Genomic epidemiology of multiple Acinetobacter baumannii outbreaks in a veterinary intensive care unit

Aldert Zomer Utrecht University

Acinetobacter baumanni

• Acinetobacter > 20 species

• greatest clinical importance : Acinetobacter baumannii

• Gram (-) coccobacilli

• Survive under dry & iron-deficient conditions

• polysaccharide capsule(s): prevent complement activation, delay phagocytosis

• Fimbriae (adhere to human bronchial epithelium)

• Pili (colonization of environmental surface to form biofilms)

What’s really special about Acinetobacter?

• It causes both invasive disease and pneumonia in animals and humans and has a high innate resistance to many antimicrobial compounds

• Sequence analysis of a multiresistant strain, combined with comparative genomics, has revealed an 86-kb ‘resistance island’ which contains a cluster of 45 different resistance genes (analogous to SCCmec) (Fournier et al., PLoS Genet. 2006 Jan;2(1):e7)

Nosocomial infections, high level of drug resistance, difficult to eradicate from the environment

A. baumanni outbreaks at the ICU veterinary faculty Utrecht University

Clinical infectiology

Companian animal clinic

A. baumanni outbreaks at the ICU

Entrance

Receiving

ER

Operating theatre

ICU

Medium care Ward

Companion animal ICU UU “Intensieve Zorgafdeling (IZa)” Two outbreaks of A. baumanni in 2012 and 2014 4 dogs infected in 2012 2 dogs infected in 2014

Timeline 2012 outbreak

June July August September October

= Days admitted to ICU

2012

Jack Russell, 2yr. Ate owners’ Baclofen (spasmolyticum). Developed total paralysis. 20/6 intubation at ICU. 22/6 Detubated, tube cultured: A. baumannii. No treatment Dachshund, 7 yr. Abscesses shoulder, Th/ amoxicillin/clavulanic acid. Developed renal insufficiency 24/7 admitted to ICU 30/7 Culture catheter urine: A. baumannii 105 bact/ml. Th/ neomycin († 3/9) Labrador, 8 yr. Joint problems, Th/ NSAIDs. Developed renal insufficiency 5/8 ICU, in isolation room (adjacent to ICU), because of leptospirosis in the DDx. 4/9 Urine culture: A. baumannii 106 bact/ml. Th/ Amikacin Chihuahua, 5 yr. Bitten by other dog. 26/8 ICU, Th/amoxicillin/clavulanic acid. 29/8 Mycoplasma cultured from wound, Th/enrofloxacin. 20/9 Culture before closing wound: A. baumannii and methicillin resistant Staphylococcus pseudintermedius.

Antibiotic resistance, typing

• MIC (broth micro dilution; mg/L)

EUCAST epidemiological cut-offs

• Disk diffusion (mm)

(Close to) Eucast clinical breakpoints

• MLST Typing Pasteur Scheme

EC-II isolates, sequence type 2

Concluding 2012 outbreak

June July August September October

= Days admitted to ICU

2012

4 dogs with indistinguishable MDR strains Extensive sanitization procedure 8/11 environment was sampled at 25 points – No Acinetobacter detected Source was not identified

Timeline 2014 outbreak

March April

2014

Kooiker dog, surgery for hernia 4/3/2014 OK 7/3/2014 A. baumanni cultured 15/3/2015† complications following surgery Miniature Dachshund 13/3/2014 emergency clinic 14/3/2014 Surgery, recovery at medium care – never at ICU 17/3/2014 A. Baumanni cultured from wound 20/3/2014 released – wound treated at outpatient clinic ICU environmental sampling ICU: kennel 9: A. baumanni ICU: treatment table: A. baumanni Identical ST and resistance pattern to 2014 outbreaks

= Days admitted to ICU

Protracted or independent outbreak(s)?

• Identical sequencetype for both 2012 and 2014 infections

• Identical sequencetype for one 2014 environmental sample

• Isolates with identical ST had identical resistances

• Extended outbreaks observed before (veterinary clinic Justus-Liebig-University Giessen* )

*Zordan S, Prenger-Berninghoff E, Weiss R, van der Reijden T, van den Broek P, Baljer G, Dijkshoorn L: Multidrug-resistant Acinetobacter baumannii in veterinary clinics, Germany. Emerging infectious diseases 2011, 17(9):1751-1754.

MLST lacks resolution Alternative is genome sequencing

Genome sequencing

All outbreak strains, three European Control strains (EC-I, -II and –III) and five environmental strains were sequenced by at UMCG by John Rossen. Illumina Miseq platform 2x250 bp, insertsize 300 bp

• Average 200 contigs

• Avg size 3.9 Mb

• 99% of sequence is present in the 50 largest contigs

WGS phylogeny

. Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and

visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15(11):524.

EC-II

WGS phylogeny

Outbreak isolates belong to EC-II cluster (st-2) Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and

visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15(11):524.

2014

2012

Ubiqitous presence of A.baumanni

EC-II

WGS phylogeny

Outbreak isolates belong to EC-II cluster (st-2) Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and

visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15(11):524.

2014

2012

400 SNP difference 400

EC-II

WGS phylogeny

Outbreak isolates belong to EC-II cluster (st-2) Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and

visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15(11):524.

EC-II

2014

2012

400 SNP difference However.. 316 SNPs are < 1 kb apart in a few large regions 75% SNPs may originate from recombination events

400

Minimal spanning tree

Instead of 400 SNPs, only 84 remaining between outbreaks. Isolates differ by 1-3 SNPs in ~3 months Can we get an estimate when these isolates diverged using molecular clock analysis?

Increase the dataset Collected 150 Acinetobacter baumanni genomes from literature. Determined SNPs in regions that are present in 95% of the genomes. Phylogenetic tree build on 7859 SNPs Determine a “reliable” set

of SNPs for divergence dating – corrected for recombination

Gardner SN, Hall BG. kSNP v2. PLoS One. 2013 Dec 9;8(12):e81760.

Correction for recombination events

9215 SNPs in recombination regions 564 SNPs in non recombination regions 545 genes have undergone HGT 2613 genes non-HGT Some very large recombination events (50-200 kb)

Correction for recombination events

• Main difficulty is choosing the right model for your situation.

• Substitution Model (HKY, GTR), Site Heterogeneity Model (gamma, invariant, both); Clock models (strict, lognormal, exponential, etc); Population (constant, exponentially growing), etc

• Assumption in my study: Mutation rate is the same for all isolates, only one clone.

• Strict clock, Bayesian skyline model population model fitted the best on my data. Bayesian skyline adjusts for differences in population size over time.

Divergence dating using BEAST

Divergence dating using BEAST

Drummond AJ et al Bayesian phylogenetics with BEAUti and the BEAST 1.7 Molecular Biology And Evolution 29: 1969-1973

Divergence dating using BEAST

Drummond AJ et al Bayesian phylogenetics with BEAUti and the BEAST 1.7 Molecular Biology And Evolution 29: 1969-1973

Divergence early 1980 Coincides with spread of MDR A. baumanni

Conclusions

• 2012 and 2014 Acinetobacter baumanni outbreaks in the companion animal ICU were not linked

• Benefit of whole genome sequencing in outbreak management

• Recombination may affect conclusions (although not in this case)

• Consider exploring time resolved phylogenetic methods

Acknowledgements

Academic Medical Center Amsterdam (AMC):

Masja Leendertse

Utrecht University; Fac. Veterinary Medicine:

Els Broens dept. Infectious Diseases & Immunology

Birgitta Duim dept. Infectious Diseases & Immunology

Jaap Wagenaar dept. Infectious Diseases & Immunology

Joost Hordijk dept. Infectious Diseases & Immunology

Joris Robben dept. Clinical Sciences of Companion Animals; ICU

University Medical Center Groningen

John Rossen

Thank you for your attention!