A Long-Term Low-Frequency Hospital Outbreak of KPC- Producing Klebsiella pneumoniae Involving Intergenus Plasmid Diffusion and a Persisting Environmental Reservoir Sta ˚le Tofteland 1 *, Umaer Naseer 2,3 , Jan Helge Lislevand 1 , Arnfinn Sundsfjord 2,3 , Ørjan Samuelsen 2 * 1 Department of Clinical Microbiology, Sørlandet Hospital HF, Kristiansand, Norway, 2 Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway, 3 Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway Abstract Background: To study the molecular characteristics of a long-term, low frequency outbreak of bla KPC-2 in a low prevalence setting involving the hospital environment. Methodology/Principal Findings: KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. Bla KPC-2 -plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ,97 kb IncFII plasmid was seen to carry bla KPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the bla KPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins. Conclusions/Significance: We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying bla KPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak. Citation: Tofteland S, Naseer U, Lislevand JH, Sundsfjord A, Samuelsen Ø (2013) A Long-Term Low-Frequency Hospital Outbreak of KPC-Producing Klebsiella pneumoniae Involving Intergenus Plasmid Diffusion and a Persisting Environmental Reservoir. PLoS ONE 8(3): e59015. doi:10.1371/journal.pone.0059015 Editor: Jan Kluytmans, Amphia Ziekenhuis, The Netherlands Received November 26, 2012; Accepted February 8, 2013; Published March 11, 2013 Copyright: ß 2013 Tofteland et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by a fellowship from SA ˜ ¸ rlandet Hospital HF and a research grant from the Northern Norway Regional Health Authority Medical Research Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: staale.tofteland@sshf.no Introduction The Klebsiella pneumoniae carbapenemase (KPC) was first identified in the USA in a Klebsiella pneumoniae isolate dated from 1996[1] and has subsequently been reported worldwide.[2] High prevalence rates of KPC have been reported from regions and countries such as USA, Israel, China, and Greece, while a number of countries have reported hospital outbreaks and sporadic cases of KPC-producing K. pneumonia. [2] KPC has been identified in various species of Enterobacteria- ceae and non-fermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii. [2] Further, bla KPC has been identified on plasmids differing in size and structure. This broad species distribution and plasmid diversity is likely due to the location of bla KPC in a functional Tn3-based transposon structure (Tn4401) with a high transposition frequency.[3] With respect to K. pneumoniae, multilocus sequence typing (MLST) has shown that the global dissemination of KPC-producing K. pneumoniae is dominated by isolates belonging to a hyperepidemic clonal complex including sequence type (ST) 258.[4] Although the dissemination and outbreaks of KPC seems to be associated with specific clones several reports are describing outbreaks where several clones and different species are involved [5,6] as well as individual patients containing different species harbouring bla KPC. [7,8,9] In many countries the emergence of KPC-producing bacteria has been associated with import of isolates from high prevalent areas. In Norway, the first case of K. pneumoniae with KPC was associated with import from Greece in 2007 at a hospital in the southern part of Norway.[10] Subsequently, five additional clinical isolates were identified at the same hospital and a nearby hospital from patients with no recent history of travel or hospitalisation abroad. In this study we describe this long-term outbreak with regards to the molecular characteristics of these isolates, plasmid PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e59015