). Characterization of mupirocin-resistant Staphylococcus aureus from different geographic areas. Antimicrobial Agents and Chemotherapy 42, 1305.
10 . Udo, E. E., Jacob, L. E. & Mokadas, E. M. (1997). Conjugative transfer of high-level mupirocin resistance from Staphylococcus haemolyticus to other staphylococci. Antimicrobial Agents and Chemotherapy 41, 6935.[Abstract]
11 . Gilbart, J., Perry, C. R. & Slocombe, B. (1993). High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases. Antimicrobial Agents and Chemotherapy 37, 328.[Abstract]
12 . Hodgson, J. E., Curnock, S. P., Dyke, K. G., Morris, R., 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]
13 . Lee, H. J., Suh, J. T., Kim, Y. S., Lenz, W., Bierbaum, G. & Schaal, K. P. (2001). Typing and antimicrobial susceptibilities of methicillin resistant Staphylococcus aureus (MRSA) strains isolated in a hospital in Korea. Journal of Korean Medical Science 16, 3815.[ISI][Medline]
14
.
Lim, J. A., Kwon, A. R., Kim, S. K., Chong, Y., Lee, K. & Choi, E. C. (2002). Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in a Korean hospital. Journal of Antimicrobial Chemotherapy 49, 48995.
15 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
16
.
Perez-Roth, E., Claverie-Martin, F., Villar, J. & Mendez-Alvarez, S. (2001). Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance. Journal of Clinical Microbiology 39, 403741.
17 . Ramsey, M. A., Bradley, S. F., Kauffman, C. A. & Morton, T. M. (1996). Identification of chromosomal location of mupA gene, encoding low-level mupirocin resistance in staphylococcal isolates. Antimicrobial Agents and Chemotherapy 40, 28203.[Abstract]
18
.
Fujimura, S., Watanabe, A. & Beighton, D. (2001). Characterization of the mupA gene in strains of methicillin-resistant Staphylococcus aureus with a low level of resistance to mupirocin. Antimicrobial Agents and Chemotherapy 45, 6412.
19 . Maniatis, N., Agel, A., Legakis, N. J. & Tzouvelekis, L. S. (2001). Mupirocin resistance in Staphylococcus aureus from Greek hospitals. International Journal of Antimicrobial Agents 18, 4078.[CrossRef][ISI][Medline]
20
.
Yanagisawa, T., Lee, J. T., Wu, H. C. & Kawakami, M. (1994). Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase. Journal of Biological Chemistry 269, 243049.
21
.
Boccazzi, P., Zhang, J. K. & Metcalf, W. W. (2000). Generation of dominant selectable markers for resistance to pseudomonic acid by cloning and mutagenesis of the ileS gene from the archaeon Methanosarcina barkeri fusaro. Journal of Bacteriology 182, 26118.
22
.
Silvian, L. F., Wang, J. & Steitz, T. A. (1999). Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin. Science 285, 10747.