Objectives: The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. Methods: Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regarding resistance genes, sequence types, virulence factors and plasmid types. Results: Statistical significance was shown by the presence of an infection if there had been a previous hospital stay; urinary catheter carriage and chronic renal disease also indicated higher probabilities of being infected. More than 95% of the isolates were non-susceptible to third-generation cephalosporins, and more than 90% were non-susceptible to quinolones. Phenotypic and genotypic methods for resistance detection were concordant and later confirmed by NGS. This is the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area. No plasmid-mediated colistin resistance was found. Tetracycline, sulfonamides and aminoglycoside resistance genes were found in almost all the isolates studied. No virulence factors were detected. Multilocus sequence typing showed more than 15 different sequence types, with ST101, ST307 and ST11 being the most prevalent. Conclusions: This study is the first to report such a large group of OXA-48 carbapenemases with clonal dissemination among carbapenem-resistant K. pneumoniae in Valencia. This is also the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area.

Klebsiella pneumoniae, a major cause of both hospital and community-acquired infections, is listed by the World Health Organization as a critical priority antibiotic- resistant bacterial pathogen. With the appearance of sequencing techniques such as Next-generation Sequencing (NGS), there is the possibility to obtain the whole genome of the bacteria, getting to know all antimicrobial resistance determinants. The purpose of this study has been to apply this new technology to clinical microbiology, in order to characterize the resistome present in carbapenem-resistant K.pneumoniae strains isolated in a tertiary hospital in Valencia, Spain. A total of 234 isolates were prepared for whole-genome sequencing with Ilumina MiSeq, and sequences were later studied for antimicrobial resistance genes, sequence-typing and plasmids. Sequence-typing showed four major circulating clones in our hospital settings: ST11, ST307, ST101 and ST147, carrying different plasmids and different resistance determinants such as OXA-48 and NDM-1 carbapenemase. Application of new technologies such as whole-genome sequencing in clinical microbiology gives advantages when it comes to rapid therapy adjustment, consequently improving the patient’s clinical outcomes.