Invasive species drive polymicrobial resistance to amoxicillin in oral biofilms through β-lactamase release

Since bacterial biofilms often cause refractory infections, antimicrobial susceptibility testing (AST) is highly desirable even for oral peri-implant biofilms. However, characterization of polymicrobial drug resistance is challenging due to high diversity and complexity of these biofilms. In this work, we developed laser-assisted AST and detected polymicrobial amoxicillin resistance in peri-implantitis. TEM-1 β-lactamase production enabled anEnterobactersp. strain SPS_532 to protect its otherwise susceptible biofilm cohabitants. To understand the β-lactamase driven cross-protection in the human microbiome we aggregated genomic (n = 200,000) and patient microbial data (n = 27,000), developed a cross-protection assay, studied a representative strain collection (n = 118) and established a complex biofilmin vitromodel (with an average of 133 species from 164 found in dental plaque). Multiple oral allochthonous species,e.g.,Enterobacter,Klebsiella,Escherichia,Staphylococcus, and only a single typical oral microorganism,Haemophilus, were able to cross-protect. Diverseblagenes conferred activity, via a high expression of chromosomal gene,e.g.,bla_AmpCgene or by the presence of plasmidic gene,e.g., bla_TEM-1gene. Invaders not only cross-protected the biofilm from the antibiotic, but also supported expansion of opportunistic pathogens likeFusobacteriumspecies. Cross-protection in complex biofilms depended on the diffusion rate and population size of the invader, which could be bio-controlled with a phage. Deciphering polymicrobial resistance might support the development of diagnostic and therapeutic approaches to combat implant-associated biofilm infections in the human mouth.