Estudo da relação clonal e dos mecanismos envolvidos na resistência à Polimixina B em isolados clínicos de Klebsiella pneumoniae obtidos em um hospital universitário da cidade de São Paulo

• Ano de defesa: 2016
• Autor(a) principal: Braun, Graziela [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Klebsiella pneumoniae; Polimixina b; Sistema de dois componentes; Mgrb; Mcr-1; Ctx-m-14
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=4988473; http://repositorio.unifesp.br/handle/11600/48642

Klebsiella pneumoniae is a ubiquitous microorganism of great importance in the hospital setting due to their ability to acquire antimicrobial resistance, including polymyxins. This study is divided in two manuscripts: Article 1 – This study aimed to assess the emergence and spread of polymyxin B-resistant K. pneumoniae clones during seven years in a Brazilian teaching hospital. A total of 54 K. pneumoniae isolates, being 36 resistant and 18 susceptible to polymyxin B, were obtained from blood cultures of hospitalized patients admitted between 2009 and 2015. The bacterial identification was carried out by MALDI-TOF MS and the antimicrobial susceptibility profile was determined by broth microdilution except for fosfomycin, to which the agar dilution method was performed. The clonal relationship of the isolates was assessed by PFGE. Detection of genes encoding beta-lactamases and 16S rRNA methyltransferases, as well the mgrB and mrc-1 genes, related to polymyxin resistance was performed by PCR using specific primers. During the study period, polymyxin B resistance rate in K. pneumoniae increased 30.6% from 2009 to 2015. Changes in mgrB gene were detected in 33.3% of polymyxin B-resistant isolates. However, none of the isolates carried the mcr-1 plasmid gene. Regarding resistance to beta-lactams, high levels of resistance were observed for carbapenems (>88.9%), 3rd and 4th generation cephalosporins (94.4%) and piperacillin/tazobactam (97.2%). The most active drug against polymyxin B-resistant K. pneumoniae isolates was gentamicin, followed by tigecycline (52.8% of sensitivity), and fosfomycin (50.0% sensitivity). The majority of polymyxin B-resistant K. pneumoniae isolates was classified as extensively drug-resistant (XDR), and one isolate was classified as pandrug resistant (PDR). The blaKPC-2 gene was observed in 77.8% of K. pneumoniae isolates resistant to polymyxin B. Distinct resistant determinants (blaSHV-like genes blaTEM-like, blaCTX-M-1/2-like, and blaCTX-M-14-like) were also detected in such isolates. In addition, the rmtB gene, related to high-level resistance to aminoglycosides, was detected in six isolates obtained from the year 2014. The analysis of clonal relationship showed the existence of ten different clonal groups among K. pneumoniae isolates circulating during 2009 to 2015. In 2014, the XDR clone C7, carrying blaSHV-like, blaTEM-like, blaKPC-2, blaCTX-M-1/2-like, blaCTX-M-14-like, and rmtB, emerged in the hospital setting and spread in the following year. Article 2 The aim was to evaluate the mechanisms involved in resistance to polymyxin B among K. pneumoniae isolates belonged to the same clonal pattern. A total of 14 isolates recovered from blood cultures of seven distinct hospitalized patients, being one isolate susceptible and another resistant to polymyxin B, were evaluated. The minimum inhibitory concentration to polymyxin B was confirmed by broth microdilution, and for the other antimicrobials, the susceptibility testing was performed by Phoenix® automated system. All 14 isolates were resistant to the broad¬spectrum cephalosporins, ciprofloxacin and combination beta-lactams betalactamase inhibitors. The blaKPC-2 gene was detected in 12/14 K. pneumoniae isolates. Both isolates from each patient had their genetic similarity confirmed by PFGE. The pmrA, pmrB, pmrD, phoB, phoQ, and mgrB genes were sequenced and compared. Among the seven polymyxin B-resistant isolates, five showed modifications in mgrB. Four of them showed the presence of insertion sequences (IS903b and ISKpn13) inactivating this gene, and one isolate presented a deletion of 12 nucleotides in the mgrB. Mutations in PhoP and PmrB proteins, which belong to the two-component systems PmrAB and PhoPQ, have also been observed separately in two polymyxin B-resistant isolates. These results demonstrated that the polymyxin B resistance may occur during therapy with this antimicrobial agent leading to the therapeutic failure, especially by mobilization of IS that inactivate the mgrB.