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              <p>Introduction. Transmission of antibiotic resistant bacteria (ARB) and opportunistic pathogensto patients and health care workers can occur by hospital environmental sources such ascolonized surfaces or bacterial biofilms present in sinks and shower drains. Hospitalwastewater (WW) receives all run-off water from hospital sinks and collects feces frompatients and hospital staff. Hospital WW could thus be an indicator of ARB and pathogenspresent in the hospital environment. Their monitoring is usually carried out by samplingsurfaces, followed by routine culturing methods, but it does not include WW or biofilmscollected from sink drains. Here, we implemented culture-dependent and culture-independentapproaches to assess hospital sink biofilms and WW biofilms as potential sources of ARBaccumulation and transmission.Methods. Sampling was performed monthly from January to May 2023 in both the neonataland the adult ICUs of the Grenoble University Hospital. Four sink biofilms from patient rooms,WW and WW biofilms were collected during each campaign in each respective unit. Ourculturing approach focused on the isolation of emerging highly resistant bacteria (EHRB) suchas carbapenemase-producing Pseudomonas aeruginosa and Enterobacteriaceae, andglycopeptide-resistant Enterococcus. Samples were cultured on selective and non-selectivemedia. The isolated species were identified by MALDI-TOF and antibiotic susceptibly testswere performed by disks diffusion method. The resistome was studied using qPCR targetingboth the class 1 integron integrase gene, which is a clinically important antibiotic resistancemarker, and the carbapenamase-like OXA-48 gene.Results. The culturable fraction revealed a low bacterial diversity, with 1/ an average of 3 [0– 6] different bacterial species per sink in both the neonatal- and the adult ICUs, and 2/ anaverage of 13 [10 – 16] different bacterial species per WW and WW biofilms in both wards. Nocarbapenemase-producing strains nor glycopeptide-resistant Enterococcus were isolated in thesampled sink biofilms in both the neonatal- and the adult ICUs. However, eightcarbapenemase producing Enterobacteriaceae could be isolated and were identified to carryOXA-48 or VIM carbapenemase in WW and WW biofilm in both the adult and the neonatal-ICUs. qPCR detected the OXA-48-like gene in all WW and WW biofilm samples, even in thosesamples from which no carbapenemase producing strains could be isolated. Interestingly, theclass 1 integron integrase resistance marker was detected in more than 85% of all sinks andin all WW and WW biofilm samples.Conclusions. Our preliminary results showed that hospital WW and WW biofilms containEHRB, which is a cause of major concern. No highly resistant bacteria were isolated from sinkbiofilms by culture. However, culture-independent analysis of the resistome showed that sinkbiofilms can act as a reservoir for the class 1 integron integrase gene, which is 1/ a geneticelement that carries antibiotic resistance genes (ARGs), 2/ that is associated with theirtransmission in Enterobacteriaceae and 3/ which is yet impossible to assess by routineculturing approaches. Correlation of these findings with the infection incidence, both theantibiotic treatments and the hygiene protocols administered at the tested ICU units willreveal whether this translates to an actual transmission risk for ARB or rather “just” anaccumulation of the latter, and whether hospital WW can be used as a monitoring tool for therisk prediction of ARB carriage in the hospital and their dissemination into the environment.</p>
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