One major concern in hospitalized patients is infections with pathogens borne on surfaces, patients, and healthcare workers. Fundamental to controlling nosocomial infections is identifying the sources of pathogens, monitoring the processes responsible for their transmission, and evaluating the efficacy of the procedures employed for restricting their transmission. Here we present a method using the bacteriophage Lambda (λ) to achieve these ends. Defined densities of multiple genetically marked λ phages were inoculated at known hotspots for contamination on high-fidelity mannequins. Healthcare workers (HCWs) then entered a pre-sanitized simulated hospital room and performed a series of patient care tasks on the mannequins. Sampling occurred on the scrubs and hands of the HCWs, as well as previously defined high-touch surfaces in hospital rooms. Following sampling, the rooms were decontaminated using procedures designed and demonstrated to be effective. Following the conclusion of the simulation, the samples were tested for the presence, identity, and densities of these Lambda phages. The data generated enabled the determination of the sources and magnitude of contamination caused by the breakdown of established infection prevention practices by HCW. This technique enabled the standardized tracking of multiple contaminants during a single episode of patient care. While our application of these methods focused on nosocomial infections and the role of HCW behaviors in their spread, these methods could be employed for identifying the sources and sites of microbial contamination in other settings.

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