- Email: [email protected]
Erythromycin-heteroresistant methicillin-resistant Staphylococcus aureus isolates from Turkey
626 Clinical Microbiology and Infection, Volume 14 Number 6, June 2008
ACKNOWLEDGEMENTS Reference strains were kindly supplied by C. Smyth (Trinity College Dublin, Ireland) (strains NY19, FRI472, FRI569, FRI913, DXD10, DX41) and A. Rossney (MRSA Reference Laboratory, St James Hospital, Dublin, Ireland) (MO50076). The colonising isolates were provided by O. Sherlock, E. Creamer and the Microbiology Laboratory at Beaumont Hospital (Dublin, Ireland). This work was funded, in part, by a Summer Research Grant to SOD from the Royal College of Surgeons in Ireland. The authors declare that they have no conflicting interests in relation to this work. S. O’Donnell, H. Humphreys and D. Hughes* Department of Clinical Microbiology, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland *E-mail: [email protected]
REFERENCES 1. Desachy A, Lina G, Vignon P et al. Role of superantigenic strains in the prognosis of community-acquired methicillin-susceptible Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2007; 13: 1131–1133. 2. Becker K, Roth R, Peters G. Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol 1998; 36: 2548–2553. 3. Diep BA, Carleton HA, Chang RF, Sensabaugh GF, Perdreau-Remington F. Roles of 34 virulence genes in the evolution of hospital- and community-associated strains of methicillin-resistant Staphylococcus aureus. J Infect Dis 2006; 193: 1495–1503. 4. Jarraud S, Mougel C, Thioulouse J et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 2002; 70: 631–641. 5. Lina G, Pie´mont Y, Godail-Gamot F et al. Involvement of Panton–Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999; 29: 1128–1132. 6. Monday SR, Bohach GA. Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J Clin Microbiol 1999; 37: 3411–3414. 7. O’Neill E, Pozzi C, Houston P et al. Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections. J Clin Microbiol 2007; 45: 1379–1388. 8. Nashev D, Toshkova K, Bizeva L et al. Distribution of enterotoxin genes among carriage- and infection-associated isolates of Staphylococcus aureus. Lett Appl Microbiol 2007; 45: 681–685. 9. Ferry T, Thomas D, Genestier AL et al. Comparative prevalence of superantigen genes in Staphylococcus aureus
isolates causing sepsis with and without septic shock. Clin Infect Dis 2005; 41: 771–777. 10. Lindsay JA, Moore CE, Day NP et al. Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J Bacteriol 2006; 188: 669–676.
Erythromycin-heteroresistant methicillinresistant Staphylococcus aureus isolates from Turkey 10.1111/j.1469-0691.2008.02004.x We read with interest the Editorial in CMI [1] concerning the heteroresistance of organisms such as Staphylococcus aureus, coagulase-negative staphylococci, Acinetobacter baumannii, Mycobacterium tuberculosis, Streptococcus pneumoniae, Enterococcus faecium and Cryptococcus neoformans. The Editorial focused especially on heteroresistant vancomycin-intermediate S. aureus and heteroresistant glycopeptide-intermediate S. aureus strains. We would like to draw attention to another heteroresistance phenomenon that we observed among methicillin-resistant S. aureus (MRSA) isolates in Turkiye Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey [2]. MRSA isolates from various clinical samples obtained from hospitalised patients at Turkiye Yuksek Ihtisas Teaching and Research Hospital were identified by Gram’s stain, tube coagulase tests and oxacillin agar screening tests. Erythromycin heteroresistance was determined using a standard disk method [3]. After realising that tiny colonies grew in the erythromycin inhibition zone, we decided to test the effect of this heteroresistance on the expression of inducible clindamycin resistance using D-tests [4]. In brief, a 15-lg erythromycin disk was placed 15 mm from the centre of a 2-lg clindamycin disk. Inhibition of the circular zone around the clindamycin disk (i.e., flattening of the zone towards the erythromycin disk) was considered to be a positive indication of inducible clindamycin resistance. The results of detailed studies with these erythromycin-heteroresistant isolates were as follows: (i) colonies growing within the erythromycin inhibition zones were so tiny and so few in number that they were hardly visible after incubation for 24 h at 37°C, and the isolate could
Ó 2008 The Authors Journal Compilation Ó 2008 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 625–627
Correspondence 627
easily have been evaluated as erythromycin-sensitive; (ii) after the same plates were incubated at 37°C for a further 24 h, the intra-zone colonies became more visible; (iii) using the double-diskapproximation method, these isolates showed an inducible clindamycin resistance phenotype, which was more visible after incubation at 37°C for 48 h; (iv) when the erythromycin disk-diffusion test was performed with a heavier inoculum (e.g., a McFarland turbidity of 1 or 2), confluent growth was observed in the erythromycin inhibition zone; and (v) the colonies growing within the erythromycin inhibition zone were of varying sizes, and there was no zone around the disk when the erythromycin disk-diffusion susceptibility test was repeated with pure cultures of these inter-zone colonies (i.e., the cultures were fully resistant to erythromycin). The size of the colonies did not influence the results, and all the colonies also showed a D-zone around a clindamycin disk. Repeated studies using the same strains yielded identical results. In conclusion, in order to identify such erythromycin-heteroresistant MRSA strains, all MRSA isolates should be tested by the disk-approximation method, using erythromycin and clindamy-
cin disks. The plates should be examined carefully for the existence of tiny colonies, with re-incubation for a further 24 h if necessary. N. Karabiber* and B. Mert Dinc Microbiology Laboratory, Turkiye Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey *E-mail: [email protected]
REFERENCES 1. Falagas ME, Makris GC, Dimopoulos G, Matthaiou DK. Heteroresistance: a concern of increasing clinical significance? Clin Microbiol Infect 2008; 14: 101–104. 2. Karabiber N, Mert Dinc B. Antibiotyping of MRSA strains isolated from various clinical samples and MRSA strains exhibiting microcolonies within the inhibition zone of erythromycin. ANKEM Derg 2007; 21: 50–58. 3. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing, 10th informational supplement, M100-S10. Wayne, PA: NCCLS, 2000. 4. Zelanzy AM, Ferraro MJ, Glennen A et al. Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in staphylococci: a CLSI collaborative study. J Clin Microbiol 2005; 43: 2613–2615.
Ó 2008 The Authors Journal Compilation Ó 2008 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 625–627
ACKNOWLEDGEMENTS Reference strains were kindly supplied by C. Smyth (Trinity College Dublin, Ireland) (strains NY19, FRI472, FRI569, FRI913, DXD10, DX41) and A. Rossney (MRSA Reference Laboratory, St James Hospital, Dublin, Ireland) (MO50076). The colonising isolates were provided by O. Sherlock, E. Creamer and the Microbiology Laboratory at Beaumont Hospital (Dublin, Ireland). This work was funded, in part, by a Summer Research Grant to SOD from the Royal College of Surgeons in Ireland. The authors declare that they have no conflicting interests in relation to this work. S. O’Donnell, H. Humphreys and D. Hughes* Department of Clinical Microbiology, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland *E-mail: [email protected]
REFERENCES 1. Desachy A, Lina G, Vignon P et al. Role of superantigenic strains in the prognosis of community-acquired methicillin-susceptible Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2007; 13: 1131–1133. 2. Becker K, Roth R, Peters G. Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol 1998; 36: 2548–2553. 3. Diep BA, Carleton HA, Chang RF, Sensabaugh GF, Perdreau-Remington F. Roles of 34 virulence genes in the evolution of hospital- and community-associated strains of methicillin-resistant Staphylococcus aureus. J Infect Dis 2006; 193: 1495–1503. 4. Jarraud S, Mougel C, Thioulouse J et al. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 2002; 70: 631–641. 5. Lina G, Pie´mont Y, Godail-Gamot F et al. Involvement of Panton–Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999; 29: 1128–1132. 6. Monday SR, Bohach GA. Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J Clin Microbiol 1999; 37: 3411–3414. 7. O’Neill E, Pozzi C, Houston P et al. Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections. J Clin Microbiol 2007; 45: 1379–1388. 8. Nashev D, Toshkova K, Bizeva L et al. Distribution of enterotoxin genes among carriage- and infection-associated isolates of Staphylococcus aureus. Lett Appl Microbiol 2007; 45: 681–685. 9. Ferry T, Thomas D, Genestier AL et al. Comparative prevalence of superantigen genes in Staphylococcus aureus
isolates causing sepsis with and without septic shock. Clin Infect Dis 2005; 41: 771–777. 10. Lindsay JA, Moore CE, Day NP et al. Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J Bacteriol 2006; 188: 669–676.
Erythromycin-heteroresistant methicillinresistant Staphylococcus aureus isolates from Turkey 10.1111/j.1469-0691.2008.02004.x We read with interest the Editorial in CMI [1] concerning the heteroresistance of organisms such as Staphylococcus aureus, coagulase-negative staphylococci, Acinetobacter baumannii, Mycobacterium tuberculosis, Streptococcus pneumoniae, Enterococcus faecium and Cryptococcus neoformans. The Editorial focused especially on heteroresistant vancomycin-intermediate S. aureus and heteroresistant glycopeptide-intermediate S. aureus strains. We would like to draw attention to another heteroresistance phenomenon that we observed among methicillin-resistant S. aureus (MRSA) isolates in Turkiye Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey [2]. MRSA isolates from various clinical samples obtained from hospitalised patients at Turkiye Yuksek Ihtisas Teaching and Research Hospital were identified by Gram’s stain, tube coagulase tests and oxacillin agar screening tests. Erythromycin heteroresistance was determined using a standard disk method [3]. After realising that tiny colonies grew in the erythromycin inhibition zone, we decided to test the effect of this heteroresistance on the expression of inducible clindamycin resistance using D-tests [4]. In brief, a 15-lg erythromycin disk was placed 15 mm from the centre of a 2-lg clindamycin disk. Inhibition of the circular zone around the clindamycin disk (i.e., flattening of the zone towards the erythromycin disk) was considered to be a positive indication of inducible clindamycin resistance. The results of detailed studies with these erythromycin-heteroresistant isolates were as follows: (i) colonies growing within the erythromycin inhibition zones were so tiny and so few in number that they were hardly visible after incubation for 24 h at 37°C, and the isolate could
Ó 2008 The Authors Journal Compilation Ó 2008 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 625–627
Correspondence 627
easily have been evaluated as erythromycin-sensitive; (ii) after the same plates were incubated at 37°C for a further 24 h, the intra-zone colonies became more visible; (iii) using the double-diskapproximation method, these isolates showed an inducible clindamycin resistance phenotype, which was more visible after incubation at 37°C for 48 h; (iv) when the erythromycin disk-diffusion test was performed with a heavier inoculum (e.g., a McFarland turbidity of 1 or 2), confluent growth was observed in the erythromycin inhibition zone; and (v) the colonies growing within the erythromycin inhibition zone were of varying sizes, and there was no zone around the disk when the erythromycin disk-diffusion susceptibility test was repeated with pure cultures of these inter-zone colonies (i.e., the cultures were fully resistant to erythromycin). The size of the colonies did not influence the results, and all the colonies also showed a D-zone around a clindamycin disk. Repeated studies using the same strains yielded identical results. In conclusion, in order to identify such erythromycin-heteroresistant MRSA strains, all MRSA isolates should be tested by the disk-approximation method, using erythromycin and clindamy-
cin disks. The plates should be examined carefully for the existence of tiny colonies, with re-incubation for a further 24 h if necessary. N. Karabiber* and B. Mert Dinc Microbiology Laboratory, Turkiye Yuksek Ihtisas Teaching and Research Hospital, Ankara, Turkey *E-mail: [email protected]
REFERENCES 1. Falagas ME, Makris GC, Dimopoulos G, Matthaiou DK. Heteroresistance: a concern of increasing clinical significance? Clin Microbiol Infect 2008; 14: 101–104. 2. Karabiber N, Mert Dinc B. Antibiotyping of MRSA strains isolated from various clinical samples and MRSA strains exhibiting microcolonies within the inhibition zone of erythromycin. ANKEM Derg 2007; 21: 50–58. 3. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing, 10th informational supplement, M100-S10. Wayne, PA: NCCLS, 2000. 4. Zelanzy AM, Ferraro MJ, Glennen A et al. Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in staphylococci: a CLSI collaborative study. J Clin Microbiol 2005; 43: 2613–2615.
Ó 2008 The Authors Journal Compilation Ó 2008 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 625–627