Aircraft lavatory wastewater surveillance for movement of antimicrobial resistance genes: a proof-of-concept study

Long-haul flight aircraft wastewater may serve as a representative microbial footprint, often of mixed country origin, offering valuable insight into the movement of pathogens and antimicrobial resistance (AMR) on a global scale. Herein, we present a proof-of-concept for aircraft-based surveillance of AMR by investigating lavatory wastewater samples from 44 repatriation flights to Australia departing from nine countries. Profiles of pathogens including ESKAPE pathogens (<i>Salmonella</i> spp., <i>Mycobacterium</i> spp., <i>Enterococcus faecium</i>, <i>Staphylococcus aureus, Klebsiella pneumoniae</i>, <i>Acinetobacter baumannii</i>, and <i>Pseudomonas aeruginosa</i>) and antibiotic resistance genes (ARGs) (<i>aph(3')-IIIa</i>, <i>bla_NDM-1, bla_{CTX_M-1}, bla_KPC, ermB, qnrS, sul1, tetM,</i> and <i>vanA</i>) were investigated along with traditional fecal indicator bacteria (<i>Escherichia coli</i> and <i>Enterococcus</i> spp.) and fecal/urine marker genes (<i>Bacteroides</i> HF183, <i>Carjivirus</i>, human polyomavirus, and a cryptic plasmid pBI143) using quantitative PCR (qPCR). Two fecal indicator bacteria (FIB) and four human fecal/urine marker genes were detected in all aircraft wastewater samples. Detection rates for ESKAPE pathogens ranged from 6.8% (<i>S. aureus</i>) to 84.1% (<i>K. pneumoniae</i>). Of all ARG targets, <i>aph(3')-IIIa</i>, <i>ermB</i>, <i>qnrS</i>, <i>sul1,</i> and <i>tetM</i> were detected in all wastewater samples, whereas <i>bla_KPC</i> and <i>vanA</i> were not detected in any of the samples. Results reflected geographic differences in ARG abundance originating from departure countries/continents and suggested a potential risk of importing ARGs that might be rare in local wastewater systems. The loss of nucleic acid targets was less than 10% over a 24 h incubation in the presence of disinfectants, suggesting that nucleic acids are resilient enough to persist in aircraft wastewater over the maximum duration of a flight.IMPORTANCEIn the context of international connectedness, aircraft-based wastewater surveillance should be viewed as a beyond-national tool to enhance global AMR management and foster international cooperation.