Abstract

Background and Objectives

Serratia marcescens is an emerging nosocomial pathogen, and the carbapenemase blaNDM has been reported in several surveys in Romania. We aimed to investigate the molecular epidemiology of S. marcescens in two Romanian hospitals over 2010–15, including a neonatal NDM-1 S. marcescens outbreak.

Methods

Isolates were sequenced using Illumina technology together with carbapenem-non-susceptible NDM-1-positive and NDM-1-negative Klebsiella pneumoniae and Enterobacter cloacae to provide genomic context. A subset was sequenced with MinION to fully resolve NDM-1 plasmid structures. Resistance genes, plasmid replicons and ISs were identified in silico for all isolates; an annotated phylogeny was reconstructed for S. marcescens. Fully resolved study NDM-1 plasmid sequences were compared with the most closely related publicly available NDM-1 plasmid reference.

Results

44/45 isolates were successfully sequenced (S. marcescens, n = 33; K. pneumoniae, n = 7; E. cloacae, n = 4); 10 with MinION. The S. marcescens phylogeny demonstrated several discrete clusters of NDM-1-positive and -negative isolates. All NDM-1-positive isolates across species harboured a pKOX_NDM1-like plasmid; more detailed comparisons of the plasmid structures demonstrated a number of differences, but highlighted the largely conserved plasmid backbones across species and hospital sites.

Conclusions

The molecular epidemiology is most consistent with the importation of a pKOX_NDM1-like plasmid into Romania and its dissemination amongst K. pneumoniae/E. cloacae and subsequently S. marcescens across hospitals. The data suggested multiple acquisitions of this plasmid by S. marcescens in the two hospitals studied; transmission events within centres, including a large outbreak on the Targu Mures neonatal unit; and sharing of the pKOX_NDM1-like plasmid between species within outbreaks.

Topic:
 
Issue Section:
 ORIGINAL RESEARCH

Introduction

Carbapenemase-producing Enterobacteriaceae (CPE) have emerged post-1990 and spread worldwide, representing a serious clinical threat.1 Although carbapenem resistance can arise through various mechanisms, acquisition of genes (blaKPC, blaNDM-1, blaOXA-48) encoding Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM) and OXA-48 carbapenemase, respectively, are currently of most concern. These genes are readily transmitted intra- and inter-species via mobile genetic elements that have facilitated their worldwide dissemination.2

Compared with Escherichia coli and K. pneumoniae, two of the most frequently identified CPE species, Serratia marcescens has been less commonly associated with any of the major transmissible carbapenemases, although acquisition of blaKPC has been described recently in China, Greece and the United States.3–5,S. marcescens has also traditionally been regarded as an opportunistic pathogen causing disease in specifically vulnerable populations, such as neonates.6,7 However, Serratia spp. have become increasingly adapted to hospital environments and have emerged as important agents of hospital-acquired infections affecting all age groups.8–10 The inherent resistance of Serratia spp. to multiple antimicrobial classes may have facilitated this adaptation, and leaves few therapeutic options open for treating carbapenem-resistant S. marcescens infections.11–13

blaNDM-1 has been increasingly reported in patients with healthcare exposure on the Indian subcontinent or in the Balkan region.1,14,15 The occurrence of blaNDM-1 in S. marcescens appears rare, with only a handful of isolates identified in Germany,16 the UK17 and Egypt.18 However, a recent faecal screening/surveillance project for carbapenemase-producing Gram-negative bacteria in three Romanian hospitals identified 17 NDM-1 S. marcescens in clinical, environmental and screening isolates,19 all of which were from the same neonatal unit at a single hospital, consistent with an outbreak. PCR-based plasmid analyses identified blaNDM-1 in association with conjugative IncFII plasmids in both S. marcescens and K. pneumoniae, suggesting inter-species transfer of blaNDM-1, possibly from K. pneumoniae into a well-established hospital-adapted S. marcescens clone.

In this study, we used Illumina (short-read) and MinION (long-read) WGS of NDM-1- and non-NDM-1 S. marcescens and other Enterobacteriaceae isolates obtained from: (i) the faecal screening/surveillance study; (ii) a previous outbreak and sporadic S. marcescens infections in this neonatal unit; and (iii) a second hospital in an adjacent county, to investigate the evidence for nosocomial and inter-hospital dissemination of NDM-1 S. marcescens and the transmission of NDM-1 plasmids amongst Enterobacteriaceae in this context in Romania