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Evolution of clonal complex 17 Enterococcus facium: The rise and fall of outbreaks

Lee, Terence Wenjun (2019) Evolution of clonal complex 17 Enterococcus facium: The rise and fall of outbreaks. PhD thesis, Murdoch University.

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Enterococcus faecium is an opportunistic pathogen which has evolved from a commensal of the mammalian gastrointestinal tract microbiome to one of the leading causes of nosocomial infections globally. Hospital-adapted E. faecium strains, identified as members of clonal complex (CC)17, typically harbour a variety of virulence factors and antimicrobial resistance genes, including resistance to vancomycin, an essential glycopeptide antimicrobial used to treat severe Gram-positive infections. In Australia, the Australian Group on Antimicrobial Resistance (AGAR) Australian Enterococcal Sepsis Outcome Program (AESOP) identified approximately 50% of E. faecium causing blood stream infections (BSI) from 2015-2017 as 48 vancomycin resistant. Compared to similar reports, the prevalence of vancomycin resistant E. faecium (VREfm) in Australia was higher than any nation within the European Economic Area (Highest, 43.9%, Cyprus, 2017) suggesting a serious and growing public health issue in Australia.

To determine if VREfm in Australia were becoming more virulent and/or antimicrobial resistant, vancomycin non-susceptible E. faecium collected as part of a 15-year surveillance program monitoring patients admitted to high risk units at Royal Perth Hospital (RPH) were whole genome sequenced (WGS) and analysed in silico. Of the 1,372 vancomycin non-susceptible E. faecium collected, 93.7% of isolates belonged to seven sequence types (STs) which were responsible for eight single strain outbreaks occurring mostly from 2010 to 2015. Except for ST173, outbreak causing STs at RPH were pandemic strains which have been previously reported internationally and across Australia. Phylogenetically, isolates were clustered according to STs with each cluster emerging from independent branches suggesting outbreak STs were distantly related. Virulence and antimicrobial profiles for each ST were shown to be unique with no increase in virulence or antimicrobial resistance observed with respect to the chronological order of outbreaks or phylogenetic order. Multiple analogous antimicrobial resistance genes were identified indicating the independent acquisition of resistance amongst the STs and suggesting its importance in strain survival.

With no increase in virulence and antimicrobial resistance observed in pandemic strains of E. faecium at RPH, subsequent investigations to determine the cause of high VREfm prevalence 68 in Australia focused on E. faecium collected by the AGAR AESOP. Each year, the AGAR AESOP monitors levels of antimicrobial resistance in Enterococci causing BSI throughout Australia. Since 2015, a WGS approach to surveillance was adopted by AGAR for AESOP sequencing all E. faecium referred by participating laboratories. Using bioinformatics, E. faecium from 2015-2017 were shown to form three main phylogenetic clusters consisting of (i) predominantly vancomycin susceptible isolates which did not harbour any van operons, (ii) predominantly VREfm isolates harbouring the vanA operon and (iii) predominantly VREfm isolates harbouring the vanB operon. Further investigations of the two VREfm dominant clusters responsible for the high prevalence of VREfm in Australia revealed isolates located on distal phylogenetic branches showed increased prevalence of the respective vanA or vanB operons compared to basal isolates. The findings suggest the development of an affinity between certain E. faecium strains with a particular van genotype. As a result, the majority of ST1421 and ST1424 isolates located on the distal branches of the vanA dominant cluster, harboured the vanA operon while the majority of ST555 and ST796 isolates located on the distal branches of the vanB dominant cluster, harboured the vanB operon. Additionally, in clusters with increased van operon prevalence, an increased 30-day all-cause patient mortality and increased number of isolates was observed.

The vanA and vanB operons conferring vancomycin resistance identified in AGAR AESOP isolates are located on mobile genetic elements which can be transferred between bacteria. To investigate the transmission of van operons, additional insertion sequences (ISs) identified between van genes were used to type each van operon. Five IS elements were identified within vanA operons while three IS elements were identified within vanB operons. In combination with van genes, eight vanA operon types and five vanB operon types were identified in the 2015-2017 AGAR AESOP collection of E. faecium isolates. The majority of IS elements were identified in intergenic regions between the regulatory, essential and accessory gene groups of the vanA and vanB operon. Phylogenetic analysis showed vanA and vanB operons clustered according to isolate STs suggesting van operons were mostly inherited via vertical transmission. The operon from vanA dominant strains ST1421 and ST1424, and the operon from vanB dominant strains ST555 and ST796 were distantly related to international reference operons suggesting that the majority of locally distributed vanA and vanB operons in Australia have evolved independently rather than from international VREfm.

In conclusion, pandemic strains of VREfm in Australia showed no indication of increasing virulence or antimicrobial resistance over time. Analysis of E. faecium causing BSI in Australia showed three phylogenetic clusters, of which, two were dominant for the vanA and vanB operon respectively and were responsible for the high VREfm prevalence observed in Australia. Both VREfm dominant clusters showed increasing prevalence of the respective vanA or vanB operons in isolates located toward the distal branches of the phylogenetic tree suggesting a strain-van operon affinity. Typing of the vanA and vanB operons suggests van operons identified in Australia were locally distributed and are distantly related to van operons from international VREfm.

Item Type: Thesis (PhD)
Murdoch Affiliation: College of Science, Health, Engineering and Education
Supervisor(s): Coombs, Geoffrey, Abraham, Sam and Pang, Stanley
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