Acinetobacter baumannii is one of the leading causes of antimicrobial-resistant (AMR) hospital-associated infections worldwide1. Various antibiotic and biocide resistance determinants coupled with an innate ability to withstand prolonged periods of desiccation makes A. baumannii extremely difficult to eradicate from both patients and the environment2. Given the ubiquity and persistence of the bacterium within healthcare facilities, intraspecies co-infections involving multiple strains of A. baumannii could occur in hospitalised patients but is likely underreported3. Furthermore, the technical difficulty of resolving multi-strain bacterial infections using standard laboratory techniques has meant molecular information on intraspecies co-infection is severely lacking4.
To address this critical research gap, we employed the genome-wide screening technique transposon-directed insertion site sequencing (TraDIS) in A. baumannii, as it is capable of strain-specific resolution. We screened for molecular drivers of intraspecies interactions by competing a dense A. baumannii ATCC 17978 TraDIS mutant library against a range of other A. baumannii strains - including pathogenic (AB5075, BAL062) and environmental (E-072658) isolates. The results revealed essential genes mediating fitness of ATCC 17978 in intraspecies co-culture, irrespective of the specific strain it was co-grown with, including siderophore-associated genes (e.g. ACX60_15845, AcsC and ACX60_09705). Conversely, several genes encoding components of Type VI secretion system (T6SS) were found to be important in only one intraspecies interaction pairing: ATCC 17978 and AB5075. Supporting this strain-specific interaction, growth in biofilm of this pairing was impeded as compared to each strain grown independently.
We also investigated how strain-specific interactions affect virulence and cross-resistance in a host, using antibiotic clearance assays in the Galleria mellonella in vivo animal model. Larvae co-infected with ATCC 17978 and AB5075 and subsequently treated with the antibiotic cefotaxime, displayed an increased survival rate compared to those with single strain infections. Taken together, these results define the mechanisms underpinning intraspecies interactions and investigate key drivers during infection, virulence states and antibiotic resistance, which will provide insight into combating intraspecies co-infections in the clinic.