Resumo

Since the initial report of carbapenemase KPC-2 in 2001, the spread of carbapenem resistant Enterobacteriaceae became one of major clinical and public health challenge. Here we report the simultaneous infection with KPC-2-producing Escherichia coli and Providencia stuartii in a 43-year-old HIV-infected patient admitted in the intensive care unit of a university hospital in Recife, Brazil, in 2011. Carbapenem-resistant Isolates were originated from blood (E. coli 670) and two urine samples (P. stuartii 388 and P. stuartii 426), collected after 8 and 11 days of E.coli isolation. PFGE was used to investigate the clonal relationship of P. stuartii isolates. The MIC determinations were carried out by broth microdilution method and interpreted according to CLSI (2013). Phenotypic screening for carbapenemases was investigated by the modified Hodge test. Plasmidial extraction was conducted according the Kieser method and used to transform quimiocompetent E. coli DH5α cells. The transformants were selected on Mueller-Hinton agar containing 100µg/mL ampicillin. The isolates and transformants were tested for the presence of class 1 integron, ESBL and class A β-lactamase genes by PCR. The amplification products were further sequenced. The characterization of transposon Tn4401 isoform was conducted by analysis of the genetic environment of blaKPC gene. PFGE analysis indicated that the P. stuartii isolates 388 and 426 were genetically indistinguishable. All donor strains were positive for carbapenemase production by the modified Hodge test and harbored blaKPC-2, blaTEM-1, class 1 integrase gene and Tn4401b isoform. Plasmid analysis revealed that the isolates P.stuartii and E.coli share one plasmid with ~65 kb, indicating the possibility of in vivo plasmid transfer. The transformant cells also showed the presence of ~65 kb plasmid, the gene blaKPC−2 and the Tn4401b isoform. MIC results showed that clinical isolates of E coli and P. stuartii were not susceptible to none of the β-lactams tested. However, E coli transformant cells, despite the presence of the blaKPC-2, were susceptible to imipenem, meropenem, cefotaxime and ceftazidime, indicating differences in gene expression levels. By contrast, P. stuartii transformant cells showed MIC values above of their correspondent clinical isolates. The presence of blaKPC-2 gene and Tn4401b harbored by a plasmid of the same size suggests the in vivo transfer of this mechanism of resistance among the species studied.