Evolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection

Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem resistant A. baumannii is considered a major public health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45-day period via Illumina and Oxford Nanopore sequencing. All isolates were multi-drug resistant, carrying two genomic islands that harboured several antibiotic resistance genes. Most isolates were genetically identical and represent a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to ≥8 μg/mL; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 μg/mL. Structural modelling of AdeB and FtsI showed that these mutations affected their drug binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacteria has not yet been exposed. DATA SUMMARY All raw genome sequences, including Illumina paired end short reads and Oxford Nanopore long reads, have been deposited in the SRA under project PRJNA396979. Individual accessions for each strain are provided in Table S1. The annotated genome assembly for strain A2, the reference genome for the founder genotype, has been submitted to GenBank under accession CP024124 (chromosome) and CP024125 (plasmid). Hybrid assemblies for strains A1, A3, A8, A13, A15, A17 and A20 are available in FigShare, doi: 10.4225/49/5987e14e9b530 (note they were not deposited in GenBank as they differ from A2 by only 1-4 SNPs as indicated). IMPACT STATEMENTS Acinetobacter baumannii is a highly drug resistant pathogen that is frequently found within intensive care units (ICUs) and especially impacts patients with severe burns. While several studies have examined the global population structure of A. baumannii, few have investigated within-host evolution of A. baumannii in direct response to antibiotic treatment in a single patient. Here, we analysed the genetic evolution of A. baumannii isolated from a patient with severe burns over the course of their stay in ICU. The A. baumannii population on this patient was highly drug resistant, carrying two distinct genomic islands encoding resistance to several antibiotics but not carbapenems. The bacterial population comprised four distinct subclades, two of which had evolved carbapenem resistance over the course of antibiotic treatment through novel mutations in genes associated with drug binding. One subclade was also transmitted to another patient on the ward. While carbapenem resistance is common in A. baumannii, this is generally attributed to horizontally transferred carbapenemase genes. These data provide evidence for carbapenem resistance arising in vivo via non-synonymous substitutions during a single infection episode, demonstrating carbapenem resistance can emerge in genetic isolation in response to exposure to carbapenems and other drugs.