In silico detection of acquired antibiotic resistance

Presented at EDAR 2015

byGisle Vestergaard

Beta-lactamases overpredicted in Genbank

• Common general functional annotation method of HMMER combined with Pfam

• Average bacterial genome in Genbank encodes 4 predicted non-metallo-beta-lactamases

Predicted Beta-lactamases in Genbank (August 2012)

  Predicted blacs SequencesPlasmids 136 104Chromosomes 6145 1501

Using Pfam HMM model PF00144 and a Eval cut-off of <1e-5

• More than 4000 of the 6411 predicted beta-lactamases are annotated as such

• Resistance genes are found in pristine environments• What is their intrensic function?

Implications of overprediction

Clustering of predicted beta-lactamases

• 92% of predicted beta-lactamases formed just 20 clusters

• The Antibiotic Resistance DataBase (ARDB) predicted 96% of cluster members as beta-lactamases

• Clusters cannot be used to predict resistance

Clustering of putative beta-lactamases• Plasmid encoded beta-lactamases form subclusters• Subclusters with known resistance genes• 18 subclusters including singlets

Distantly related beta-lactamasesconserved gene synteny

Conserved predicted beta-lactamasedistinct gene synteny

Beta-lactamases and housekeeping genes

• Beta-lactamases show significant similarity to various house-keeping genes

• House-keeping genes with the potential to become resistance genes.

Esterase Protease Transpeptidase

Resfams• Curated database of antibiotic resistance HMMs• Significantly less false positives than Pfam

Gibson et al., (2015) Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology. The ISME Journal.

Betalactamases Tetracycline Quinolone SequencesPlasmids 1840 586 71 5507

Chromosomes 4331 933 62 3067Using resfams provided gathering thresholds. Genbank as of March 2015

Quinolone resistance subcluster from Resfams hits

• Sensitive Serratia species• Resistance genes• 88% identical residues• Cluster also contains sensitive Aeromonas strains

Sequence similarity enough?• Genbank contains a large amount of dubious resistance

genes• Some resistance genes show significant similarity to

housekeeping genes• A novel functional annotation method used for

metagenomics is still overpredicting resistance

Chromosomal conservation and plasmid hypervariability

• MGE proximal to 90% of plasmid-encoded beta-lactamases and only 10% of chromosomally-encoded

Integrating gene synteny in antibiotic resistance detection

• Sensitive superfamily based HMMs• Proximal genes suggesting resistance

– MGEs– Genomic island– Antibiotics sensitive regulator

• Proximal genes suggesting other intrensic function– Typical operon structure

Hurdles in metagenomic context analysis

• Bioinformatical tools– Hypothesis driven assembler as opposed to data-driven

• Current assemblers are black boxes• ...and have problems with repetitive elements• TriMetAss by Johan Bengtsson-Palme

– Gene synteny and co-occurence analysis tools• Extract flanking genes and functions• Determine co-occurence

– Integrating genetic context into risk assessment

• High-throughput sequencers – Longer reads– Better coverage

Transitions are rare, but crucial• Resistance genes form subgroups• Each subgroup caused by one transition from proto-

resistance gene to resistance gene• Maximum of 18 transitions in Genbank• The genesis of a novel resistance gene of clinical

relevance is rare, but spread is rapid

McKenna, Nature, 2013

Acknowledgements• Joseph Nesme• Lars Hestbjerg Hansen• Søren Sørensen• Michael Schloter