First Report of Linezolid-resistant Staphylococcal Clinical Isolates in Mexico Caused by cfr: Evidence of in. Rodrigo E.

Transcription

1 JCM Accepts, published online ahead of print on 2 June 2010 J. Clin. Microbiol. doi: /jcm Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 2 First Report of Linezolid-resistant Staphylococcal Clinical Isolates in Mexico Caused by cfr: Evidence of in vivo cfr Mobilization Intended category: New-data Letter to the Editor. Keywords: Cfr, plasmid, dissemination, Mexico. Rodrigo E. Mendes 1*, Lalitagauri Deshpande 1, Eduardo Rodriguez-Noriega 2 James E. Ross 1 Ronald N. Jones 1 and Rayo Morfin-Otero 2, 3, 1 JMI Laboratories, North Liberty, Iowa, USA. 2 Instituto de Patología Infecciosa y Experimental, Centro Universitario Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico. 3 Infectología, Microbiología, Hospital Civil de Guadalajara Fray Antonio Alcalde, Guadalajara, Jalisco, Mexico *Corresponding Author: Rodrigo E. Mendes, Ph.D. JMI Laboratories 345 Beaver Kreek Centre, Suite A North Liberty, Iowa USA Phone: (319) Fax: (319) rodrigo-mendes@jmilabs.com 1

2 An oxazolidinone resistance mechanism (Cfr) was recently described in human isolates of staphylococci (18). Cfr causes post-transcriptional methylation of the 23S rrna (A2503) affecting drugs belonging to several antimicrobial classes (10). cfr-carrying isolates recovered from human clinical specimens are still rare (4, 6); however, cases were described in the United States (12), Colombia (18) and Spain (15). Here, we report the first cases of human clinical infections caused by Cfr-producing Staphylococcus spp. in Mexico and demonstrate evidences of interspecies cfr mobilization. Three linezolid-resistant (MIC, 32 µg/ml) Staphylococcus spp. were submitted to a central monitoring laboratory (JMI Laboratories) as part of the SENTRY Antimicrobial Surveillance Program in These strains were collected from hospitalized patients at the Hospital Civil de Guadalajara. Staphylococcus cohnii (10842A) was found in a blood culture (August/2009) from a 30-year-old man admitted with multiple trauma. The Staphylococcus epidermidis (12898A) was also recovered (October/2009) from a bacteremia in a 50-year-old female admitted with a diagnosis of Guillain-Barre Syndrome. Both isolates were cultured within 48 hours after patients had developed clinical signs of sepsis (i.e. systemic inflammatory response syndrome; SIRS). The third organism was a S. epidermidis (5873X) cultured (October/2009) from an abdominal fluid in a 36-year-old male presenting with multiple trauma. Bacterial identification was confirmed by 16S rrna sequencing (3). Isolates were tested for susceptibility by the reference broth microdilution method (1). MIC interpretations were performed based on Clinical and Laboratory Standards Institute criteria (2), except for retapamulin MIC values (19). Quality control strains included Staphylococcus aureus ATCC and Enterococcus faecalis ATCC (2). Isolates were screened for cfr and mutations in the 23S rrna as described previously (12). L3- and L4- encoding genes were PCR amplified (13), amplicons sequenced on both strands and putative proteins compared with those from linezolid-susceptible S. epidermidis ATCC and S. cohnii ATCC Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were performed on S. epidermidis isolates (11, 14). After extraction (Plasmid DNA Mini Kit; Qiagen GmbH, Hilden, Germany), plasmid DNA were digested (Hind III and XbaI), separated on 1% agarose gel and transferred onto a nylon membrane by Southern blot (17). Membranes were hybridized using a cfr-specific probe (Roche 60 Diagnostics GmbH, Mannheim, Germany). 2

3 Linezolid-resistant isolates had the identification confirmed as S. epidermidis (isolates 12898A and 5873X) and S. cohnii (isolate 10842A). Isolates were oxacillin-resistant (MIC, >2 µg/ml) and exhibited elevated MIC results for linezolid (32 µg/ml), quinupristin/dalfopristin (1 4 µg/ml), retapamulin ( 8 µg/ml), chloramphenicol (16 32 µg/ml) and clindamycin (>64 µg/ml; Table 1). Isolates were susceptible to tetracycline, tigecycline, daptomycin and glycopeptides. All strains were PCR-positive for cfr and wild-type for 23S rrna and L4, except for S. cohnii that showed L4 substitutions (Asn20Ser, Ala133Thr and Val155Ile). L3 Ser158Tyr, Asp159Tyr and Leu101Val mutations were noted on both S. epidermidis, while Ser158Phe and Asp159Tyr were observed in S. cohnii. The L3 Leu101Val substitution was previously detected in a linezolid-susceptible clinical isolate (data on file, JMI Laboratories). However, Gly155 and Ala157 were previously implicated on disturbing the linezolid binding (8, 9). Thus, due to the proximity of these amino acid substitutions with those found in this study, the L3 mutations coupled with cfr, may act synergistically and possibly contribute to the elevated linezolid MIC results. An Asn158Ser mutation in L4 was previously noted in a linezolidsusceptible S. epidermidis (20). Therefore the fact that Val155Ile is close to Asn158 and the alterations found in L4 are not within a conserved region, they likely do not represent resistance mutations; however, additional experiments are needed. S. epidermidis (12898A and 5873X) displayed identical PFGE profiles (data not shown) and were ST-23. S. epidermidis strains also demonstrated identical plasmid patterns, which were different from 10842A (Figure 1). All strains showed similar hybridization signal patterns, suggesting similar cfr genetic contexts. These results indicate that recombination events may have mobilized the cfr gene into different plasmids, which were later acquired by these species, as previously proposed (7). However, additional experiments are required to determine the genetic elements responsible for these events. Cfr-encoding genes are still rare among human isolates (4, 6) and the relevance of coagulasenegative staphylococci (CoNS) isolates in clinical specimens can be difficult to assess (5). Nevertheless, the CoNS role as pathogens has increasingly been recognized, especially among immunocompromised patients, with indwelling or implanted foreign bodies (5, 16). S. cohnii (10842A) and S. epidermidis (12898A) were cultured from blood of patients associated with SIRS, and were therefore considered clinically relevant. In addition, these staphylococcal species may, at the least, act as a reservoir for 89 resistance gene determinants in this nosocomial environment. 3

4 Acknowledgements Expert technical support was kindly provided by D. Biedenbach, P. Rhomberg, G. Moet, A. Small, M. Stillwell and M. Janechek Funding No specific funding was received for this study. Transparency declarations R. N. Jones has received research/education grants in the last two years from: API, Anacor, Arpida, Astellas, AstraZeneca, Bayer, biomerieux, Cadence, Cempra, Cerexa, Cubist, Enanta, Forest, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), Novartis, Optimer, Pfizer, Protez, Shionogi, The Medicines Company, Theravance and TREK Diagnostics. Downloaded from on October 23, 2018 by guest 4

5 References 1. Clinical and Laboratory Standards Institute (CLSI) M07-A8. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically Approved standard. Eighth edition. Wayne, PA, USA. 2. Clinical and Laboratory Standards Institute (CLSI) M100-S20. Performance standards for antimicrobial susceptibility testing. 20 th Informational Supplement. Wayne, PA, USA. 3. Devulder, G., G. Perriere, F. Baty, and J. P. Flandrois BIBI, a bioinformatics bacterial identification tool. J. Clin. Microbiol. 41: Farrell, D. J., R. E. Mendes, J. E. Ross, and R. N. Jones Linezolid surveillance program results for 2008 (LEADER Program for 2008). Diagn. Microbiol. Infect. Dis. 65: Huebner, J., and D. A. Goldmann Coagulase-negative staphylococci: Role as pathogens. Annu. Rev. Med. 50: Jones, R. N., J. E. Ross, J. M. Bell, U. Utsuki, I. Fumiaki, I. Kobayashi, and J. D. Turnidge Zyvox Annual Appraisal of Potency and Spectrum program: Linezolid surveillance program results for Diagn. Microbiol. Infect. Dis. 65: Kehrenberg, C., F. M. Aarestrup, and S. Schwarz IS insertion sequences are involved in the mobility of the multiresistance gene cfr. Antimicrob. Agents Chemother. 51: Locke, J. B., M. Hilgers and K. J. Shaw Novel ribosomal mutations in Staphylococcus aureus strains identified through selection with the oxazolidinones linezolid and torezolid (TR-700). Antimicrob. Agents Chemother. 53: Locke, J. B., M. Hilgers and K. J. Shaw Mutations in ribosomal protein L3 are associated with oxazolidinone resistance in staphylococci of clinical origin. Antimicrob. Agents Chemother. 53: Long, K. S., J. Poehlsgaard, C. Kehrenberg, S. Schwarz, and B. Vester The cfr rrna methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics. Antimicrob. Agents Chemother. 50: McDougal, L. K., C. D. Steward, G. E. Killgore, J. M. Chaitram, S. K. McAllister, and F. C. Tenover Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: Establishing a national database. J. Clin. Microbiol. 41: Mendes, R. E., L. M. Deshpande, M. Castanheira, J. DiPersio, M. A. Saubolle, and R. N. Jones First report of cfr-mediated resistance to linezolid in human staphylococcal clinical isolates recovered in the United States. Antimicrob. Agents Chemother. 52:

6 13. Miller, K., C. J. Dunsmore, C. W. Fishwick, and I. Chopra Linezolid and tiamulin cross-resistance in Staphylococcus aureus mediated by point mutations in the peptidyl transferase center. Antimicrob. Agents Chemother. 52: Miragaia, M., J. C. Thomas, I. Couto, M. C. Enright, and H. de Lencastre Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data. J. Bacteriol. 189: Morales, G., J. J. Picazo, E. Baos, F. J. Candel, A. Arribi, B. Peláez, R. Andrade, M. A. de la Torre, J. Fereres, and M. Sánchez-García Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant Staphylococcus aureus. Clin. Infect. Dis. 50: Piette, A., and G. Verschraegen Role of coagulase-negative staphylococci in human disease. Vet. Microbiol. 134: Sambrook, J., and D. W. Russell Molecular cloning: A laboratory manual. 3 rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 18. Toh, S. M., L. Xiong, C. A. Arias, M. V. Villegas, K. Lolans, J. Quinn, and A. S. Mankin Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. Molec Microbiol. 64: Traczewski, M. M., and S. D. Brown Proposed MIC and disk diffusion microbiological cutoffs and spectrum of activity of retapamulin, a novel topical antimicrobial agent. Antimicrob. Agents Chemother. 52: Wong, A., S. P. Reddy, D. S. Smyth, M. E. Aguero-Rosenfeld, G. Sakoulas, and D. A. Robinson Polyphyletic emergence of linezolid-resistant staphylococci in the United States. Antimicrob. Agents Chemother. 54:

7 Table 1. Antimicrobial susceptibility profile and molecular findings among cfr-carrying Staphylococcus spp. recovered from clinical specimens of hospitalized patients in Guadalajara (Mexico). Antimicrobial agent MIC (µg/ml) [susceptibility category] a S. cohnii 10842A S. epidermidis 12898A S. epidermidis 5873X Linezolid 32 [R] 32 [R] 32 [R] Quinopristin/dalfopristin 4 [R] 2 [I] 1 [S] Retapamulin >8 [R] 8 [R] >8 [R] Chloramphenicol 32 [R] 16 [I] 16 [I] Clindamycin >64 [R] >64 [R] >64 [R] Tigecycline 0.06 [S] 0.12 [S] 0.25 [S] Tetracycline 0.12 [S] 2 [S] 1 [S] Doxycycline 0.12 [S] 0.5 [S] 1 [S] Daptomycin 0.25 [S] 0.5 [S] 0.5 [S] Vancomycin 1 [S] 2 [S] 2 [S] Teicoplanin 2 [S] 8 [S] 8 [S] Oxacillin >2 [R] >2 [R] >2 [R] Ciprofloxacin >4 [R] >4 [R] >4 [R] Erythromycin >2 [R] >2 [R] >2 [R] Gentamicin >8 [R] >8 [R] >8 [R] Trimethoprim/sulfamethoxazole 0.5 [S] >2 [R] >2 [R] Molecular findings cfr Positive Positive Positive 23S rrna WT WT WT L3 Ser158Phe/Asp159Tyr Ser158Tyr/Asp159Tyr/Leu101Val Ser158Tyr/Asp159Tyr/Leu101Val L4 Asn20Ser/Ala133Thr/Val155Ile WT WT a MIC interpretive criteria as published by CLSI M100-S20 (2). Retapamulin MIC results were interpreted according to parameters reported by Traczewski et al. (2008)(19). S, susceptible; I, intermediate; and R, resistant; WT, wildtype. 7

8 Downloaded from Figure 1. Plasmid profile analysis of cfr-carrying strains. A. λ represents 1-kb DNA Ladder used as negative control plasmid DNA (New England Biolabs, Ipswich, MA, USA). Lanes 1, 2 and 3 represent S. cohnii 10842A, S. epidermidis 12898A and S. epidermidis 5738X, respectively. Band patterns of uncut, and HindIII- and XbaI-digested plasmid preparations. B. Hybridization profiles with a cfr-specific probe. Horizontal arrow indicates hybridization signals. on October 23, 2018 by guest 8