Epidemiologia de la carbapenemasa OXA-48 en aïllats de Klebsiella pneumoniae a Catalunya

Abstract

The OXA-48 carbapenemase is widely spread around the world and found mainly in Enterobacteriaceae. In Catalonia, OXA-48-producing Klebsiella pneumoniae was first detected in 2009, since then, a growing prevalence has been observed in different hospitals, which prompted the aim of this multicenter study, which includes 11 regional hospitals and the Hospital de Santa Creu i Sant Pau, is the characterization, both epidemiological and molecular, of OXA-48–producing K. pneumoniae strains isolated throughout 2012. The study included all K. pneumoniae strains with an unusual pattern of resistance to beta-lactam antibiotics. Selected strains underwent a modified Hodge test (MHT) to detect any enzyme activity against carbapenemics. A total of 3,901 K. pneumoniae strains, 171 MHT-positive strains (4.4%) were selected. PCR confirmed that 85 strains carried OXA-48 (49.7%). Eighty nine percent of the 85 OXA-48–producing strains coexpressed the ESBL CTX-M-15, with or without beta-lactamases OXA-1 and TEM-1. The clonality of these 85 OXA-48–producing strains was studied by macrorestriction analysis (PFGE) and multilocus sequence typing (MLST). Five clones were observed by PFGE: A, B, C, D and E. These five clones were perfectly correlated with the five sequence types found, ST101, ST17I, ST1233, ST14 and ST405. ST1233 is described for the first time in this study. The blaOXA-48 gene was found in a conjugative plasmid of incompatibility group IncL (aprox. 62 Kb) and located in Tn1999.2 (91.7%). The other beta-lactamase genes of these strains were not found in this plasmid. Of the 85 OXA-48–producing strains, 75 (88.23%) harboured the qnrB gene. Genes involved in enzyme-mediated resistance against aminoglycosides were also studied. Of the 85 OXA-48–producing strains, 82 showed resistance to some of the aminoglycosides studied. The major phenotype showed resistance to kanamycin, gentamicin and tobramycin (KTG), and it was explained by the presence of aac(3')-IIa (KTGN) and aac(6’)-Ib (KTAN) genes. We observed that genetically related strains (that is, closely related PFGE, identical ST and the presence of the same resistance genes) showed different levels of resistance. These differences couldn’t be due to alterations in the expression of the efflux pump AcrAB or in porins. Results obtained by classical techniques (PCR, sequencing, PFGE and MLST) in 37 strains selected were compared with those of the new techniques of massive sequencing of microbial genomes (Whole Genome Sequencing: WGS) and cgMLST (core genome MultiLocus Sequence Typing). A good correlation was found between PFGE-MLST and cgMLST, and four strains were identical by cgMLST but not by PFGE. After uploading the sequences obtained by WGS to the PlasmidFinder and ResFinder online search tools, we were able to identify recently classified plasmids and determine the presence of other resistance genes, such as: strA / strB, oqxA / oqxB, drfA, sul2, fosA, catB3, tet(A) and tet(D). The use of WGS allowed us to obtain sequences of the targets of the quinolones, the QRDR. Surprisingly, only alterations described as responsible for this resistance were detected in all three ST101 strains. In conclusion, the increase in the prevalence of OXA-48-carrying K. pneumoniae in Catalonia is due to the expansion of the ST405 and ST101 clones in all hospitals where strains were isolated. No differences in the genetic background of the blaOXA-48 gene were found, as reported elsewhere. Moreover, WGS was found to be a useful tool for the molecular and epidemiological analysis of outbreaks caused by multidrug-resistant strains.

Date of Award	14 Jun 2016
Original language	Catalan
Awarding Institution	Hospital de la Santa Creu i Sant Pau
Supervisor	Elsa Miró Cardona (Director) & Ferran Navarro Risueño (Director)