Research letters of M. morganii, was detected in two isolates of E. coli. DHA-1 is an inducible plasmid-mediated AmpC b-lactamase whose emergence raises concerns because the mortality of patients infected with organisms that produce DHA-1 has been shown to be higher than that of patients infected with organisms that produce CMY-1.2 In conclusion, we have demonstrated that plasmid-mediated AmpC b-lactamases have emerged in Switzerland. The prevalence of 0.16% is still low. However, the occurrence of DHA-1, an inducible type of enzyme, raises clinical concerns. Additionally, a novel plasmid-mediated AmpC b-lactamase, which was designated CMY-31, was found.
Funding This study was funded by the Department of Laboratory Medicine, University Hospital Basel.
None to declare.
References 1. Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC b-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40: 2153– 62. 2. Pai H, Kang CI, Byeon JH et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-b-lactamaseproducing Klebsiella pneumoniae. Antimicrob Agents Chemother 2004; 48: 3720–8. 3. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16. CLSI, Wayne, PA, USA, 2006. 4. Winokur PL, Brueggemann A, DeSalvo DL et al. Animal and human multidrug-resistant, cephalosporin-resistant salmonella isolates expressing a plasmid-mediated CMY-2 AmpC b-lactamase. Antimicrob Agents Chemother 2000; 44: 2777– 83. 5. Muratani T, Kobayashi T, Matsumoto T. Emergence and prevalence of b-lactamase-producing Klebsiella pneumoniae resistant to cephems in Japan. Int J Antimicrob Agents 2006; 27: 491–9. 6. Liebana E, Gibbs M, Clouting C et al. Characterization of b-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from foodproducing animals in the United Kingdom. Microb Drug Resist 2004; 10: 1 – 9.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm483 Advance Access publication 21 December 2007
Prevalence and characterization of macrolide-lincomycin-streptogramin B-resistant Staphylococcus aureus in Korean hospitals Young-Hee Jung1, Kwang Wook Kim1, Kyeong Min Lee1, Jae Il Yoo1, Gyung Tae Chung1, Bong Soo Kim2, Hyo-Sun Kwak3 and Yeong Seon Lee1*
Division of Antimicrobial Resistance, Center for Infectious Disease Research, National Institute of Health, 194, TongilLo, Eunpyung-Gu, Seoul 122-701, Republic of Korea; 2 Division of Biodefence Research, Center for Infectious Disease Research, National Institute of Health, 194, TongilLo, Eunpyung-Gu, Seoul 122-701, Republic of Korea; 3Food Microbiology Team, Center for Food Safety Evaluation, Korea Food and Drug Administration, 194, Tongil-Lo, Eunpyung-Gu, Seoul 122-704, Republic of Korea Keywords: macrolide resistance genes, double disc diffusion, non-tertiary hospitals *Corresponding author. Tel: þ82-2-380-1478; Fax: þ82-2-3801550; E-mail:
[email protected] Sir, Resistance to macrolide, lincosamide and streptogramin B (MLSB) antibiotics in Staphylococcus spp. is mediated by a methylase encoded by erythromycin ribosome methylation (erm) genes or ATP transporter efflux pumps encoded by the msr or mef genes. The methylation of the 50S ribosomal subunit by erm gene products causes constitutive or inducible resistance to MLSB antibiotics. Constitutively resistant MLSB (MLSBc) strains and inducibly resistant MLSB (MLSBi) strains express erm genes, erm(A) or/and erm(C). In this study, we investigated and characterized the constitutive and inducible resistance, and the susceptibility to clindamycin of Staphylococcus aureus isolated from non-tertiary hospitals. We collected 508 S. aureus strains from 157 hospitals nationwide from January to June 2005 in Korea. Antimicrobial susceptibility to oxacillin, erythromycin, clindamycin and quinupristin/ dalfopristin and the MICs were evaluated according to the guidelines of the CLSI. Of the 508 S. aureus isolates, 46.1% (234 isolates) were resistant to oxacillin, 55.1% (280 isolates) were resistant to erythromycin, which comprised 98.3% (230/234) methicillin-resistant S. aureus (MRSA) strains and 18.2% (50/274) methicillin-susceptible S. aureus (MSSA) strains, and 37.4% (190 isolates) were resistant to clindamycin. No strains were susceptible to erythromycin and resistant to clindamycin. One hundred and eighty-six MRSA (80.9%) and four MSSA (8.0%) were MLSBc (resistant to erythromycin and clindamycin; Table 1). D-test according to Steward’s method was used for isolates with erythromycin resistance and clindamycin susceptibility.1 Forty-three MRSA (18.7%) and 40 MSSA (80.0%) were MLSBi (showing the D form on the D-test), and 1 MRSA (0.4%) and 6 MSSA (12.0%) were MLSBs (susceptible to clindamycin). The MLSB resistance genes, erm(A), erm(B), erm(C), msr(A), msr(B) and mef, were detected using a PCR method.2 erm(A) was detected in 250 strains, erm(C) in 14 strains, erm(B) in 1 strain, msr(A) in 3 strains, erm(A) and erm(C) in 2 strains, erm(A) and msr(A) in 3 strains and no genes in 7 strains. Most MLSBc and MLSBi strains carried the erm(A) or erm(C) gene (Table 1). The erm(A) gene (in 250 isolates) was detected far more frequently than erm(C) (in 14 strains). However, the erm(C) gene was distributed more frequently in MLSBi strains (12 isolates) than in MLSBc strains (2 isolates). Using OLIGO primers designed by Arthur et al.,3 the PCR products were detected in three MLSBc or MLSBi strains lacking erm(A), erm(B) or erm(C). It was possible that the three strains carry an erm gene besides erm(A), erm(B) or erm(C). msr(A)
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Transparency declarations
1
Research letters Table 1. Phenotypes, genotypes and MICs of erythromycin-resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) Phenotype
MIC (mg/L)
230 MRSA isolates (%) Genotype erm(A) erm(B) erm(C) msr(A) erm(A) erm(C) erm(A) msr(A) erm a Mef Non-typeable Total
50 MSSA isolates (%)
Cr-con
Cr-ind
Cs
Cr-con
Cr-ind
Cs
ERY
183 (79.6) — — — 1 (0.4) — 2 (0.9) — — 186 (80.9)
34 (14.8) — 7 (3.0) — 1 (0.4) — 1 (0.4) — — 43 (18.7)
— — — 1 (0.4) — — — — — 1 (0.4)
1 (2.0) 1 (2.0) 2 (4.0) — — — — — — 4 (8.0)
32 (64.0) — 5 (10.0) — — 3 (6.0) — — — 40 (80.0)
— — — 2 (4.0) — —
8 or 256 256 256 4 or 64 256 256 256 — 8 4 to 256
— 4 (8.0) 6 (12.0)
CLI 1 or 64 1 or 1 1 or 1 1 or — 1 1 or
64 64 64 64
64
was detected in one MRSA isolate and five MSSA isolates, three MLSBi isolates carried msr(A) together with erm(A) and three MLSBs contained only msr(A). We also found msr(A) in three of seven MLSBs isolates but did not detect the msr(A) gene in four MLSBs strains. To determine whether the four MLSBs isolates carried the msr(A) gene, Southern hybridization was performed by the Amersham ECL Direct Nucleic Acid Labeling and Detection system (Amersham, GE Healthcare Life Science, USA), but no fragments were detected (data not shown). Also, any resistance genes encoding ATP transporter efflux pumps [msr(B), msr(C) and msr(D)], mef(A/E) genes and the phosphorylase mph(C) gene were not detected in the four strains (data not shown). The MICs of erythromycin for MLSBc and MLSBi isolates were 256 mg/L, with the exception of one strain with an MIC of 8 mg/L. The MICs of erythromycin for the MLSBs strains were 4, 8 or 64 mg/L. The MICs of clindamycin for clindamycin-susceptible and clindamycin-resistant strains, determined by agar dilution, were 1 and 64 mg/L, respectively (Table 1). It has been reported that MLSBc and MLSBi isolates in Staphylococcus carry erm(A), erm(B) or erm(C), and at times erm together with msr(A), but MLSBs carry the msr(A) gene.4 Although msr(A) and erm(A) were detected together in MLSBi, the msr(A) gene was detected in MLSBs. In Streptococcus, MLSBc and MLSBi strains carried erm(A) and had MICs of erythromycin of 16– 256 mg/L; and MLSBs or M phenotype strains had mef(A) and MICs of 16 –48 or 8– 32 mg/L.5 We propose that MICs of erythromycin of 4 –64 mg/L are insufficient to induce resistance to clindamycin, and the erm genes were expressed at high concentrations of erythromycin and the msr(A) gene at low concentrations. If the msr and erm genes are detected together, the erm gene may be expressed more rapidly than the msr gene and the phenotype of the strain may be MLSBc or MLSBi.
In conclusion, the erythromycin resistance rate in S. aureus was high at 55.1%, and 99.6% of erythromycin-resistant MRSA and 88.0% of MSSA strains were MLSBc or MLSBi. Therefore, the treatment of infections caused by erythromycin-resistant S. aureus with clindamycin in non-tertiary hospitals is not effective.
Acknowledgements We would like to thank the researchers in Seoul Clinical Laboratories (SCL) for sending the Staphylococcus isolates isolated from non-tertiary hospitals to our laboratory.
Funding This study was supported by a research grant from the Korea Food and Drug Administration (KFDA) in Republic of Korea in 2005.
Transparency declarations None to declare.
References 1. Steward CD, Raney PM, Morrell AK et al. Testing for induction of clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus. J Clin Microbiol 2005; 43: 1716–21. 2. Lim JA, Kwon AR, Kim SK et al. Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in a Korean hospital. J Antimicrob Chemother 2002; 49: 489 –95. 3. Arthur M, Molinas C, Mabilat C et al. Detection of erythromycin resistance by the polymerase chain reaction using primers in conserved regions of erm rRNA methylase genes. Antimicrob Agents Chemother 1990; 34: 2024–6.
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Cr-con, MLSB-constitutive; Cr-ind, MLSB-inducible; Cs, MLSB-susceptible; ERY, erythromycin; CLI, clindamycin. a erm, conserved primer designed among erm(A), erm(C), erm(BC) and erm(G) to identify erm genes, by Arthur et al.3 Three isolates lacking erm(A), erm(B) or erm(C) were detected by the erm primers using the PCR method.
Research letters 4. Chavez-Bueno S, Bozdogan B, Katz K et al. Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus in Dallas, Texas. Antimicrob Agents Chemother 2005; 49: 2283–8. 5. Hesenbein ME, Warner JE, Lambert KG et al. Detection of multiple macrolide- and lincosamide-resistant strains of Streptococcus pyogenes from patients in the Boston area. J Clin Microbiol 2004; 42: 1559–63.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm488 Advance Access publication 19 December 2007
In vitro activity of ceftobiprole against Burkholderia pseudomallei Visanu Thamlikitkul* and Suwanna Trakulsomboon
Keywords: B. pseudomallei, melioidosis, cephalosporins *Corresponding author. Tel/Fax: þ66-2-412-5994; E-mail:
[email protected] Sir, Burkholderia pseudomallei, a Gram-negative bacterium, causes a disease called melioidosis in humans and animals.1 The bacterium is a soil organism found mainly in Southeast Asia and Northern Australia. Antibiotics currently recommended for therapy of melioidosis are ceftazidime, imipenem, meropenem, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, doxycycline and chloramphenicol.1 A development of resistance of B. pseudomallei to the aforementioned antibiotics has been recognized;2,3 hence, a search for new agents effective against B. pseudomallei is needed. Ceftobiprole is a novel parenteral cephalosporin whose broad spectrum of activity includes most clinically important Gram-positive and Gram-negative bacteria.4 One hundred and fifteen strains of ceftazidime-susceptible B. pseudomallei from different infected patients were selected from our collection. All strains were identified as B. pseudomallei by API 20NE (bioMe´rieux, France). In vitro susceptibility was determined by Kirby-Bauer disc diffusion for all 115 strains and Etest for 5 randomly chosen strains. Paper discs containing 30 mg ceftobiprole per disc (MASTDISC) and Etest strips of ceftobiprole at concentrations of 0.016 – 256 mg/L were provided by Janssen-Cilag (Thailand). The disc diffusion test was repeated for six strains of B. pseudomallei in order to determine
Acknowledgements We thank Janssen-Cilag (Thailand) and The Thailand Research Fund for supporting the study.
Funding V. T. is a recipient of Senior Researcher Scholar of the Thailand Research Fund. Janssen-Cilag (Thailand) provided ceftobiprole susceptibility discs and Etest strips for this study.
Transparency declarations None to declare.
References 1. White NJ. Melioidosis. Lancet 2003; 361: 1715–22. 2. Dance DA, Wuthiekanun V, Chaowagul W et al. Development of resistance to ceftazidime and co-amoxiclav in Pseudomonas pseudomallei. J Antimicrob Chemother 1991; 28: 321 –4.
Table 1. Distribution of ceftobiprole inhibition zone diameter for 115 strains of B. pseudomallei No. of strains (%) for which the inhibition zone diameter was 13 mm 1 (0.9)
15 mm 5 (4.3)
16 mm 8 (7.0)
17 mm 25 (21.7)
18 mm 19 (16.5)
19 mm 11 (9.6)
460
20 mm 30 (26.1)
21 mm 7 (6.1)
22 mm 3 (2.6)
23 mm 4 (3.5)
25 mm 2 (1.7)
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Division of Infectious Disease and Tropical Medicine, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
the reproducibility of the test. The methodology for susceptibility testing was performed by direct colony suspension according to guidelines suggested by the CLSI.5 Quality control was performed by testing susceptibility of Pseudomonas aeruginosa ATCC 27853. The proposed breakpoints for inhibition zone diameters of ceftobiprole are 20 mm for susceptible, 17–19 mm for intermediate and 16 mm for resistant. The proposed breakpoints for MICs of ceftobiprole are 4 mg/L for susceptible, 8 mg/L for intermediate and 16 mg/L for resistant. The inhibition zone diameter of ceftobiprole against P. aeruginosa ATCC 27853 was within the reference limits. The distribution of inhibition zone diameters of ceftobiprole against B. pseudomallei is shown in Table 1. Inhibition zone diameters of 20, 17 –19 and 16 mm were observed in 46 (40%), 55 (47.8%) and 14 (12.2%) strains, respectively. Four strains of B. pseudomallei with inhibition zone diameters of 15 –19 mm on the initial disc diffusion test had identical inhibition zone diameters on the second test. Another two strains with an inhibition zone diameter of .20 mm had 1 mm difference in inhibition zone diameter on the second test, but the inhibition zone diameters from both tests were still within susceptible values. Four B. pseudomallei strains with an inhibition zone diameter of 17 – 19 mm had MICs of ceftobiprole of 6 – 8 mg/L, whereas a strain with an inhibition zone diameter of 16 mm had an MIC of 16 mg/L. Our findings indicate that the in vitro activity of ceftobiprole against B. pseudomallei determined by Kirby-Bauer disc diffusion is reproducible and correlates with that determined by Etest. Ceftobiprole has less in vitro activity than ceftazidime against B. pseudomallei, and only 40% of B. pseudomallei strains are susceptible to ceftobiprole.
