Short-Lived Success: Repeated Peroxide-Peracetic Acid-Based Foam Disinfection Selects for Carbapenem-Resistant Enterobacterales in Hospital Sinks Drains

Short-Lived Success: Repeated Peroxide-Peracetic Acid-Based Foam Disinfection Selects for Carbapenem-Resistant Enterobacterales in Hospital Sinks Drains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Short-Lived Success: Repeated Peroxide-Peracetic Acid-Based Foam Disinfection Selects for Carbapenem-Resistant Enterobacterales in Hospital Sinks Drains Shireen M Kotay, Aubrey E Hetzler, Sharvari Narendra, Kevin K Chau, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9163989/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Sink-drains/traps in healthcare facilities are recognized reservoirs of drug-resistant Gram-negative bacilli, yet effective remediation strategies remain uncertain. Using culture-based and metagenomic approaches, we evaluated the impact of a peroxide peracetic-acid (PPAA)-foam disinfectant applied at 3-, 5- or 7-day intervals over four-weeks in a controlled Sinklab and a hospital setting. Across all application frequencies and repeated applications, PPAA-foam was ineffective in reducing carbapenemase-producing Enterobacterales counts from baseline over 28days. Instead, treatment induced pronounced microbiome dysbiosis in sink-drains and traps, characterized by reduced community diversity, enrichment of Enterobacterales, and amplification of resistance determinants, including bla KPC and bla NDM . Hospital sinks exhibited comparable transient effects following PPAA-foam treatment, with rapid post-treatment recovery of both microbial communities and resistome. Together, these findings demonstrate that repeated chemical disinfection in established plumbing systems may destabilize drain microbiomes and paradoxically reinforce the persistence of high-risk pathogens and antimicrobial resistance, underscoring the need for ecologically informed alternatives to chemical-only interventions. Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology hospital sinks drain biofilms CPE disinfectants microbiome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Antimicrobial resistance (AMR) is a major health threat and key contributor to global mortality. Healthcare-Acquired Infections (HAI) are often caused by multidrug-resistant Gram-negative bacilli (MDR-GNB), including Carbapenemase-producing Enterobacterales (CPE), making them hard to treat 1,2 . Healthcare-associated wastewater interfaces, like sink-drains, represent major reservoirs for MDR-GNB and are associated with transmission to patients, patient colonization, and subsequent HAI, but mitigation of this risk is challenging 3-5 . The main risk to patients is thought to be linked to the growth of biofilm from sink-traps allowing upward growth to the sink-drain surface, with bacterial pathogens then dispersed out from the sink during faucet use through water turbulence impacting contaminated drain surfaces and resulting in droplet generation 6,7 . Although targeting sink-drains for decolonization rather than sink-traps is important given they are the focal point for pathogen dispersal to the surroundings 6,8 , sink-traps are reservoirs for the transfer of AMR genes and selection of drug-resistant pathogens which contribute to this biofilm, representing an additional important target for mitigation 9,10 . The optimum strategy for cleaning hospital sink-drains and sink-traps to minimize pathogen and AMR gene selection in these reservoirs and avoid transmission remains unclear 3 . Several types of cleaning products and disinfectants are used for environmental cleaning in hospitals, which often include one or more of the following; chlorine, peracetic/hydroxy-peracetic acid, hydrogen peroxide and quaternary acid compounds. These chemical disinfectants are inconsistently effective at eliminating pathogens in environmental biofilms, even at recommended concentrations 11 . As a result, biofilm-associated pathogens frequently persist or recolonize surfaces after disinfection, highlighting the need for alternative approaches beyond standard chemical disinfection. Most commercially available cleaning products are conventionally poured down the sink-drain as liquids, and often lack adequate contact time to disrupt established biofilms inside plumbing systems 12-15 . Novel approaches targeting biofilms show promise, but their efficacy remains to be established 3,4 , including an evaluation of possible adverse effects (e.g., selection for potential pathogens). The application of foam-based cleaning agents containing broad-spectrum disinfectant, bactericidal and anti-biofilm properties, with increased contact time with pipework, has reportedly been more effective in reducing hospital-sink-drain pathogen colonization than bleach and other disinfectants 16-20 . While foam-based, broad-spectrum cleaning may offer advantages, the durability of its effects and the optimal frequency of application have not been determined. For this study, we aimed to determine an effective frequency of application for such a product Virasept™, Ecolab, a peroxide-peracetic acid (PPAA) foam-based, broad-spectrum cleaning agent. We sought to understand the impact of this intervention on the sink-trap and sink-drain microbiomes with a focus on CPE, AMR gene amplification, and dysbiosis, using culture-based and metagenomic methods in both experimental and hospital settings. RESULTS PPAA-foam treatment selectively enriches Enterobacterales, including antimicrobial-resistant strains, in experimental sink-drains and sink-traps We first evaluated the impact of repeated PPAA-foam treatment on sink-drain and sink-trap biofilms using a controlled experimental SinkLab system, integrating quantitative culture and shotgun metagenomics. We then assessed whether these effects translated to a clinical environment by applying the same intervention to hospital sink-drains and monitoring microbial and resistome dynamics longitudinally (Fig. 1 ). To evaluate the effect of PPAA-foam treatment frequency on CPE colonization of sink-drains, sink-drains were seeded with a known CPE consortium and left undisturbed for 28 days (Fig. 1 .a, SinkLab biofilm establishment) prior to invention. Subsequently, across three experimental arms, sink-drain biofilms were treated with PPAA-foam either every 3 days [T3], 5 days [T5] or 7 days [T7] (5 sink-drains for each arm) over a 28-day period, alongside untreated controls (Fig. 1 a). Systematic sampling was undertaken at baseline (D-3) and at defined intervals post-treatment at D0, D3, D7, D14, D28 and D35. We quantified CPE burden at the drain level via quantitative culture (see Methods), which revealed an immediate but non-durable reduction in CPE burden following PPAA-foam exposure. Across all treatment regimens, CPE cumulative counts showed 2–5 log reductions relative to baseline (D-3) immediately after the first PPAA-foam exposure (D0; Fig. 2 a). In the T3 group, (three-day treatment intervals), CPE abundance declined further after the second treatment (D3), but rebounded thereafter despite continued PPAA-foam applications every three days. In the T5 or T7 groups (5- & 7-day application intervals), CPE levels rebounded to near-baseline levels between treatments, declined transiently after the second application (at D5 and D7 respectively), and then progressively increased to near-baseline levels despite of continued PPAA-foam application, as with the T3 group (Fig. 2 a). Regardless of application frequency or total number of applications (T3 [n = 10], T5 [n = 6] and T7 [n = 5]), PPAA-foam was ineffective in reducing CPE counts from baseline over 28 days. By Day 35 (eight days after final treatment), CPE abundance did not differ significantly from pre-treatment baseline in any treatment groups (Wilcoxon signed-rank test with Bonferroni correction p adj =1.00, Fig. 2 a). A summary of baseline and endpoint comparisons, effect sizes and adjusted p-values is provided in Table S1 . In contrast, untreated control sinks exhibited a significant increase in CPE abundance over the same period (Fig. 2 a, Table S1 ). However, endpoint (D35) CPE abundance in the control sinks was comparable to those observed in all treatment group endpoints, with the observed increase likely indicating natural maturation and upward trend in CPE abundance over time in the absence of any intervention. Additionally, while some variability in CPE abundance was observed in the control sinks (IQR: 7.28–8.28 log 10 CFU/ml, max: 8.95), the overall variation was substantially lower than that observed in the treatment groups over the course of the study (T3:1.70–6.98, max: 9.18; T5: 3.73–7.26, max: 9.18; T7: 2.85–7.232, max:9.079). The patterns observed for sink-drain CPE counts were mirrored for counts of all Gram-negative bacilli, including lactose- and non-lactose-fermenting organisms (Fig. S1 ). Metagenomic analysis corroborated culture-based results, and revealed treatment-associated community restructuring. In treated sinks, Enterobacterales relative abundance decreased transiently following PPAA-foam application but only up to D14 (Fig. 2 b). In T5 and T7 sinks on D3 (i.e. a sampling timepoint between treatments), Enterobacterales populations briefly recovered followed by transient suppression again after the second PPAA-foam treatment. After D14, however, Enterobacterales relative abundance increased steadily across treated sinks regardless of treatment interval or total treatments administered, dominating the sink-drain biofilm communities by D35 (mean relative abundance: 0.741 [SD: +/-0.161]). This resurgence in Enterobacterales in treated sink-drains coincided with a marked decrease in relative abundance in several taxa commonly associated with stable drain biofilms, including the orders Micrococcales, Pseudomonadales, Brevundimonadales and Agrococcales (Fig. 2 b). In contrast, control sink-drains maintained relatively stable community profiles, with Enterobacterales decreasing over time and remaining a minor community component at the end point (D35 mean relative abundance: 0.034 [SD: +/-0.018]; Fig. 2 b). PPAA-foam treatment induces pronounced restructuring of the sink-drain microbial communities in the SinkLab model Consistent with the post-treatment enrichment and eventual dominance of Enterobacterales, PPAA-foam application led to marked reduction in sink-drain microbial diversity over time. Alpha-diversity metrics (Chao1 richness, Pielou’s Evenness and Shannon Diversity) show marked reductions in overall microbiome diversity by D28 across treated sink-drains. This reduction in overall microbiome diversity was significant for treatment intervals T3 (Shannon, Welch’s Two-Sample t-test p = 0.0023), and T5 (Shannon, p = 0.001), but not for T7 (Shannon, p = 0.0798) (Fig. 2 c). In contrast, controls sink-drains that did not receive PPAA-foam treatment showed no significant changes in any diversity metric over the study period, indicating relative community stability in the absence of intervention (Fig. 2 c). Comparisons of pre-treatment baseline (D-3) and post-treatment endpoints (D28/D35) revealed significant overall community restructuring across all sink-drains, irrespective of treatment frequency as evidenced by shifts in beta-diversity (Fig. 2 d; Welch’s Two-Sample t-test, T3- p = 0.005, T5- p = 0.010 & T7- p = 0.045 for D-3vsD35). During the biofilm establishment, experimental sinks were inoculated with a defined mix of nine CPE strains (six genera; see Methods). Longitudinal metagenomic profiling enabled tracking of the abundance of the six genera corresponding to which the spiked-in species belong. Across the all PPAA-foam treatment groups, four genera associated with the inoculum ( Citrobacter , Enterobacter , Escherichia , and Klebsiella ) exhibited upward trends in relative abundance following treatment (Fig. 3 b, Fig.S2), consistent with selective enrichment rather than sustained suppression. Comparing baseline (D-3) and post-treatment timepoints (D35), the increase in relative abundance was significant for Citrobacter in T5 sink-drains ( p = 0.0107), whereas other genera showed variable, non-uniform responses across treatment groups (Fig. 3 b). Notably, statistically significant reductions were observed for Enterobacter in T7 sink-drains on D28, but with a recovering trend by D35. In contrast, Serratia populations declined significantly in both treated and untreated sink-drains. Comparable dynamics were observed at the sink-trap level. Cumulative counts of CPE and Aeromonas as well as Gram-negative bacteria rebounded strongly in treated sink-traps after D14, despite continued PPAA-foam application (Fig.S3a-c). Metagenomic analysis further demonstrated reductions in genus-level richness and clear compositional shifts in treated sink-traps relative to the baseline (Fig.S2d&e mirroring patterns observed in corresponding sink-drains. PPAA-foam treatment increases sink-drain resistome burden, including the major carbapenemases , bla KPC and bla NDM , in experimental sink-drains To assess the impact of PPAA-foam on antimicrobial resistance dynamics, we quantified the sink-drain resistome using shotgun metagenomics, expressing ARG abundance as transcripts per million (TPM) aggregated at the AMR gene family level. The experimental biofilms were initially seeded with carbapenemase-producing Enterobacterales carrying bla KPC and bla NDM (see Methods), enabling longitudinal tracking of these clinically relevant resistance determinants alongside global resistome changes. Across all treatment regimens, the total resistome load decreased by several orders of magnitude immediately following the first PPAA-foam application but recovered progressively with continued treatment (Fig. 4 a). By the post-treatment endpoints (D28/D35), the total resistome load exceeded baseline in all treated sink-drains, reaching statistical significance in T3 group (Fig. 4 a; Welch Two Sample t-test p = 0.025). In contrast, untreated control sink-drains showed a steady decline in resistome loads over time, resulting in a significantly lower ARG abundance at D35 compared with baseline for all PPAA-foam-treated groups (Fig. 4 a; p = 0.0251). At the AMR gene family-level, PPAA-foam treatment was associated with reduced resistome richness and evenness (Fig.S4), coinciding with an increase in the relative abundance of key carbapenemase genes, bla KPC and bla NDM (Fig. 4 b; D- 3 vs by D28/D35). Notably, bla KPC abundance increased significantly in T5 and T7 sink-drains by D28/D35 (Fig. 4 c; Welch Two Sample t-test p = 0.0150 and 0.0012), while bla NDM displayed concordant upward trends the corresponding treatment groups it was not found significant. Despite the overall increase in resistome burden, NMDS ordination based on Bray–Curtis dissimilarities revealed more modest separation between baseline and post-treatment samples at the AMR gene-family level than observed for corresponding taxonomic profiles, suggesting selective amplification of dominant resistance determinants rather than wholesale restructuring of the resistome (Fig.S4c). Comparable patterns of resistome recovery and carbapenemase enrichment were observed in sink-traps (Fig. S4b&d). In hospital sink-drains, PPAA-foam treatment exerts only transient suppression of carbapenemase-producing Enterobacterales and reshapes resident microbial communities To determine whether the effects of PPAA-foam treatment observed in experimental sinks translated to a ‘real-world’ hospital setting, we conducted a pilot intervention in four hospital sinks, applying PPAA-foam at a 7-day interval (T7) over a 28-day period, with sampling immediately before and after each treatment (Fig. 1 b). In contrast to the experimental SinkLab system, hospital surveillance relied on qualitative culture-based detection of CPE (presence/absence), reflecting routine infection prevention workflows and precluding direct quantitative comparisons. Further, historical CPE surveillance data (culture-based CPE- presence/absence) were available for these sinks, allowing qualitative comparison of pre- and post-treatment colonization status. Consistent with the transient effects observed in the SinkLab, PPAA-foam application in hospital sinks produced a short-lived suppression of detectable CPE colonization (Fig. 5 a). Following the first treatment, one of three initially CPE-positive sinks remained culture-positive, while all three were culture-negative after the second treatment. However, this suppression was not sustained after the third treatment again yielded CPE, and by the fourth application all four sinks, including the two that were CPE culture-negative prior to intervention were CPE culture-positive (Fig. 5 a). While qualitative in nature, these findings parallel the rebound dynamics observed in the experimental model and suggest that repeated foam-based cleaning does not prevent, and may ultimately coincide with, CPE recolonization in hospital sink-drains. Shotgun metagenomic profiling provided higher-resolution insight into community-level responses to the treatment. Over the 28-day intervention period, the relative abundance of Enterobacterales increased progressively compared with baseline (Fig. 5 b), accompanied by declines in the relative abundance of Sphingomonadales (although not significant, Welch Two Sample t-test p = 0.1452 and 0.09475 respectively). Importantly, this shift contrasted with the stability of the order-level community structure across the three historical pre-treatment timepoints spanning 213 days, indicating that the observed perturbations were PPAA-foam treatment-associated rather than a natural community drift. Alpha-diversity analyses showed an initial reduction in the genus-level richness and evenness immediately after PPAA-foam treatment with recovery toward baseline values by D28 (Fig. 5 c), consistent with a transient disturbance rather than sustained suppression. NMDS ordination confirmed significant shifts in genus-level composition over the treatment period (Fig. 5 d), characterized by increased representation of Citrobacte r, Enterobacter , Klebsiella and Pseudomonas genera (Supplemental Fig.S5). Changes in the log10 abundance of dominant Enterobacterales genera pre- and post-treatment following individual PPAA-foam treatment events were modest, reinforcing that community restructuring occurred gradually over repeated applications rather than as an acute effect. Repeated PPAA-foam treatment produces limited and short-lived perturbation of the hospital sink-drain resistome Longitudinal metagenomic profiling demonstrated that PPAA-foam treatment exerted only short-lived effects on hospital sink-drain resistome. The total ARG abundance decreased immediately following the first treatment but rebounded to near-baseline levels within seven days (Fig. 6 a, D0 vs D7B). Similar transient reductions were observed immediately after each subsequent treatment, with ARG loads consistently recovering between treatment cycles and frequently exceeding the historical mean baseline. By D28, total ARG abundance was comparable to or higher than pre-treatment values (Fig. 6 a, D-47 vs D28), indicating that repeated PPAA-foam disinfection did not achieve sustained suppression of the resistance determinants within the hospital drain biofilms. Analysis of clinically relevant carbapenemase genes revealed a modest, non-significant increases in the mean normalized bla KPC abundance by D28, while bla NDM remained largely unchanged throughout the study period (Fig. 6 b). These findings suggest persistence of key carbapenemase determinants despite repeated disinfectant exposure, rather than effective depletion. At the ARG family level, resistome richness and diversity increased modestly over time (Fig. 6 b), consistent with selective turnover rather than overall contraction of resistance gene classes. Bray–Curtis ordination demonstrated a detectable shift in resistome composition by D28 compared with historical baseline values (Fig. 6 d), although the magnitude of divergence was limited. Heatmap-based profiling of the top 20 ARG families further supported this interpretation, revealing transient post-treatment decreases in several β-lactamase families immediately following the first PPAA-foam application, but these genes rebounded by D28 alongside a modest enrichment of efflux-associated genes and macrolide- and aminoglycoside-resistance determinants (Fig.S6). Comparable temporal patterns were observed in sink-traps, reinforcing the inference that the hospital sink resistome exhibit substantial resilience and structural stability despite repeated chemical disruption using PPAA-foam. DISCUSSION We set out to determine the impact of a foam-based, peroxide–peracetic acid disinfectant on the microbiology and ecology of sink-drain biofilms when applied at different frequencies over time. We chose to focus on effects at the sink-drain level as this presents the most immediate risk of dissemination to patients 6 , but observed similar effects at the sink-trap level (Figs S1 , S3, S4, S6 & S7). We demonstrate that PPAA-foam exerts unintended and potentially counter-productive ecological effects. Across both controlled SinkLab experiments and hospital pilot testing, repeated PPAA-foam exposure consistently destabilized resident microbial communities, promoted Enterobacterales dominance, and favored the persistence and amplification of CPE. These observed effects occurred within short periods of time (28 days), and irrespective of the application intervals tested (3, 5 and 7 days), indicating that frequency modulation alone does not mitigate adverse ecological outcomes. Although interventions targeting sink-drains are intended to reduce transmission risk, our findings indicate that they alter the drain-biofilm microbial communities associated with amplification and persistence of resistance determinants within both sink-drains as well as sink-traps. A key finding of this study is that PPAA-foam induces pronounced microbiome dysbiosis rather than sustained microbial suppression. In experimental sinks, repeated exposure resulted in marked loss of community richness and evenness, accompanied by depletion of taxa commonly implicated as foundational biofilm members (including Micrococcales, Pseudomonadales, Brevundimonadales and Agrococcales). Biofilms are structured, mixed microbial communities embedded within a self-produced extracellular matrix that contributes disproportionately to community stability, nutrient cycling, and protection from stressors, with model taxa such as Pseudomonadales (e.g., Pseudomonas spp. ) known to produce key matrix components central to biofilm architecture and function 21 , 22 . Their selective loss likely compromises the ecological scaffolding of the biofilm, creating vacant niches that facilitate recolonization by fast-growing, stress-tolerant opportunists such as Enterobacterales. Such restructuring represents a classical form of dysbiosis in surface-attached microbial assemblages and provides a plausible mechanistic basis for the observed pathogen enrichment and resistance persistence. 23 By integrating quantitative culture-based approaches focused on Enterobacterales with longitudinal shotgun metagenomics, our study extends beyond prior work that has primarily relied on short-term culture-based endpoints or overall bacterial burden. Previous studies have highlighted that disinfection of sink-drains may have limited efficacy and result in a transient reduction of Gram-negative bacilli followed by recolonization within 5–7 days after single PPAA-foam applications, and noted a progressive decrease in the bacterial burden with repeated treatments every 3 days. However, these studies examined shorter follow-up intervals (≤ 13 days) or focused on overall bacterial burden rather than resistance ecology 16 , 17 , 24 . Our data substantially extend these observations by demonstrating that repeated PPAA-foam application does not only fail to achieve durable suppression of Enterobacterales or ARG loads beyond the immediate post-treatment period, and further suggests PPAA-foam may become ineffective after prolonged use. Another study testing different PPAA-foam application frequencies on the burden of P. aeruginosa and S. maltophilia in ICU room sink drains found that applications 5 times a week was more effective than a weekly application 18 . However, they similarly noted a resurgence and/or persistence of these organisms in the post-intervention phases, and that this frequency of application may not be routinely sustainable in hospital settings. A fourth study reported regrowth of Gram-negative bacilli using PPAA-foam treatment intervals of 2, 3 and 5 days and suggested that daily application may be required to reduce the concentration of Gram-negative bacilli to 2 log10 CFU/mL, whilst recognizing that the “safe” abundance threshold remains unknown 25 . Findings from a recent randomized controlled trial in renovated inpatient sinks evaluating peroxide-peracetic acid foam disinfectant using culture-based screening, demonstrated delayed colonization in the treated sinks and site-dependent reductions in drain and tailpipe contamination, with re-establishment of Gram-negative organisms after disinfection 26 . We did not attempt daily application which could be a noted limitation of the current work, but previous reports question the practicality and sustainability of such intensive cleaning regimens in healthcare settings. Repeated PPAA-foam exposure functioned as a recurring ecological disturbance, triggering loss of foundational taxa and rapid biofilm reassembly dominated by opportunistic and resistant organisms. These findings are likely relevant to other engineered wet environments where biofilms experience repeated chemical disturbance, including wastewater infrastructure. A notable strength of this study lies in its ability to capture both potentially high-risk pathogens of interest (e.g., CPE) and wider microbiome-level responses. We observed that the dysbiosis induced in sink-drain and sink-trap microbiomes in response to PPAA-foam treatment appears to have contributed to the overgrowth of drug-resistant potential pathogens. This was evident from the marked reductions in the alpha-diversity indices and community-level shifts observed over time in the sinks that received treatment that diminished competition and favored opportunistic pathogen expansion. This phenomenon was more pronounced in the experimental sinks (SinkLab), potentially reflecting the absence of the continual microbial seeding and physicochemical/environmental fluctuations present in the hospital setting. For example, hospital sinks may have been exposed to different biotic and abiotic factors as a result of sink usage by and inputs from patients, patient-care providers and the interconnected plumbing network that likely provide diverse inocula that influence recolonization kinetics 27 . Nevertheless, the directionality of effects was consistent across SinkLab and hospital settings, indicating that similar ecological mechanisms operate under both controlled and real-world conditions. Although this study offers valuable insights, some limitations merit consideration. The numbers of sinks within each experimental treatment group was modest, and although we identified some significant changes at the order-, genus- and AMR gene-level, our statistical power to detect subtler taxonomic or functional shifts may be limited. In the hospital setting, only a 7-day treatment interval was assessed as it was considered most feasible, and was limited to relatively small number of sinks in a single facility due to the available resources and the logistics of implementation. Our findings however, were consistent with those observed in our experimental set-up, albeit less marked. This convergence of findings across independent models strengthens their generalizability. It is possible that PPAA-foam cleaning would work differently in different contexts, given the variability observed in previous studies of sink microbiomes and usage 16 , 17 ; however, our findings are consistent with effects observed in the limited number of other studies evaluating sink-drain/trap disinfection with the PPAA-foam product across different contexts. Similarly, there may be other combinations of chemical disinfectants or chemical disinfection strategies that have different effects that we have not evaluated here. Moreover, in contrast to our hospital study, our experimental sink study did not include immediate pre- and post- application sampling. In retrospect, this strategy was valuable and could have further clarified short-term recovery kinetics. Although hospital sink-drains differ from the SinkLab model in baseline complexity and surveillance resolution, both systems exhibit convergent qualitative patterns in response to PPAA-foam treatment: transient microbial suppression followed by recovery or enrichment of Enterobacterales and associated microbiome restructuring. Importantly, the qualitative nature of our hospital environmental culture data limits inference on magnitude, but the consistency in temporal trajectories strengthens the conclusion that foam-based cleaning alone is insufficient to durably control sink-associated CPE in clinical environments. In conclusion, we show that sink-drain and sink-trap disinfection with foam-based, broad-spectrum cleaning products such as PPAA-foam may paradoxically select for the very organisms these interventions aim to eradicate. The remarkable ecological resilience of these complex microbial reservoirs poses a substantial challenge to routine infection-prevention practices, particularly in healthcare environments where colonization of wastewater plumbing systems are driven by several factors. These constraints underscore the limitations of relying solely on chemical disinfection to suppress pathogen and resistance burdens in these wet environments and highlight that monitoring for unintended microbial shifts following chemical disinfection strategies is important. Moving forward, effective mitigation will likely require integrated and ecologically informed strategies that combine structural redesign of sink components, targeted microbial competition, and enhanced surveillance technologies capable of detecting and responding to early shifts in microbial and resistome composition. Addressing these challenges will be essential to ensure that practices reduce rather than inadvertently propagate pathogen reservoirs and antimicrobial resistance within the hospital built environment. ONLINE METHODS Experimental SinkLab setup All experiments under this study were conducted in the University of Virginia (UVA) SinkLab, a Biosafety Level 2 facility equipped with four independent sink rigs, each housing a set of ten sinks. The drain line on each sink is comprised of a cast grid drain (P/N 760-1 Dearborn Brass®-Oatey, Cleveland, Ohio), a chrome coated over brass 6-inch tailpipe, 1¼-inch P-trap and trap-arm (P/N 701-1, Dearborn Brass®-Oatey, Cleveland, Ohio). All sinks on a rig drain into a shared 2-inch PVC pipe (Charlotte Pipe Charlotte, NC) leading into a 60-gallon high-density polyethylene resin (HDPE) holding water tank (Ronco Plastics, Tustin, CA). Cold-water delivery was automated using a ½” Brass Electric solenoid valve (JFSV00006, US Solid) with 4 mm orifice installed on faucet connectors and controlled via Raspberry Pi microcontroller (Sparkfun, Niwot, CO) with a custom MOSFET-enabled shield. These together are programmed to deliver water into each sink for 30 seconds every four hours (~ 475 ml per flush; 5.1 Lmin − 1 flow rate). Wastewater was collected into holding tanks, disinfected using chlorine tabs prior to disposal into a common floor drain. Experimental Sink (SinkLab): Inoculation and Biofilm establishment To establish polymicrobial biofilms comprising carbapenemase-producing Enterobacterales, sinks were inoculated with a mixture of nine strains previously isolated from hospital sinks, wastewater or patients (Table S2). Prior to experiments, frozen stocks (− 80°C) were revived on Trypic Soy Broth (TSB) and pooled in equal volumes to generate an inoculum cocktail. Each of the sinks was inoculated by pouring 25 ml of this cocktail over the sink drain (~ 10 Log CFUs of each strain). To support biofilm establishment and maturation, 25ml TSB was poured over the sink-drain daily with an average 2h dwell time before routine water flush. Biofilms were allowed to establish for 28 days prior to PPAA-foam intervention. PPAA-foam application and Experimental sink sampling We sought to understand the impact of a foam-based, broad-spectrum disinfectant product, Virasept™ (EcoLab) containing acetic acid, hydrogen peroxide, octanoic acid, and peroxyacetic acid on sink-drains/traps in an experimental and a hospital setting. PPAA-foam foam was applied through the drain holes filling the vertical void space above the p-trap water until foam was visible over the drain, as per the manufacturer’s instructions. A five-minute contact time was maintained (per manufacturer’s instructions) before flushing with faucet water for one minute. We evaluated three different PPAA-foam application regimens (3-day interval [T3], 5-day interval [T5], 7-day interval [T7]). Control sinks did not undergo treatment but were flushed with water alone for one minute (Fig. 1 a). Sampling occurred at baseline (D-3) and post-treatment at D0, D3, D7, D14, D28 (immediate post-treatment) and D35 (eight days post treatment). Drain biofilm was sampled using ESwabs® and 50ml P-trap water were aspirated using a 50mL syringe attached to a cannula adapter and IV tubing (further details reported previously 28 ). Hospital sinks: Treatment and Sampling Four bathroom sinks in individual intensive-care rooms with prior CPE-positivity were selected for in-hospital testing. PPAA-foam foam was applied every seven days for 28 days (Fig. 1 b). Sink-drain and sink-trap water were sampled at weekly intervals. For each sink sampling event, drain samples were collected immediately before and after PPAA-foam treatment, except on day 0 when only a post-treatment sample was taken. Sink-trap samples were collected after each PPAA-foam treatment only. Sinks remained in clinical use, resulting in variable patient occupancy and water-use patterns over the study period. Microbial culture All samples were processed immediately upon collection. Sink-trap samples were centrifuged at 2,460g for 15 minutes using an Eppendorf 5810 R Refrigerated Centrifuge with a 10cm rotor radius. The supernatant was decanted to minimize any residual PPAA-foam product in the culture sample and two mL of 0.9% sterile saline solution was added to the pellet. The mixture was then vortexed to resuspend the pellet. Drain samples (ESwab) were vortexed for 10 seconds and then transferred to a two mL microtube. To increase the sample volume for culture and extraction, one mL of 0.9% sterile saline solution was added to the ESwab tube. The ESwab tube was vortexed again for 10 seconds, and the contents were transferred to the two mL microtube and vortexed to combine. Culture was prepared using CHROMagar™ mSuperCARBA™ (CHROM) (NEL Scientific Waterville, ME), a selective and differential (chromogenic) media for the detection of carbapenem-resistant Enterobacteriaceae, and McConkey (MAC) Agar, selective for gram -negative bacilli, and differential for lactose-fermenting organisms. Quantitative culture was prepared using the methods previously described 29 . To increase sensitivity, an enriched culture was prepared by inoculating 500µl of each sample into 4.5mL of TSB containing a 10µg Ertapenem (ETP) BD BBL™ Sensi-Disc. After 12 hours at 35°C a 10µl inoculating loop was used to streak the cultures on CHROM, which were subsequently incubated for 12 hours at 35°C. MAC quantitative culture was enumerated to identify the growth of lactose fermenters (pink colonies), and non-lactose fermenters (white or clear colonies). The CHROM quantitative culture was enumerated for unique colonies, based on color and morphology. Additionally, the enriched culture was assessed for the presence of unique colonies using the same criteria (Fig. 1 c). Metagenomic sequencing DNA from sink drain biofilm samples was extracted using DNeasy PowerSoil® Kit (Qiagen) following manufacturer’s protocol with modifications to increase gDNA yield. The tubes were centrifuged at 16,000g for two minutes. 400µL of the sample, prioritizing the concentrated pellet, was transferred to a powerbead tube and combined with 600µl of CD1. Groups of 24 PowerBead tubes were vortexed for 20 minutes using a horizontal microtube holder. The tubes were centrifuged at 15,000g for one minute and transferred to a heat block set at 70 o C for 10 minutes. After cooling to room temperature, 800µl of supernatant was combined with 200µl of CD2. Following centrifugation, 700µl of lysate was combined with 675µl of CD3. The lysate was transferred to the column in two 730 µl increments, centrifuging at 15,000g and discarding the flow through each time. Solution EA and Solution C5 were added per the manufacturer's protocol. The provided C6 buffer was used to elute 50µl of gDNA. DNA quality and yield (ng/µl) for each sample were assessed with a Qubit 4 Fluorometer using the 1X dsDNA HS Qubit Assay (Invitrogen). gDNA was stored at − 20 o C until shipment at ambient temperature. Extracts were sequenced on Illumina platforms, using a commercial provider (Azenta GeneWiz, UK). Bioinformatics analysis Raw sequencing reads were processed using the ResPipe v1.6.1 pipeline 30 , with taxonomic and resistance estimation. Adapter trimming and quality filtering were performed with TrimGalore v0.6.4 ( https://github.com/FelixKrueger/TrimGalore ) 31302928 , removing reads with below average Phred score of Q25, lengths < 75 bp, and the first 13 bp of Illumina adapters. Taxonomic profiling was conducted with Kraken2 v2.1.3 32 using a reference database of NCBI’s RefSeq bacterial and viral genomes (accessed in January 2024, version: k2_pluspf_20240112). For each taxonomic level, relative abundances were estimated using Bracken (v2.9) 33 . For AMR gene abundance estimation, the filtered metagenomics reads were mapped using BBMAP v38.34 34 , against the Comprehensive Antibiotic Resistance Database (CARD v3.2.4) 35 . Pseudo-abundance estimation was performed for normalization using (transcripts per million (TPM)) reads. Statistical Analysis Log 10 -transformed total CPE counts were analyzed longitudinally across treatment groups (Control, T3, T5, T7). Zero counts generating –∞ values were replaced with zero prior to downstream analyses. Descriptive statistics (mean, standard deviation, standard error, median, and interquartile range) were calculated for each group-timepoint combination. Pairwise comparisons across timepoints were conducted using the Wilcoxon signed-rank test with Bonferroni correction for multiple testing. Statistical significance was determined at α = 0.05. Analyses were conducted in R (v.4.2.2) using the rstatix (v.0.7.2) package and ggplot2 (v.3.5.1). Within-sample alpha diversity was measured at each taxonomic-level by assessing overall diversity (Shannon Diversity Index), community richness (Chao1), and community evenness (Pielou). Beta diversity compositional dissimilarities between samples was calculated using the Bray-Curtis dissimilarities and visualized using non-metric multidimensional scaling (NMDS). Taxonomic alpha diversity indices were calculated using phyloseq v1.44.0. AMR alpha diversity indices and all beta diversity indices were calculated using vegan v2.6.4. Visualizations were generated with ggplot2 v3.5.0; analysis used R version v4.3.0. Correlation was calculated and visualized using ggpubr v0.6.0. Statistical differences in Shannon diversity indices between groups were evaluated using Welch’s two-sample t-test, which accounts for unequal variances and is appropriate for comparisons between groups with heterogeneous dispersion. Data availability The metagenomic sequences are available at NCBI’s Sequence Read Archive (SRA) under the BioProject ID: PRJNA1280267. Declarations Author Contributions SMK, AJM, NS conceived and planned the execution of the experimental strategy and intervention in the hospital. AEH, and SMK performed the experiments, sampling, and laboratory analysis. AEH, SMK, SN and AJM worked on data and metadata curation. SN, KKC, SMK and SCB performed and refined the microbiome analysis methods and analyzed the microbiome data. SN, SMK, SCB, AEH, NS, and AJM reviewed the statistical approaches and contributed to the interpretation of the results. SMK, KKC, NS and AJM took the lead in writing the manuscript. Funding was secured by SMK, NS and AJM. All authors provided critical feedback and helped shape the research, analysis, and manuscript. Acknowledgements This study was supported by the National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance (NIHR200915, NIHR207397), a partnership between the UK Health Security Agency (UKHSA) and the University of Oxford, and 8also by the NIHR Oxford Biomedical Research Centre (BRC). The views expressed are those of the authors and not necessarily those of the NHS, NIHR, UKHSA or the UK Department of Health and Social Care. Competing interests The authors declare no competing interests. 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Infect Control Hosp Epidemiol , 1–3 (2024). https://doi.org/10.1017/ice.2024.58 Vanstokstraeten, R. et al. Evaluation of a peracetic-acid-based sink drain disinfectant on the intensive care unit of a tertiary care centre in Belgium. Journal of Hospital Infection 154, 45–52 (2024). https://doi.org/https://doi.org/10.1016/j.jhin.2024.09.008 Bamford, N. C., MacPhee, C. E. & Stanley-Wall, N. R. Microbial Primer: An introduction to biofilms - what they are, why they form and their impact on built and natural environments. Microbiology (Reading) 169 (2023). https://doi.org/10.1099/mic.0.001338 Mann, E. E. & Wozniak, D. J. Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol Rev 36, 893–916 (2012). https://doi.org/10.1111/j.1574-6976.2011.00322.x Penesyan, A., Paulsen, I. T., Kjelleberg, S. & Gillings, M. R. 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Spread from the Sink to the Patient: in situ Study Using Green Fluorescent Protein (GFP) Expressing- Escherichia coli to Model Bacterial Dispersion from Hand Washing Sink Trap Reservoirs. Appl Environ Microbiol (2017). https://doi.org/10.1128/AEM.03327-16 Mathers, A. J. et al. Intensive Care Unit Wastewater Interventions to Prevent Transmission of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Organisms. Clin Infect Dis 67, 171–178 (2018). https://doi.org/10.1093/cid/ciy052 Loudermilk, E. M. et al. Tracking Klebsiella pneumoniae carbapenemase gene as an indicator of antimicrobial resistance dissemination from a hospital to surface water via a municipal wastewater treatment plant. Water Research 213, 118151 (2022). https://doi.org/https://doi.org/10.1016/j.watres.2022.118151 Gweon, H. S. et al. The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples. bioRxiv , 593301 (2019). https://doi.org/10.1101/593301 Krueger, F. TrimGalore- A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files. (2015). %3Chttps://github.com/FelixKrueger/TrimGalore%3E . Wood, D. E. & Salzberg, S. L. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 15, R46 (2014). https://doi.org/10.1186/gb-2014-15-3-r46 Lu, J., Breitwieser, F. P., Thielen, P. & Salzberg, S. L. Bracken: estimating species abundance in metagenomics data. PeerJ Computer Science 3, e104 (2017). https://doi.org/10.7717/peerj-cs.104 Bushnell, B. BBMap: A Fast, Accurate, Splice-Aware Aligner , < https://escholarship.org/uc/item/1h3515gn%3E (2014). Jia, B. et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45, D566-D573 (2017). Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 29 Apr, 2026 Reviews received at journal 28 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviewers agreed at journal 05 Apr, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 21 Mar, 2026 First submitted to journal 18 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9163989","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":615391984,"identity":"75e58763-5867-4a46-a148-b357e1e2053a","order_by":0,"name":"Shireen M Kotay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBAC+Rn5zx/zVDDwMDAwNjAkgIQOENBicOMMmzHPGZK0SPawSfO2IQsR1CLNe0yad95hGfP+xW0PHtQwyPHdSMCvRX4eX5o077bDPDI3HrYbJBxjMJYkpIXhHoMZUEsaj4TEwTaJBDaGxA0EtdwGaZkD0/KPoZ6wlps9QC0NNjwS/I1tEoltDAkGhLQY3DiWZjjjGFCLBCNQS5+E4cwzD/BrkZ+RfPzBhxoJewn+488kf3yzkec7TshhcCABVilBrHIQ4D9AiupRMApGwSgYSQAAwaxExfI5/98AAAAASUVORK5CYII=","orcid":"","institution":"University of Virginia Health System","correspondingAuthor":true,"prefix":"","firstName":"Shireen","middleName":"M","lastName":"Kotay","suffix":""},{"id":615391986,"identity":"d51a06eb-dc79-4828-90cd-c289fe1d18f5","order_by":1,"name":"Aubrey E Hetzler","email":"","orcid":"","institution":"University of Virginia Health System","correspondingAuthor":false,"prefix":"","firstName":"Aubrey","middleName":"E","lastName":"Hetzler","suffix":""},{"id":615391988,"identity":"fdf9e351-d64f-486c-a798-a6f261a31f36","order_by":2,"name":"Sharvari Narendra","email":"","orcid":"","institution":"University of Virginia Health System","correspondingAuthor":false,"prefix":"","firstName":"Sharvari","middleName":"","lastName":"Narendra","suffix":""},{"id":615391990,"identity":"0cf23cc8-acdf-4022-bcb5-8b686bbd762e","order_by":3,"name":"Kevin K Chau","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"K","lastName":"Chau","suffix":""},{"id":615391992,"identity":"21e926a1-c0d4-4238-b9dc-b5c7f703171b","order_by":4,"name":"Salvador Castañeda-Barba","email":"","orcid":"","institution":"University of Virginia Health System","correspondingAuthor":false,"prefix":"","firstName":"Salvador","middleName":"","lastName":"Castañeda-Barba","suffix":""},{"id":615391994,"identity":"53406356-bf70-4b21-8769-67ca7bb91303","order_by":5,"name":"Nicole Stoesser","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Stoesser","suffix":""},{"id":615391996,"identity":"142bca57-4f36-4c23-bae9-e46e9edaf55e","order_by":6,"name":"Amy J Mathers","email":"","orcid":"","institution":"University of Virginia Health System","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"J","lastName":"Mathers","suffix":""}],"badges":[],"createdAt":"2026-03-19 02:24:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9163989/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9163989/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105957949,"identity":"1c49f847-b8f4-41ec-bf54-05bcef94ddec","added_by":"auto","created_at":"2026-04-01 21:17:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":740366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the experimental strategy and sample processing workflow.\u003c/strong\u003e Timeline of PPAA-foam application and sampling in a) experimental (SinkLab) and b) hospital settings. Red dotted lines indicate PPAA-foam treatment days; green solid lines denote sink sampling timepoints. c) Sample processing workflow for sink-drain biofilm and sink-trap water using culture-based and shotgun metagenomic sequencing approaches. (Data for sink-traps presented in Supplementary Figures).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/4b316c1d42a1ad14b31cc4a2.png"},{"id":106093460,"identity":"b99d36ea-ad12-4f6c-b24b-7b1a4870cb89","added_by":"auto","created_at":"2026-04-03 11:37:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":856784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of repeated PPAA-foam-based cleaning treatment on bacterial populations in experimental sink-drains.\u003c/strong\u003e A) Quantitative culture of carbapenemase-producing Enterobacterales (CPE) in sink-drains across treatment groups (Control=untreated, T3=3-day PPAA-foam application interval, T5=5-day PPAA-foam application interval, T7=7-day PPAA-foam application interval), B) Temporal changes in bacterial community composition at the order level. Relative abundance of bacterial taxa at the order-level in sink-drains across time and treatment groups, C) Alpha diversity indices for sink-drain microbiomes across collection timepoints and treatment groups. Error bars represent standard deviation; statistical significance was assessed using non-parametric tests as indicated in text. D) Non-metric multidimensional scaling (NMDS) of genus-level profiles based on Bray–Curtis dissimilarities comparing baseline (D-3) with post-treatment timepoints (D28, D35).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/cadfabe0deb14621644315d9.png"},{"id":105957951,"identity":"3a0f247b-8921-4e1f-8e96-4d0513a43d0c","added_by":"auto","created_at":"2026-04-01 21:17:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":985726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative sink-drain abundance profiles in response to foam-based cleaning treatment of the subset of six genera corresponding to the CPE species inoculated in the experimental sinks during the biofilm establishment phase.\u003c/strong\u003e a) Profiles are shown over time stratified by treatment group (Control=untreated, T3=3-day PPAA-foam application interval, T5=5-day PPAA-foam application interval, T7=7-day PPAA-foam application interval). b) Mean relative abundance at the genus-level at baseline (D-3), early post-treatment phase (D28) and 8 days post-treatment phase (D35).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/a9f8e1fee4476ebee939364a.png"},{"id":105957953,"identity":"ff69bdda-f5e2-4f8b-9e4c-2bd8a2a99b01","added_by":"auto","created_at":"2026-04-01 21:17:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":834609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the known resistome in response to foam-based cleaning treatment, including total resistome load and the carbapenemases \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eKPC\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eNDM\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003eProfiles of the longitudinal change in\u003cstrong\u003e \u003c/strong\u003ea)\u003cstrong\u003e \u003c/strong\u003etotal sink-drain resistome load and b) sink-drain \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e abundance across the treatment groups; g) Mean normalized sink-drain \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e abundance at baseline (D-3) versus post-treatment (D28 \u0026amp; D35).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/c50d1be91ae1bd1c9b3746b9.png"},{"id":106093943,"identity":"9ef3331f-637f-40c9-8748-7210f4c19671","added_by":"auto","created_at":"2026-04-03 11:40:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":477521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of PPAA-foam treatment on bacterial populations in hospital sink-drains.\u003c/strong\u003e \u0026nbsp;a) Qualitative CPE culture results across time by sink; b) trend of relative abundance among bacterial orders across historical and after-treatment timepoints; c) profile of alpha-diversity indices across time; and d) non-metric multidimensional scaling using Bray–Curtis dissimilarities at the genus-level comparing baseline (mean historical) to post-treatment (D28) timepoints.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/0a29c81a5131d66ca52d4d75.png"},{"id":106402201,"identity":"16f61183-306e-4d66-91d5-913f44941dc2","added_by":"auto","created_at":"2026-04-08 09:11:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":416185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistome in hospital sink-drains in response to PPAA-foam treatment with a 7-day application interval. \u003c/strong\u003eA) Profiles of the\u003cstrong\u003e \u003c/strong\u003elog-scaled\u003cstrong\u003e \u003c/strong\u003etotal resistome load across 4 sink-drains evaluated, B) normalized mean \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e abundance in historical baseline samples and at D28, C) alpha diversity indices across time in relation to treatment application and, D) non-metric multi-dimensional scaling analysis using Bray–Curtis dissimilarities at the ARG family-level comparing baseline (all historical samples) to post-treatment samples (D28).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/132b8c2da28fafcd101cdc4b.png"},{"id":106405475,"identity":"14623a96-7a99-49d6-853a-eac837601906","added_by":"auto","created_at":"2026-04-08 09:26:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5540447,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/7622f890-8047-43f2-b197-e0b9758fe924.pdf"},{"id":105957948,"identity":"4791ca61-77c6-4053-9e35-7b3f091c73d4","added_by":"auto","created_at":"2026-04-01 21:17:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1370008,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9163989/v1/e730e19ae794236c629ebc14.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Short-Lived Success: Repeated Peroxide-Peracetic Acid-Based Foam Disinfection Selects for Carbapenem-Resistant Enterobacterales in Hospital Sinks Drains","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) is a major health threat and key contributor to global mortality. Healthcare-Acquired Infections (HAI) are often caused by multidrug-resistant Gram-negative bacilli (MDR-GNB), including Carbapenemase-producing Enterobacterales (CPE), making them hard to treat\u003csup\u003e1,2\u003c/sup\u003e. Healthcare-associated wastewater interfaces, like sink-drains, represent major reservoirs for MDR-GNB and are associated with transmission to patients, patient colonization, and subsequent HAI, but mitigation of this risk is challenging\u003csup\u003e3-5\u003c/sup\u003e. The main risk to patients is thought to be linked to the growth of biofilm from sink-traps allowing upward growth to the sink-drain surface, with bacterial pathogens then dispersed out from the sink during faucet use through water turbulence impacting contaminated drain surfaces and resulting in droplet generation\u003csup\u003e6,7\u003c/sup\u003e. Although targeting sink-drains for decolonization rather than sink-traps is important given they are the focal point for pathogen dispersal to the surroundings\u003csup\u003e6,8\u003c/sup\u003e, sink-traps are reservoirs for the transfer of AMR genes and selection of drug-resistant pathogens which contribute to this biofilm, representing an additional important target for mitigation\u003csup\u003e9,10\u003c/sup\u003e. The optimum strategy for cleaning hospital sink-drains and sink-traps to minimize pathogen and AMR gene selection in these reservoirs and avoid transmission remains unclear\u003csup\u003e3\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Several types of cleaning products and disinfectants are used for environmental cleaning in hospitals, which often include one or more of the following; chlorine, peracetic/hydroxy-peracetic acid, hydrogen peroxide and quaternary acid compounds. These chemical disinfectants are inconsistently effective at eliminating pathogens in environmental biofilms, even at recommended concentrations\u003csup\u003e11\u003c/sup\u003e. As a result, biofilm-associated pathogens frequently persist or recolonize surfaces after disinfection, highlighting the need for alternative approaches beyond standard chemical disinfection. Most commercially available cleaning products are conventionally poured down the sink-drain as liquids, and often lack adequate contact time to disrupt established biofilms inside plumbing systems\u003csup\u003e12-15\u003c/sup\u003e. Novel approaches targeting biofilms show promise, but their efficacy remains to be established\u003csup\u003e3,4\u003c/sup\u003e, including an evaluation of possible adverse effects (e.g., selection for potential pathogens). The application of foam-based cleaning agents containing broad-spectrum disinfectant, bactericidal and anti-biofilm properties, with increased contact time with pipework, has reportedly been more effective in reducing hospital-sink-drain pathogen colonization than bleach and other disinfectants\u003csup\u003e16-20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;While foam-based, broad-spectrum cleaning may offer advantages, the durability of its effects and the optimal frequency of application have not been determined. For this study, we aimed to determine an effective frequency of application for such a product Virasept\u0026trade;, Ecolab, a peroxide-peracetic acid (PPAA) foam-based, broad-spectrum cleaning agent. We sought to understand the impact of this intervention on the sink-trap and sink-drain microbiomes with a focus on CPE, AMR gene amplification, and dysbiosis, using culture-based and metagenomic methods in both experimental and hospital settings.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003ePPAA-foam treatment selectively enriches Enterobacterales, including antimicrobial-resistant strains, in experimental sink-drains and sink-traps\u003c/h2\u003e \u003cp\u003eWe first evaluated the impact of repeated PPAA-foam treatment on sink-drain and sink-trap biofilms using a controlled experimental SinkLab system, integrating quantitative culture and shotgun metagenomics. We then assessed whether these effects translated to a clinical environment by applying the same intervention to hospital sink-drains and monitoring microbial and resistome dynamics longitudinally (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To evaluate the effect of PPAA-foam treatment frequency on CPE colonization of sink-drains, sink-drains were seeded with a known CPE consortium and left undisturbed for 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.a, SinkLab biofilm establishment) prior to invention. Subsequently, across three experimental arms, sink-drain biofilms were treated with PPAA-foam either every 3 days [T3], 5 days [T5] or 7 days [T7] (5 sink-drains for each arm) over a 28-day period, alongside untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Systematic sampling was undertaken at baseline (D-3) and at defined intervals post-treatment at D0, D3, D7, D14, D28 and D35.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe quantified CPE burden at the drain level via quantitative culture (see Methods), which revealed an immediate but non-durable reduction in CPE burden following PPAA-foam exposure. Across all treatment regimens, CPE cumulative counts showed 2\u0026ndash;5 log reductions relative to baseline (D-3) immediately after the first PPAA-foam exposure (D0; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In the T3 group, (three-day treatment intervals), CPE abundance declined further after the second treatment (D3), but rebounded thereafter despite continued PPAA-foam applications every three days. In the T5 or T7 groups (5- \u0026amp; 7-day application intervals), CPE levels rebounded to near-baseline levels between treatments, declined transiently after the second application (at D5 and D7 respectively), and then progressively increased to near-baseline levels despite of continued PPAA-foam application, as with the T3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Regardless of application frequency or total number of applications (T3 [n\u0026thinsp;=\u0026thinsp;10], T5 [n\u0026thinsp;=\u0026thinsp;6] and T7 [n\u0026thinsp;=\u0026thinsp;5]), PPAA-foam was ineffective in reducing CPE counts from baseline over 28 days. By Day 35 (eight days after final treatment), CPE abundance did not differ significantly from pre-treatment baseline in any treatment groups (Wilcoxon signed-rank test with Bonferroni correction \u003cem\u003ep\u003c/em\u003e\u003csub\u003eadj\u003c/sub\u003e=1.00, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A summary of baseline and endpoint comparisons, effect sizes and adjusted p-values is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, untreated control sinks exhibited a significant increase in CPE abundance over the same period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, endpoint (D35) CPE abundance in the control sinks was comparable to those observed in all treatment group endpoints, with the observed increase likely indicating natural maturation and upward trend in CPE abundance over time in the absence of any intervention. Additionally, while some variability in CPE abundance was observed in the control sinks (IQR: 7.28\u0026ndash;8.28 log\u003csub\u003e10\u003c/sub\u003e CFU/ml, max: 8.95), the overall variation was substantially lower than that observed in the treatment groups over the course of the study (T3:1.70\u0026ndash;6.98, max: 9.18; T5: 3.73\u0026ndash;7.26, max: 9.18; T7: 2.85\u0026ndash;7.232, max:9.079). The patterns observed for sink-drain CPE counts were mirrored for counts of all Gram-negative bacilli, including lactose- and non-lactose-fermenting organisms (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetagenomic analysis corroborated culture-based results, and revealed treatment-associated community restructuring. In treated sinks, Enterobacterales relative abundance decreased transiently following PPAA-foam application but only up to D14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In T5 and T7 sinks on D3 (i.e. a sampling timepoint between treatments), Enterobacterales populations briefly recovered followed by transient suppression again after the second PPAA-foam treatment. After D14, however, Enterobacterales relative abundance increased steadily across treated sinks regardless of treatment interval or total treatments administered, dominating the sink-drain biofilm communities by D35 (mean relative abundance: 0.741 [SD: +/-0.161]). This resurgence in Enterobacterales in treated sink-drains coincided with a marked decrease in relative abundance in several taxa commonly associated with stable drain biofilms, including the orders Micrococcales, Pseudomonadales, Brevundimonadales and Agrococcales (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, control sink-drains maintained relatively stable community profiles, with Enterobacterales decreasing over time and remaining a minor community component at the end point (D35 mean relative abundance: 0.034 [SD: +/-0.018]; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePPAA-foam treatment induces pronounced restructuring of the sink-drain microbial communities in the SinkLab model\u003c/h2\u003e \u003cp\u003eConsistent with the post-treatment enrichment and eventual dominance of Enterobacterales, PPAA-foam application led to marked reduction in sink-drain microbial diversity over time. Alpha-diversity metrics (Chao1 richness, Pielou\u0026rsquo;s Evenness and Shannon Diversity) show marked reductions in overall microbiome diversity by D28 across treated sink-drains. This reduction in overall microbiome diversity was significant for treatment intervals T3 (Shannon, Welch\u0026rsquo;s Two-Sample t-test \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0023), and T5 (Shannon, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), but not for T7 (Shannon, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0798) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In contrast, controls sink-drains that did not receive PPAA-foam treatment showed no significant changes in any diversity metric over the study period, indicating relative community stability in the absence of intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Comparisons of pre-treatment baseline (D-3) and post-treatment endpoints (D28/D35) revealed significant overall community restructuring across all sink-drains, irrespective of treatment frequency as evidenced by shifts in beta-diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed; Welch\u0026rsquo;s Two-Sample t-test, T3-\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005, T5-\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010 \u0026amp; T7-\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045 for D-3vsD35).\u003c/p\u003e \u003cp\u003eDuring the biofilm establishment, experimental sinks were inoculated with a defined mix of nine CPE strains (six genera; see Methods). Longitudinal metagenomic profiling enabled tracking of the abundance of the six genera corresponding to which the spiked-in species belong. Across the all PPAA-foam treatment groups, four genera associated with the inoculum (\u003cem\u003eCitrobacter\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eEscherichia\u003c/em\u003e, and \u003cem\u003eKlebsiella\u003c/em\u003e) exhibited upward trends in relative abundance following treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Fig.S2), consistent with selective enrichment rather than sustained suppression.\u003c/p\u003e \u003cp\u003eComparing baseline (D-3) and post-treatment timepoints (D35), the increase in relative abundance was significant for \u003cem\u003eCitrobacter\u003c/em\u003e in T5 sink-drains (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0107), whereas other genera showed variable, non-uniform responses across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, statistically significant reductions were observed for \u003cem\u003eEnterobacter\u003c/em\u003e in T7 sink-drains on D28, but with a recovering trend by D35. In contrast, \u003cem\u003eSerratia\u003c/em\u003e populations declined significantly in both treated and untreated sink-drains.\u003c/p\u003e \u003cp\u003eComparable dynamics were observed at the sink-trap level. Cumulative counts of CPE and \u003cem\u003eAeromonas\u003c/em\u003e as well as Gram-negative bacteria rebounded strongly in treated sink-traps after D14, despite continued PPAA-foam application (Fig.S3a-c). Metagenomic analysis further demonstrated reductions in genus-level richness and clear compositional shifts in treated sink-traps relative to the baseline (Fig.S2d\u0026amp;e mirroring patterns observed in corresponding sink-drains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePPAA-foam treatment increases sink-drain resistome burden, including the major carbapenemases\u003c/b\u003e, \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eKPC\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eNDM\u003c/b\u003e\u003c/sub\u003e, \u003cb\u003ein experimental sink-drains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the impact of PPAA-foam on antimicrobial resistance dynamics, we quantified the sink-drain resistome using shotgun metagenomics, expressing ARG abundance as transcripts per million (TPM) aggregated at the AMR gene family level. The experimental biofilms were initially seeded with carbapenemase-producing Enterobacterales carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (see Methods), enabling longitudinal tracking of these clinically relevant resistance determinants alongside global resistome changes. Across all treatment regimens, the total resistome load decreased by several orders of magnitude immediately following the first PPAA-foam application but recovered progressively with continued treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). By the post-treatment endpoints (D28/D35), the total resistome load exceeded baseline in all treated sink-drains, reaching statistical significance in T3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; Welch Two Sample t-test \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025). In contrast, untreated control sink-drains showed a steady decline in resistome loads over time, resulting in a significantly lower ARG abundance at D35 compared with baseline for all PPAA-foam-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0251). At the AMR gene family-level, PPAA-foam treatment was associated with reduced resistome richness and evenness (Fig.S4), coinciding with an increase in the relative abundance of key carbapenemase genes, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; D-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e vs by D28/D35). Notably, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e abundance increased significantly in T5 and T7 sink-drains by D28/D35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec; Welch Two Sample t-test \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0150 and 0.0012), while \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e displayed concordant upward trends the corresponding treatment groups it was not found significant.\u003c/p\u003e \u003cp\u003eDespite the overall increase in resistome burden, NMDS ordination based on Bray\u0026ndash;Curtis dissimilarities revealed more modest separation between baseline and post-treatment samples at the AMR gene-family level than observed for corresponding taxonomic profiles, suggesting selective amplification of dominant resistance determinants rather than wholesale restructuring of the resistome (Fig.S4c). Comparable patterns of resistome recovery and carbapenemase enrichment were observed in sink-traps (Fig. S4b\u0026amp;d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn hospital sink-drains, PPAA-foam treatment exerts only transient suppression of carbapenemase-producing Enterobacterales and reshapes resident microbial communities\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the effects of PPAA-foam treatment observed in experimental sinks translated to a \u0026lsquo;real-world\u0026rsquo; hospital setting, we conducted a pilot intervention in four hospital sinks, applying PPAA-foam at a 7-day interval (T7) over a 28-day period, with sampling immediately before and after each treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast to the experimental SinkLab system, hospital surveillance relied on qualitative culture-based detection of CPE (presence/absence), reflecting routine infection prevention workflows and precluding direct quantitative comparisons. Further, historical CPE surveillance data (culture-based CPE- presence/absence) were available for these sinks, allowing qualitative comparison of pre- and post-treatment colonization status.\u003c/p\u003e \u003cp\u003eConsistent with the transient effects observed in the SinkLab, PPAA-foam application in hospital sinks produced a short-lived suppression of detectable CPE colonization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Following the first treatment, one of three initially CPE-positive sinks remained culture-positive, while all three were culture-negative after the second treatment. However, this suppression was not sustained after the third treatment again yielded CPE, and by the fourth application all four sinks, including the two that were CPE culture-negative prior to intervention were CPE culture-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). While qualitative in nature, these findings parallel the rebound dynamics observed in the experimental model and suggest that repeated foam-based cleaning does not prevent, and may ultimately coincide with, CPE recolonization in hospital sink-drains.\u003c/p\u003e \u003cp\u003eShotgun metagenomic profiling provided higher-resolution insight into community-level responses to the treatment. Over the 28-day intervention period, the relative abundance of Enterobacterales increased progressively compared with baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), accompanied by declines in the relative abundance of Sphingomonadales (although not significant, Welch Two Sample t-test \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.1452 and 0.09475 respectively). Importantly, this shift contrasted with the stability of the order-level community structure across the three historical pre-treatment timepoints spanning 213 days, indicating that the observed perturbations were PPAA-foam treatment-associated rather than a natural community drift. Alpha-diversity analyses showed an initial reduction in the genus-level richness and evenness immediately after PPAA-foam treatment with recovery toward baseline values by D28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), consistent with a transient disturbance rather than sustained suppression. NMDS ordination confirmed significant shifts in genus-level composition over the treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), characterized by increased representation of \u003cem\u003eCitrobacte\u003c/em\u003er, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e genera (Supplemental Fig.S5). Changes in the log10 abundance of dominant Enterobacterales genera pre- and post-treatment following individual PPAA-foam treatment events were modest, reinforcing that community restructuring occurred gradually over repeated applications rather than as an acute effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRepeated PPAA-foam treatment produces limited and short-lived perturbation of the hospital sink-drain resistome\u003c/h3\u003e\n\u003cp\u003eLongitudinal metagenomic profiling demonstrated that PPAA-foam treatment exerted only short-lived effects on hospital sink-drain resistome. The total ARG abundance decreased immediately following the first treatment but rebounded to near-baseline levels within seven days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, D0 vs D7B). Similar transient reductions were observed immediately after each subsequent treatment, with ARG loads consistently recovering between treatment cycles and frequently exceeding the historical mean baseline. By D28, total ARG abundance was comparable to or higher than pre-treatment values (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, D-47 vs D28), indicating that repeated PPAA-foam disinfection did not achieve sustained suppression of the resistance determinants within the hospital drain biofilms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of clinically relevant carbapenemase genes revealed a modest, non-significant increases in the mean normalized \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e abundance by D28, while \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e remained largely unchanged throughout the study period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These findings suggest persistence of key carbapenemase determinants despite repeated disinfectant exposure, rather than effective depletion. At the ARG family level, resistome richness and diversity increased modestly over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), consistent with selective turnover rather than overall contraction of resistance gene classes. Bray\u0026ndash;Curtis ordination demonstrated a detectable shift in resistome composition by D28 compared with historical baseline values (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), although the magnitude of divergence was limited. Heatmap-based profiling of the top 20 ARG families further supported this interpretation, revealing transient post-treatment decreases in several β-lactamase families immediately following the first PPAA-foam application, but these genes rebounded by D28 alongside a modest enrichment of efflux-associated genes and macrolide- and aminoglycoside-resistance determinants (Fig.S6). Comparable temporal patterns were observed in sink-traps, reinforcing the inference that the hospital sink resistome exhibit substantial resilience and structural stability despite repeated chemical disruption using PPAA-foam.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe set out to determine the impact of a foam-based, peroxide\u0026ndash;peracetic acid disinfectant on the microbiology and ecology of sink-drain biofilms when applied at different frequencies over time. We chose to focus on effects at the sink-drain level as this presents the most immediate risk of dissemination to patients\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, but observed similar effects at the sink-trap level (Figs \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S3, S4, S6 \u0026amp; S7). We demonstrate that PPAA-foam exerts unintended and potentially counter-productive ecological effects. Across both controlled SinkLab experiments and hospital pilot testing, repeated PPAA-foam exposure consistently destabilized resident microbial communities, promoted Enterobacterales dominance, and favored the persistence and amplification of CPE. These observed effects occurred within short periods of time (28 days), and irrespective of the application intervals tested (3, 5 and 7 days), indicating that frequency modulation alone does not mitigate adverse ecological outcomes. Although interventions targeting sink-drains are intended to reduce transmission risk, our findings indicate that they alter the drain-biofilm microbial communities associated with amplification and persistence of resistance determinants within both sink-drains as well as sink-traps.\u003c/p\u003e \u003cp\u003eA key finding of this study is that PPAA-foam induces pronounced microbiome dysbiosis rather than sustained microbial suppression. In experimental sinks, repeated exposure resulted in marked loss of community richness and evenness, accompanied by depletion of taxa commonly implicated as foundational biofilm members (including Micrococcales, Pseudomonadales, Brevundimonadales and Agrococcales). Biofilms are structured, mixed microbial communities embedded within a self-produced extracellular matrix that contributes disproportionately to community stability, nutrient cycling, and protection from stressors, with model taxa such as Pseudomonadales (e.g., \u003cem\u003ePseudomonas spp.\u003c/em\u003e) known to produce key matrix components central to biofilm architecture and function\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Their selective loss likely compromises the ecological scaffolding of the biofilm, creating vacant niches that facilitate recolonization by fast-growing, stress-tolerant opportunists such as Enterobacterales. Such restructuring represents a classical form of dysbiosis in surface-attached microbial assemblages and provides a plausible mechanistic basis for the observed pathogen enrichment and resistance persistence.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBy integrating quantitative culture-based approaches focused on Enterobacterales with longitudinal shotgun metagenomics, our study extends beyond prior work that has primarily relied on short-term culture-based endpoints or overall bacterial burden. Previous studies have highlighted that disinfection of sink-drains may have limited efficacy and result in a transient reduction of Gram-negative bacilli followed by recolonization within 5\u0026ndash;7 days after single PPAA-foam applications, and noted a progressive decrease in the bacterial burden with repeated treatments every 3 days. However, these studies examined shorter follow-up intervals (\u0026le;\u0026thinsp;13 days) or focused on overall bacterial burden rather than resistance ecology\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Our data substantially extend these observations by demonstrating that repeated PPAA-foam application does not only fail to achieve durable suppression of Enterobacterales or ARG loads beyond the immediate post-treatment period, and further suggests PPAA-foam may become ineffective after prolonged use. Another study testing different PPAA-foam application frequencies on the burden of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. maltophilia\u003c/em\u003e in ICU room sink drains found that applications 5 times a week was more effective than a weekly application\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, they similarly noted a resurgence and/or persistence of these organisms in the post-intervention phases, and that this frequency of application may not be routinely sustainable in hospital settings. A fourth study reported regrowth of Gram-negative bacilli using PPAA-foam treatment intervals of 2, 3 and 5 days and suggested that daily application may be required to reduce the concentration of Gram-negative bacilli to 2 log10 CFU/mL, whilst recognizing that the \u0026ldquo;safe\u0026rdquo; abundance threshold remains unknown\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFindings from a recent randomized controlled trial in renovated inpatient sinks evaluating peroxide-peracetic acid foam disinfectant using culture-based screening, demonstrated delayed colonization in the treated sinks and site-dependent reductions in drain and tailpipe contamination, with re-establishment of Gram-negative organisms after disinfection\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We did not attempt daily application which could be a noted limitation of the current work, but previous reports question the practicality and sustainability of such intensive cleaning regimens in healthcare settings. Repeated PPAA-foam exposure functioned as a recurring ecological disturbance, triggering loss of foundational taxa and rapid biofilm reassembly dominated by opportunistic and resistant organisms. These findings are likely relevant to other engineered wet environments where biofilms experience repeated chemical disturbance, including wastewater infrastructure.\u003c/p\u003e \u003cp\u003eA notable strength of this study lies in its ability to capture both potentially high-risk pathogens of interest (e.g., CPE) and wider microbiome-level responses. We observed that the dysbiosis induced in sink-drain and sink-trap microbiomes in response to PPAA-foam treatment appears to have contributed to the overgrowth of drug-resistant potential pathogens. This was evident from the marked reductions in the alpha-diversity indices and community-level shifts observed over time in the sinks that received treatment that diminished competition and favored opportunistic pathogen expansion. This phenomenon was more pronounced in the experimental sinks (SinkLab), potentially reflecting the absence of the continual microbial seeding and physicochemical/environmental fluctuations present in the hospital setting. For example, hospital sinks may have been exposed to different biotic and abiotic factors as a result of sink usage by and inputs from patients, patient-care providers and the interconnected plumbing network that likely provide diverse inocula that influence recolonization kinetics\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the directionality of effects was consistent across SinkLab and hospital settings, indicating that similar ecological mechanisms operate under both controlled and real-world conditions.\u003c/p\u003e \u003cp\u003eAlthough this study offers valuable insights, some limitations merit consideration. The numbers of sinks within each experimental treatment group was modest, and although we identified some significant changes at the order-, genus- and AMR gene-level, our statistical power to detect subtler taxonomic or functional shifts may be limited. In the hospital setting, only a 7-day treatment interval was assessed as it was considered most feasible, and was limited to relatively small number of sinks in a single facility due to the available resources and the logistics of implementation. Our findings however, were consistent with those observed in our experimental set-up, albeit less marked. This convergence of findings across independent models strengthens their generalizability. It is possible that PPAA-foam cleaning would work differently in different contexts, given the variability observed in previous studies of sink microbiomes and usage\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; however, our findings are consistent with effects observed in the limited number of other studies evaluating sink-drain/trap disinfection with the PPAA-foam product across different contexts. Similarly, there may be other combinations of chemical disinfectants or chemical disinfection strategies that have different effects that we have not evaluated here. Moreover, in contrast to our hospital study, our experimental sink study did not include immediate pre- and post- application sampling. In retrospect, this strategy was valuable and could have further clarified short-term recovery kinetics. Although hospital sink-drains differ from the SinkLab model in baseline complexity and surveillance resolution, both systems exhibit convergent qualitative patterns in response to PPAA-foam treatment: transient microbial suppression followed by recovery or enrichment of Enterobacterales and associated microbiome restructuring. Importantly, the qualitative nature of our hospital environmental culture data limits inference on magnitude, but the consistency in temporal trajectories strengthens the conclusion that foam-based cleaning alone is insufficient to durably control sink-associated CPE in clinical environments.\u003c/p\u003e \u003cp\u003eIn conclusion, we show that sink-drain and sink-trap disinfection with foam-based, broad-spectrum cleaning products such as PPAA-foam may paradoxically select for the very organisms these interventions aim to eradicate. The remarkable ecological resilience of these complex microbial reservoirs poses a substantial challenge to routine infection-prevention practices, particularly in healthcare environments where colonization of wastewater plumbing systems are driven by several factors. These constraints underscore the limitations of relying solely on chemical disinfection to suppress pathogen and resistance burdens in these wet environments and highlight that monitoring for unintended microbial shifts following chemical disinfection strategies is important. Moving forward, effective mitigation will likely require integrated and ecologically informed strategies that combine structural redesign of sink components, targeted microbial competition, and enhanced surveillance technologies capable of detecting and responding to early shifts in microbial and resistome composition. Addressing these challenges will be essential to ensure that practices reduce rather than inadvertently propagate pathogen reservoirs and antimicrobial resistance within the hospital built environment.\u003c/p\u003e"},{"header":"ONLINE METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental SinkLab setup\u003c/h2\u003e \u003cp\u003eAll experiments under this study were conducted in the University of Virginia (UVA) SinkLab, a Biosafety Level 2 facility equipped with four independent sink rigs, each housing a set of ten sinks. The drain line on each sink is comprised of a cast grid drain (P/N 760-1 Dearborn Brass\u0026reg;-Oatey, Cleveland, Ohio), a chrome coated over brass 6-inch tailpipe, 1\u0026frac14;-inch P-trap and trap-arm (P/N 701-1, Dearborn Brass\u0026reg;-Oatey, Cleveland, Ohio). All sinks on a rig drain into a shared 2-inch PVC pipe (Charlotte Pipe Charlotte, NC) leading into a 60-gallon high-density polyethylene resin (HDPE) holding water tank (Ronco Plastics, Tustin, CA). Cold-water delivery was automated using a \u0026frac12;\u0026rdquo; Brass Electric solenoid valve (JFSV00006, US Solid) with 4 mm orifice installed on faucet connectors and controlled via Raspberry Pi microcontroller (Sparkfun, Niwot, CO) with a custom MOSFET-enabled shield. These together are programmed to deliver water into each sink for 30 seconds every four hours (~\u0026thinsp;475 ml per flush; 5.1 Lmin\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e flow rate). Wastewater was collected into holding tanks, disinfected using chlorine tabs prior to disposal into a common floor drain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Sink (SinkLab): Inoculation and Biofilm establishment\u003c/h2\u003e \u003cp\u003eTo establish polymicrobial biofilms comprising carbapenemase-producing Enterobacterales, sinks were inoculated with a mixture of nine strains previously isolated from hospital sinks, wastewater or patients (Table S2). Prior to experiments, frozen stocks (\u0026minus;\u0026thinsp;80\u0026deg;C) were revived on Trypic Soy Broth (TSB) and pooled in equal volumes to generate an inoculum cocktail. Each of the sinks was inoculated by pouring 25 ml of this cocktail over the sink drain (~\u0026thinsp;10 Log CFUs of each strain). To support biofilm establishment and maturation, 25ml TSB was poured over the sink-drain daily with an average 2h dwell time before routine water flush. Biofilms were allowed to establish for 28 days prior to PPAA-foam intervention.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePPAA-foam application and Experimental sink sampling\u003c/h3\u003e\n\u003cp\u003eWe sought to understand the impact of a foam-based, broad-spectrum disinfectant product, Virasept\u0026trade; (EcoLab) containing acetic acid, hydrogen peroxide, octanoic acid, and peroxyacetic acid on sink-drains/traps in an experimental and a hospital setting. PPAA-foam foam was applied through the drain holes filling the vertical void space above the p-trap water until foam was visible over the drain, as per the manufacturer\u0026rsquo;s instructions. A five-minute contact time was maintained (per manufacturer\u0026rsquo;s instructions) before flushing with faucet water for one minute. We evaluated three different PPAA-foam application regimens (3-day interval [T3], 5-day interval [T5], 7-day interval [T7]). Control sinks did not undergo treatment but were flushed with water alone for one minute (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Sampling occurred at baseline (D-3) and post-treatment at D0, D3, D7, D14, D28 (immediate post-treatment) and D35 (eight days post treatment). Drain biofilm was sampled using ESwabs\u0026reg; and 50ml P-trap water were aspirated using a 50mL syringe attached to a cannula adapter and IV tubing (further details reported previously\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eHospital sinks: Treatment and Sampling\u003c/h3\u003e\n\u003cp\u003eFour bathroom sinks in individual intensive-care rooms with prior CPE-positivity were selected for in-hospital testing. PPAA-foam foam was applied every seven days for 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Sink-drain and sink-trap water were sampled at weekly intervals. For each sink sampling event, drain samples were collected immediately before and after PPAA-foam treatment, except on day 0 when only a post-treatment sample was taken. Sink-trap samples were collected after each PPAA-foam treatment only. Sinks remained in clinical use, resulting in variable patient occupancy and water-use patterns over the study period.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial culture\u003c/h2\u003e \u003cp\u003eAll samples were processed immediately upon collection. Sink-trap samples were centrifuged at 2,460g for 15 minutes using an Eppendorf 5810 R Refrigerated Centrifuge with a 10cm rotor radius. The supernatant was decanted to minimize any residual PPAA-foam product in the culture sample and two mL of 0.9% sterile saline solution was added to the pellet. The mixture was then vortexed to resuspend the pellet. Drain samples (ESwab) were vortexed for 10 seconds and then transferred to a two mL microtube. To increase the sample volume for culture and extraction, one mL of 0.9% sterile saline solution was added to the ESwab tube. The ESwab tube was vortexed again for 10 seconds, and the contents were transferred to the two mL microtube and vortexed to combine. Culture was prepared using CHROMagar\u0026trade; mSuperCARBA\u0026trade; (CHROM) (NEL Scientific Waterville, ME), a selective and differential (chromogenic) media for the detection of carbapenem-resistant Enterobacteriaceae, and McConkey (MAC) Agar, selective for gram -negative bacilli, and differential for lactose-fermenting organisms. Quantitative culture was prepared using the methods previously described\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To increase sensitivity, an enriched culture was prepared by inoculating 500\u0026micro;l of each sample into 4.5mL of TSB containing a 10\u0026micro;g Ertapenem (ETP) BD BBL\u0026trade; Sensi-Disc. After 12 hours at 35\u0026deg;C a 10\u0026micro;l inoculating loop was used to streak the cultures on CHROM, which were subsequently incubated for 12 hours at 35\u0026deg;C.\u003c/p\u003e \u003cp\u003eMAC quantitative culture was enumerated to identify the growth of lactose fermenters (pink colonies), and non-lactose fermenters (white or clear colonies). The CHROM quantitative culture was enumerated for unique colonies, based on color and morphology. Additionally, the enriched culture was assessed for the presence of unique colonies using the same criteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMetagenomic sequencing\u003c/h2\u003e \u003cp\u003eDNA from sink drain biofilm samples was extracted using DNeasy PowerSoil\u0026reg; Kit (Qiagen) following manufacturer\u0026rsquo;s protocol with modifications to increase gDNA yield. The tubes were centrifuged at 16,000g for two minutes. 400\u0026micro;L of the sample, prioritizing the concentrated pellet, was transferred to a powerbead tube and combined with 600\u0026micro;l of CD1. Groups of 24 PowerBead tubes were vortexed for 20 minutes using a horizontal microtube holder. The tubes were centrifuged at 15,000g for one minute and transferred to a heat block set at 70\u003csup\u003eo\u003c/sup\u003eC for 10 minutes. After cooling to room temperature, 800\u0026micro;l of supernatant was combined with 200\u0026micro;l of CD2. Following centrifugation, 700\u0026micro;l of lysate was combined with 675\u0026micro;l of CD3. The lysate was transferred to the column in two 730 \u0026micro;l increments, centrifuging at 15,000g and discarding the flow through each time. Solution EA and Solution C5 were added per the manufacturer's protocol. The provided C6 buffer was used to elute 50\u0026micro;l of gDNA. DNA quality and yield (ng/\u0026micro;l) for each sample were assessed with a Qubit 4 Fluorometer using the 1X dsDNA HS Qubit Assay (Invitrogen). gDNA was stored at \u0026minus;\u0026thinsp;20\u003csup\u003eo\u003c/sup\u003eC until shipment at ambient temperature. Extracts were sequenced on Illumina platforms, using a commercial provider (Azenta GeneWiz, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eRaw sequencing reads were processed using the ResPipe v1.6.1 pipeline\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, with taxonomic and resistance estimation. Adapter trimming and quality filtering were performed with TrimGalore v0.6.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/FelixKrueger/TrimGalore\u003c/span\u003e\u003cspan address=\"https://github.com/FelixKrueger/TrimGalore\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e31302928\u003c/sup\u003e, removing reads with below average Phred score of Q25, lengths\u0026thinsp;\u0026lt;\u0026thinsp;75 bp, and the first 13 bp of Illumina adapters. Taxonomic profiling was conducted with Kraken2 v2.1.3\u003csup\u003e32\u003c/sup\u003e using a reference database of NCBI\u0026rsquo;s RefSeq bacterial and viral genomes (accessed in January 2024, version: k2_pluspf_20240112). For each taxonomic level, relative abundances were estimated using Bracken (v2.9)\u003csup\u003e33\u003c/sup\u003e. For AMR gene abundance estimation, the filtered metagenomics reads were mapped using BBMAP v38.34\u003csup\u003e34\u003c/sup\u003e, against the Comprehensive Antibiotic Resistance Database (CARD v3.2.4)\u003csup\u003e35\u003c/sup\u003e. Pseudo-abundance estimation was performed for normalization using (transcripts per million (TPM)) reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eLog\u003csub\u003e10\u003c/sub\u003e-transformed total CPE counts were analyzed longitudinally across treatment groups (Control, T3, T5, T7). Zero counts generating \u0026ndash;\u0026infin; values were replaced with zero prior to downstream analyses. Descriptive statistics (mean, standard deviation, standard error, median, and interquartile range) were calculated for each group-timepoint combination. Pairwise comparisons across timepoints were conducted using the Wilcoxon signed-rank test with Bonferroni correction for multiple testing. Statistical significance was determined at α\u0026thinsp;=\u0026thinsp;0.05. Analyses were conducted in R (v.4.2.2) using the rstatix (v.0.7.2) package and ggplot2 (v.3.5.1).\u003c/p\u003e \u003cp\u003eWithin-sample alpha diversity was measured at each taxonomic-level by assessing overall diversity (Shannon Diversity Index), community richness (Chao1), and community evenness (Pielou). Beta diversity compositional dissimilarities between samples was calculated using the Bray-Curtis dissimilarities and visualized using non-metric multidimensional scaling (NMDS). Taxonomic alpha diversity indices were calculated using phyloseq v1.44.0. AMR alpha diversity indices and all beta diversity indices were calculated using vegan v2.6.4. Visualizations were generated with ggplot2 v3.5.0; analysis used R version v4.3.0. Correlation was calculated and visualized using ggpubr v0.6.0. Statistical differences in Shannon diversity indices between groups were evaluated using Welch\u0026rsquo;s two-sample t-test, which accounts for unequal variances and is appropriate for comparisons between groups with heterogeneous dispersion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe metagenomic sequences are available at NCBI\u0026rsquo;s Sequence Read Archive (SRA) under the BioProject ID: PRJNA1280267.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSMK, AJM, NS conceived and planned the execution of the experimental strategy and intervention in the hospital. AEH, and SMK performed the experiments, sampling, and laboratory analysis. AEH, SMK, SN and AJM worked on data and metadata curation. SN, KKC, SMK and SCB performed and refined the microbiome analysis methods and analyzed the microbiome data. SN, SMK, SCB, AEH, NS, and AJM reviewed the statistical approaches and contributed to the interpretation of the results. SMK, KKC, NS and AJM took the lead in writing the manuscript. Funding was secured by SMK, NS and AJM. All authors provided critical feedback and helped shape the research, analysis, and manuscript.\u003c/p\u003e\n\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance (NIHR200915, NIHR207397), a partnership between the UK Health Security Agency (UKHSA) and the University of Oxford, and 8also by the NIHR Oxford Biomedical Research Centre (BRC). The views expressed are those of the authors and not necessarily those of the NHS, NIHR, UKHSA or the UK Department of Health and Social Care.\u003c/p\u003e\n\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eHuman Ethics and Consent to Participate declarations: not applicable\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMurray, C. J. 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Using culture-based and metagenomic approaches, we evaluated the impact of a peroxide peracetic-acid (PPAA)-foam disinfectant applied at 3-, 5- or 7-day intervals over four-weeks in a controlled Sinklab and a hospital setting. Across all application frequencies and repeated applications, PPAA-foam was ineffective in reducing carbapenemase-producing Enterobacterales counts from baseline over 28days. Instead, treatment induced pronounced microbiome dysbiosis in sink-drains and traps, characterized by reduced community diversity, enrichment of Enterobacterales, and amplification of resistance determinants, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e. Hospital sinks exhibited comparable transient effects following PPAA-foam treatment, with rapid post-treatment recovery of both microbial communities and resistome. Together, these findings demonstrate that repeated chemical disinfection in established plumbing systems may destabilize drain microbiomes and paradoxically reinforce the persistence of high-risk pathogens and antimicrobial resistance, underscoring the need for ecologically informed alternatives to chemical-only interventions.\u003c/p\u003e","manuscriptTitle":"Short-Lived Success: Repeated Peroxide-Peracetic Acid-Based Foam Disinfection Selects for Carbapenem-Resistant Enterobacterales in Hospital Sinks Drains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 21:17:04","doi":"10.21203/rs.3.rs-9163989/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T05:59:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T00:29:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T15:19:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T09:16:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2737317497999735369869567259173444535","date":"2026-04-05T15:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238392368521995663924287416555573473222","date":"2026-03-31T00:53:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264376817107341299936466392893332807341","date":"2026-03-31T00:18:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T22:48:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T19:20:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-22T00:44:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Antimicrobials and Resistance","date":"2026-03-19T02:12:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"131ce44b-dd8c-4b9d-acde-a836f7c70a5c","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":65480549,"name":"Earth and environmental sciences/Environmental sciences"},{"id":65480550,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-29T06:11:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 21:17:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9163989","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9163989","identity":"rs-9163989","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}