Tracing Klebsiella pneumoniae in intensive care units using genomic methods and a new rapid Sequence Type assignment technique: Three years of molecular epidemiology results

Introduction: Carbapenem-resistant Klebsiella pneumoniae is an opportunistic pathogen causing outbreaks in healthcare settings. In hospitals with high K. pneumoniae prevalence, identifying outbreaks is particularly difficult because of recurrent carbapenem-resistant strains. In this study we aimed to describe K. pneumoniae circulation in four intensive care units (ICUS) of a large teaching hospital using a new PCR-based method for rapid detection of major sequence types (STs). Materials and methods: A comparative genome study was conducted to identify unique genes in the most relevant STs circulating in Italy (ST11, ST37, ST101, ST147, ST258/ST512, ST307, ST395). These genes were used to set up multiplex-PCR assays (Rapid STyping) to quickly identify the STs. Results were compared to Multi-Locus Sequence Typing (MLST) and pulsed-field gel electrophoresis. Representative strains underwent whole genome sequencing (WGS) to confirm their relatedness and enable in-depth genomic and epidemiological analyses. Results: A total of 179 strains were collected over a three-year period; of these, 125 were carbapenem-resistant. Rapid STyping accurately identified ST37, ST101, ST307, and ST512, as confirmed by MLST. Single Nucleotide polymorphism (SNP) analysis of representative strains identified six clonally related clusters. ST512 clone was the most prevalent, but SNP analysis suggested several separated introductions along time. Other relevant outbreaks were sustained by ST307 and ST101 strains. Conclusions: The new approach was proven able to accurately assign isolates to specific STs with reduced time and costs. Results of the analyses showed that outbreak-causing strains often spread across multiple wards. Enhanced surveillance and better containment measures are needed to avoid cross-transmission.