Comparação genotípica e fenotípica de Enterococcus faecalis resistentes à vancomicina isolados nos anos de 2009 e 2011 em um hospital de Minas Gerais
Thaís Panhan Merlo
AGRADECIMENTOS À Profa. Dra. Ilana L. B. C. Camargo, pelos ensinamentos, paciência, suporte e auxílio nos momentos de dificuldade. Aos meus pais, Alexandre e Maria, e ao meu irmão Tiago, pelo apoio e suporte durante os anos de graduação e pós. Ao meu namorado e sempre companheiro Matheus, pelo apoio em todas as minhas decisões, a paciência e as longas conversas. À minha avó Alice e meus tios, Marilice, Renato e Mariângela, sem os quais minha chegada até aqui teria sido muito mais difícil.
... ço pelo carinho e confiança. À minha prima e amiga Fátima e minha tia Mariana, pelas conversas, conselhos e risadas. Às minhas queridas amigas de graduação Ana Paula, Jaqueline e Mariana, que sempre me incentivaram e apoiaram. Às minhas colegas e amigas do LEMiMo, Simoni, Jéssica e Nicoli. Obrigada por me auxiliarem quando precisei, estarem sempre dispostas a discutir todos os assuntos e dúvidas e pelas noites de descontração depois do trabalho. Aos amigos Evandro e Paola, que passaram logos períodos comigo no laboratório, tentando resolver meus muitos problemas com proteína. À Susana, que sempre estava disposta a me ajudar no tempo que fiquei no Laboratório de Cristalografia. Aos funcionários da Pós-graduação, biblioteca e gráfica do IFSC, por serem sempre tão prestativos em tudo o que precisei. À FAPESP e ao CNPq pelo auxílio financeiro. ABSTRACT MERLO, T. P. Genotypic and phenotypic comparison of vancomycin resistant Enterococcus faecalis isolated in 2009 and 2011 in a hospital in Minas Gerais. 2013. 97 p. Dissertação (Mestrado em Ciências)-Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, 2013. Enterococci are Gram-positive cocci occurring isolated, in pairs (diplococci) or chains and belong to the intestinal tract of a wide variety of hosts, from mammalian to insect. Enterococcus organisms were originally considered of little clinical importance, but have proved important nosocomial pathogens. The fact that Enterococcus possess intrinsic and acquired resistance to multiple antibiotics complicates the treatment of infections caused by them. Enterococcus faecalis is usually the predominant species among enterococci isolates, with 80% to 90% of the clinical samples. The emergence of vancomycin-resistant Enterococcus (VRE) significantly reduced the treatment options. The aim of this study was to identify and compare samples of vancomycin-resistant E. faecalis (VREfs) in patients in the years 2009 and 2011, during a surveillance program in Hospital Risoleta Tolentino Neves, Belo Horizonte, MG. Species identification was done by multiplex-PCR with primers speciesspecific and E. faecalis were selected for this study. Tests were conducted for the presence of genes elrA, cylL L , esp and gelE, the determination of the genotype responsible for the resistance and characterization of transposon containing the resistance gene, all by PCR. The minimum inhibitory concentration (MIC) was determined according to the CLSI (2013) to vancomycin, linezolid, daptomycin and tigecycline and tests for resistance to high concentrations of aminoglycosides. Finally, the samples typing was performed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). It was found that 22.2 % of VRE isolates in 2009 and 61.7 % of isolates in 2011 belong to the species E. faecalis and these were used in the study. There was emergence of tigecycline resistance in 2011, with 10 resistant isolates, after the introduction of this drug in the hospital. One isolate from 2011 showed intermediate resistance to linezolid. All isolates were sensitive to daptomycin and highly vancomycin resistant, with MICs greater than 256 mg / mL. This high resistance to vancomycin is consistent with the vanA genotype found in all samples. The virulence profile prevalent in VREfs in 2009 was elrA + gelE + , belonging to the pulsotypes A1 and A4 and ST103. Only one isolate from 2009, presented pulsotype A1 and the virulence profile cyl + elrA + gelE + . In 2011, the prevalent virulence profile was cyl + esp + elrA + gelE + , and the 12 isolates with this profile belonged to pulsotypes B and C and ST6, which occurred only in 2011 isolates. Eleven isolates from 2011 have the profile elrA + gelE + and were classified as pulsotype A, six isolates have profile cyl + elrA + gelE + and are of pulsotypes B and D. PFGE results show the insertion of ST6 lineage in the hospital, a multiresistant clone widely disseminated in hospitals in Italy, Portugal, Spain and the United States. It was concluded that there were changes in the profile of E. faecalis resistant to vancomycin in the hospital over the two years, with increased resistance and more virulent strains, which are of concern.