Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome

The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.