Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Accurate Identification of Enterococcus lactis Causing Bacteraemia by MALDI-TOF Mass Spectrometry and Multiplex qPCR (104363)

Marhami Fahriani 1 , Geoffrey W Coombs 1 2 3 , Princy Shoby 1 , Haley Hood 1 , Denise Daley 2 3 , Christopher A Mullally 1 4 , Shakeel Mowlaboccus 1 2 3 4
  1. Antimicrobial Resistance and Infectious Diseases Research Laboratory, Murdoch University, Murdoch, Western Australia
  2. Department of Microbiology, PathWest Laboratory Medicine-WA, Fiona Stanley Hospital, , Murdoch, Western Australia
  3. Australian Group on Antimicrobial Resistance, Fiona Stanley Hospital, Murdoch, Western Australia
  4. School of Biomedical Sciences, The University of Western Australia, Perth, Western Australia

Background: Enterococcus faecium is responsible for a significant proportion of hospital-associated infections. Historically, E. faecium has been categorised into two phylogenetically distinct clades, A and B. However, in 2021, E. faecium clade B isolates were reclassified as Enterococcus lactis, a different species. Most diagnostic laboratories still report clinical E. lactis isolates as E. faecium because the MALDI Biotyper® lacks a main spectrum profile for E. lactis. This study aims to construct an in-house E. lactis MALDI Biotyper® database and additionally design a multiplex qPCR assay to rapidly identify and differentiate E. lactis from E. faecium.

Methods: A total of 51 E. faecium and 37 E. lactis isolates identified from bacteraemia cases across Australia were included. Short-read whole genome sequencing (Illumina) was used to confirm species identification. The in-house E. lactis MALDI Biotyper® database was constructed using the ethanol/formic acid/acetonitrile protocol and validated using the extended direct transfer protocol. The gdh locus was used as target for the multiplex qPCR assay which was performed on the QuantStudio™ 6 Flex platform with a cut-off Ct-value of <35. A total of 1 ng DNA was used per reaction.

Results: The E. faecium and E. lactis isolates were genetically diverse, represented by 51 and 21 unique sequence types (STs), respectively. All E. lactis isolates were misidentified as E. faecium by the original BioTyper® reference library (2022). Following implementation of the constructed in-house database, all E. lactis isolates were accurately identified to the species level with a score >2.000. Primers binding to the gdh locus in E. faecium and E. lactis generated a 103-bp and 131-bp amplicon, respectively. The HEX probe was used for E. faecium and the FAM probe was used for E. lactis. All E. lactis and E. faecium isolates were accurately identified using the multiplex qPCR assay, with Ct-values ranging from 18.51±0.74 to 20.60±0.55 and 18.08±0.27 to 20.58±0.34, respectively.

Conclusion: We have successfully constructed a custom MALDI Biotyper® database and a multiplex qPCR assay to differentiate E. lactis from E. faecium.