Application of Next-Generation Sequencing to Enterobacter Hormaechei Subspecies Analysis during a Neonatal Intensive Care Unit Outbreak

Patrick Morhart; Roman G Gerlach; Caroline Kunz; Jürgen Held; Giuseppe Valenza; Joachim Wölfle; Heiko Reutter; Gregor J Hanslik; Fabian B Fahlbusch

doi:10.3390/children10101696

Application of Next-Generation Sequencing to Enterobacter Hormaechei Subspecies Analysis during a Neonatal Intensive Care Unit Outbreak

Children (Basel). 2023 Oct 16;10(10):1696. doi: 10.3390/children10101696.

Authors

Patrick Morhart¹, Roman G Gerlach², Caroline Kunz², Jürgen Held², Giuseppe Valenza², Joachim Wölfle³, Heiko Reutter¹, Gregor J Hanslik¹, Fabian B Fahlbusch⁴

Affiliations

¹ Division of Neonatology and Paediatric Intensive Care Medicine, Department of Paediatrics and Adolescent Medicine, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
² Institute of Microbiology-Clinical Microbiology, Immunology and Hygiene, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
³ Department of Paediatrics and Adolescent Medicine, Friedrich-Alexander-University of Erlangen-Nürnberg, 91054 Erlangen, Germany.
⁴ Neonatology and Pediatric Intensive Care, Faculty of Medicine, University of Augsburg, 86156 Augsburg, Germany.

Abstract

Introduction: The Enterobacter cloacae complex (ECC) species are potential neonatal pathogens, and ECC strains are among the most commonly encountered Enterobacter spp. associated with nosocomial bloodstream infections. Outbreaks caused by ECC can lead to significant morbidity and mortality in susceptible neonates. At the molecular level, ECC exhibits genomic heterogeneity, with six closely related species and subspecies. Genetic variability poses a challenge in accurately identifying outbreaks by determining the clonality of ECC isolates. This difficulty is further compounded by the limitations of the commonly used molecular typing methods, such as pulsed field gel electrophoresis, which do not provide reliable accuracy in distinguishing between ECC strains and can lead to incorrect conclusions. Next-generation sequencing (NGS) offers superior resolution in determining strain relatedness. Therefore, we investigated the clinical pertinence of incorporating NGS into existing bundle measures to enhance patient management during an outbreak of ECC in a level-3 neonatal intensive care unit (NICU) in Germany.

Methods: As the standard of care, all neonates on the NICU received weekly microbiological swabs (nasopharyngeal and rectal) and analysis of endotracheal secretion, where feasible. During the 2.5-month outbreak, colonisation with ECC was detected in n = 10 neonates. The phylogenetic relationship and potential antimicrobial resistance genes as well as mobile genetic elements were identified via bacterial whole-genome sequencing (WGS) using Illumina MiSeq followed by in silico data analysis.

Results: Although all ECC isolates exhibited almost identical antimicrobial susceptibility patterns, the WGS data revealed the involvement of four different ECC clones. The isolates could be characterised as Enterobacter hormaechei subspecies steigerwaltii (n = 6, clonal), subsp. hoffmannii (n = 3, two clones) and subsp. oharae (n = 1). Despite the collection of environmental samples, no source of this diffuse outbreak could be identified. A new standardised operating procedure was implemented to enhance the management of neonates colonised with MRGN. This collaborative approach involved both parents and medical professionals and successfully prevented further transmission of ECC.

Conclusions: Initially, it was believed that the NICU outbreak was caused by a single ECC clone due to the similarity in antibiotic resistance. However, our findings show that antibiotic susceptibility patterns can be misleading in investigating outbreaks of multi-drug-resistant ECC. In contrast, bacterial WGS accurately identified ECC at the clonal level, which significantly helped to delineate the nature of the observed outbreak.

Keywords: ECC; Enterobacter; MRGN; NGS; NICU; WGS.

Grants and funding

This study was supported by a grant (BBST-D-20-00029) from the B. Braun foundation, Melsungen, Germany, for infection control (inpatient/outpatient). We gratefully acknowledge the generous support of Manfred Roth-Stiftung, Fürth, Germany (to R.G.G). The funding sources were neither involved in the design and execution of this study nor in the data analysis, interpretation or decision to publish the results. We further acknowledge the financial support from “Deutsche Forschungsgemeinschaft” and Friedrich-Alexander-University of Erlangen-Nürnberg within the funding programs “Open Access Publication Funding”.