Sir, We describe herein the microbiological characterization of what was, to our knowledge, the first Cfr-producing MSSA strain (048-45547X) causing a fatal pneumonia in an oncology patient in São Paulo, Brazil. The index Staphylococcus aureus 048-45547X was recovered from blood cultures and species identification and susceptibility testing were performed locally using the BD Phoenix™ Automated Microbiology System. S. aureus was submitted to a central monitoring laboratory (JMI Laboratories, North Liberty, IA, USA) as part of the 2012 SENTRY Antimicrobial Surveillance Program, where identification was confirmed by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). The index strain was further tested for susceptibility by CLSI broth microdilution (BMD) and also interpreted by the CLSI criteria, when available.1 Strain 048-45547X was susceptible to oxacillin (MIC, 0.25–0.5 mg/L) and linezolid (MIC, 2–4 mg/L) by the BD system and BMD as shown in Table 1. The 048-45547X strain was screened for the presence of cfr, erm(A–C), mecA, mecC and mutations in the 23S rRNA and ribosomal proteins (L3 and L4).2 The cfr and erm(B) genes were detected in the 048-45547X strain. No mutations were observed in the domain V region of the 23S rRNA gene or L3, but a V142I amino acid substitution was detected in L4. The mecA and mecC genes were not detected, as expected. Antimicrobial susceptibility profiles and molecular findings for the S. aureus clinical strain (048-45547X), recipient and transformant (carrying the cfr-harbouring plasmid p45547X) strains and E. coli DH5α and DH5α carrying the aadY-containing plasmid aMIC interpretive criteria by CLSI (2014), when available. S, susceptible; I, intermediate; R, resistant. bInitial susceptibility testing was performed by the submitting local site using the BD Phoenix™ Automated Microbiology System. cReference BMD was performed by the central monitoring site (JMI Laboratories, North Liberty, IA, USA). Antimicrobial susceptibility profiles and molecular findings for the S. aureus clinical strain (048-45547X), recipient and transformant (carrying the cfr-harbouring plasmid p45547X) strains and E. coli DH5α and DH5α carrying the aadY-containing plasmid aMIC interpretive criteria by CLSI (2014), when available. S, susceptible; I, intermediate; R, resistant. bInitial susceptibility testing was performed by the submitting local site using the BD Phoenix™ Automated Microbiology System. cReference BMD was performed by the central monitoring site (JMI Laboratories, North Liberty, IA, USA). Southern blot/hybridization experiments revealed cfr and erm(B) signals from a single band of ∼48.5 kb, indicating these genes were located in the same plasmid DNA. These results were confirmed by PCR amplification of regions between erm(B) and cfr (∼15 kb each). Amplicons were sequenced and results were submitted to EMBL/GenBank/DDBJ (accession number KJ192337). cfr was plasmid located (p45547X) and bracketed by recombination/mobilization proteins, a context identical to that of pBS-03 detected in a Bacillus spp. (BS-03; JQ394981) recovered from a nasal swab of a pig in Shandong, China.3 Several insertion sequences are widely disseminated in Gram-positive and -negative bacteria and have been associated with the translocation of the cfr gene between different plasmids as well as its integration into chromosomal DNA.4,5 In addition, an aminoglycoside 6-adenyltransferase (aadY-like) was observed, which was most closely related to that of the pBS-03 plasmid.3 However, these adenyltransferases shared only 80.5% amino acid identity. The tet(L), aadD, rep(U)-like, tnpB, rep and erm(B) genes were located further downstream of cfr and the fragment (3003 bp) containing tet(L), aadD and rep(U)-like was identical to that of pKKS825 detected in ST398 MRSA collected from pigs in Germany.6 Moreover, the usually erm(B)-associated Tn917 or Tn551 was truncated by the insertion of a resolvase commonly found among staphylococci, which was closely related (99.0%) to that observed in a Staphylococcus lentus (NC_015432). Conjugation experiments failed to transfer p45547X, suggesting that these genes were probably located in a non-conjugative plasmid. p45547X was transferred by electroporation into S. aureus RN4220 and the transformant strain exhibited MICs of linezolid, chloramphenicol, clindamycin and pleuromutilins ≥4-fold higher than RN4220. In addition, the transformant displayed higher MICs of amikacin (8-fold), tobramycin (256-fold), kanamycin (64-fold) and streptomycin (128-fold). However, when aadY-like was cloned into pJET1.2 vector (Thermo Scientific, Waltham, MA, USA) and transformed into Escherichia coli DH5α, this strain showed an elevated MIC of streptomycin only (Table 1). These results confirm the functionality of the aadD-like gene as a streptomycin resistance gene on p45547X.7 048-45547X was grouped under ST398, an ST not previously reported among Brazilian isolates. Currently, MRSA ST398 has been considered a livestock-associated clone since it is widely disseminated in animals, especially among industrially raised pigs.8 Strain 048-45547X was spa t034, which is highly related to t571, and the ST398/t571 MSSA clone was recently reported as responsible for colonizing the anterior nares of Texan detainees.9 In summary, to our knowledge, this is the first report of a Cfr-producing S. aureus in Brazil. It is interesting to notice that it was detected in a livestock-associated MSSA clone (ST398) susceptible to linezolid, which was isolated from a patient without previous linezolid exposure, suggesting coselection probably by the use of other antimicrobial agents. A linezolid MIC value indicative of susceptibility and the absence of other markers may render the recognition of a Cfr-associated phenotype difficult. Further surveillance is warranted to determine the extent of cfr and ST398 dissemination among MSSA and MRSA in Brazil. This study was carried out using internal JMI Laboratories funding. JMI Laboratories, Inc. (L. M. D. and R. E. M.) received research and educational grants in 2009–12 from American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, Cempra, Cerexa, Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), LegoChem Biosciences Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Pfizer (Wyeth), Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, The Medicines Co., Theravance and ThermoFisher. A. C. G. is a researcher from the National Council for Science and Technological Development (CNPq), Ministry of Science and Technology, Brazil (Process number: 307816/2009-5) and has participated in advisory boards for AstraZeneca and Merck. A. G. de S. and A. C. C. P.: none to declare. With regards to stock options – all authors: none to declare. We wish to thank the following staff members at JMI Laboratories (North Liberty, IA, USA) for technical support and manuscript assistance: S. Benning, M. Castanheira, A. Costello and S. Costello. The S. aureus strain was obtained through the ZAAPS Program, which is sponsored by educational/research grants from Pfizer Inc., Specialty Business Unit (Collegeville, PA, USA).
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