Multidrug-resistant Klebsiella pneumoniae harboring extended spectrum β-lactamase encoding genes isolated from human septicemias

Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum beta -lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of beta -lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZ(R)) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (bla(CTX-M), bla(SHV), bla(TEM), bla(KPC), bla(NDM,)bla(VIM,)bla(OXA-48,)bla(CMY-2), bla(DHA-1,)bla(FOX,)bla(MOX,) and bla(ACC)) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6')-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZ(R)K. pneumoniae isolates and 11 of them carried the bla(CTX-M-15) gene (together with bla(TEM))(,) and the remaining isolate carried the bla(SHV-106) gene. All the bla(CTX-M-15) harboring isolates also contained a bla(SHV) gene (bla(SHV-1), bla(SHV-11) or bla(SHV-27) variants). Both bla(SHV-27) and bla(SHV-106) genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (bla(KPC2/3) and bla(OXA-48)) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.