KPC-3-Producing Klebsiella pneumoniae in Portugal Linked to Previously Circulating Non-CG258 Lineages and Uncommon Genetic Platforms (Tn4401d-IncFIA and Tn4401d-IncN)

Carla Rodrigues,José Amorim,Luísa Peixe,Jan Bavlovič,Elisabete Machado,Ângela Novais

doi:10.3389/fmicb.2016.01000

Abstract

KPC-3-producing bacteria are endemic in many countries but only recently became apparent their wide distribution in different Portuguese hospitals. The aim of this study is to characterize genetic backgrounds associated with blaKPC−3 among Klebsiella pneumoniae isolates recently identified on non-hospitalized patients in Portugal. Twenty KPC-producing K. pneumoniae identified between October 2014 and November 2015 in three different community laboratories were characterized. Isolates were mainly from patients from long-term care facilities (n = 11) or nursing homes (n = 6), most of them (75%) previously hospitalized in different Portuguese hospitals. Standard methods were used for bacterial identification and antibiotic susceptibility testing. Carbapenemase production was assessed by the Blue-Carba test, and identification of bla genes was performed by PCR and sequencing. Epidemiological features of KPC-producing K. pneumoniae included population structure (XbaI-PFGE, MLST and wzi sequencing), genetic context (mapping of Tn4401), and plasmid (replicon typing, S1-PFGE, and hybridization) analysis. All K. pneumoniae isolates produced KPC-3, with two MDR K. pneumoniae epidemic clones representing 75% of the isolates, namely ST147 (wzi64/K14.64, February–November 2015) and ST15 (two lineages exhibiting capsular types wzi19/K19 or wzi93/K60, July-November 2015). Other sporadic clones were detected: ST231 (n = 3; wzi104), ST348 (n = 1; wzi94) and ST109 (n = 1, wzi22/K22.37). blaKPC−3 was identified within Tn4401d in all isolates, located in most cases (80%) on cointegrated plasmids (repAFIA+repAFII+oriColE1;105-250 kb) or in 50 kb IncN plasmids. In conclusion, this study highlights a polyclonal structure of KPC-3-producing K. pneumoniae and the predominance of the ST147 clone among non-hospitalized patients in Portugal, linked to platforms still unnoticed in Europe (blaKPC−3-Tn4401d-IncFIA) or firstly reported (blaKPC−3-Tn4401d-IncN). This scenario underlines the recent penetration of successful mobile genetic elements in previously circulating MDR K. pneumoniae lineages (mainly ST147 and ST15) in Portugal, rather than the importation of the global lineages from clonal group 258.

Highlights

In the last years, carbapenem-resistant Enterobacteriaceae have spread globally, being responsible for high rates of morbidity and mortality among healthcare-associated infections, mainly due to the depletion of effective therapeutic options (WHO, 2014; Albiger et al, 2015, http://www.cdc.gov/drugresistance/threat-report-2013/)
All isolates produced Klebsiella pneumoniae carbapenemases (KPCs)-3 and demonstrated resistance or intermediate phenotypes to ertapenem (MIC = 1 to 16 mg/L), and susceptible, intermediate or resistance phenotypes to imipenem (MIC = 2 to 16 mg/L) and meropenem (MIC = 1 to 8 mg/L), with colonies growing within the inhibition zone of all carbapenems tested, a hetero-resistance phenotype usually observed for KPC producers (Nordmann et al, 2009; Table 2)
For some isolates the MIC values for imipenem and meropenem were interpreted as susceptible by the clinical breakpoints defined by EUCAST, in all cases they were above the epidemiological cut-off values (ECOFFs) defined for K. pneumoniae

Summary

Introduction

Carbapenem-resistant Enterobacteriaceae have spread globally, being responsible for high rates of morbidity and mortality among healthcare-associated infections, mainly due to the depletion of effective therapeutic options (WHO, 2014; Albiger et al, 2015, http://www.cdc.gov/drugresistance/threat-report-2013/). The blaKPC genes are commonly located on Tn4401, a 10 kb Tn3-like transposon delimited by two 39-bp imperfect inverted repeat sequences harboring blaKPC, transposase and resolvase genes, and insertion sequences ISKpn (upstream blaKPC) and ISKpn (downstream blaKPC; Chen et al, 2014b). It is recognized as a highly active transposon enhancing the spread of blaKPC genes to different plasmid scaffolds (Cuzon et al, 2011). The acquisition of blaKPC by plasmids from different incompatibility groups (IncFIIK2, IncFIA, IncI2, IncN, IncX3, ColE), favored a quick intra- and inter-species dissemination (Chen et al, 2014b)

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