Efficacy of locally-available cleaning methods in removing biofilms from taps and surfaces of household water storage containers

Globally, 785 million people lack access to a basic drinking water source and approximately 1.8 billion are exposed to fecally contaminated sources. Safe, on-plot water is a goal of the SDGs, and in the interim, household water treatment and safe storage (HWTS) are promoted to reduce waterborne disease burdens. HWT improves microbiological quality and reduces diarrheal disease, while safe storage aims to prevent recontamination via physical barriers and safe dispensing. Literature indicates that unsafe water transport and storage are associated with higher odds of cholera, and safe practices are protective. Field evaluations of container disinfection show short-term improvements in chlorine residual and microbiological indicators but rapid regrowth. Stored household water often shows higher contamination than source water, indicating risks during transport and storage. Although biofilms are frequently cited as a cause of recontamination, prior studies seldom measured biofilms directly. Biofilms—microorganism aggregates within an extracellular polymeric substance (EPS) matrix—are known to develop on surfaces in contact with non-sterile water and can harbor pathogens. They have been widely studied in healthcare settings and drinking water distribution systems (DWDS), including taps, where contamination can originate from the system or retrograde contamination. Various tap materials and components show differing biofilm loads, and remediation approaches include removing parts, proprietary cleaners, or antimicrobial taps. In storage containers, surrogate indicators and microscopy have documented biofilm presence and associated water quality deterioration. However, direct measurement of biofilm growth and rigorous evaluation of household-feasible cleaning strategies have been limited. Research question: Which locally-available cleaning agents and methods are most efficacious at removing biofilms from taps and storage container surfaces?

Evidence from case-control and field studies shows that safe water transport and storage reduce cholera risk (OR ~0.55) and unsafe practices increase it (OR ~2.8). Emergency evaluations of jerrican disinfection (2.5 mg/L to 5% chlorine) report transient improvements with rapid regrowth. Some interventions reduced diarrheal prevalence, though results vary by container design. Stored water often becomes more contaminated than source water, with meta-analyses confirming post-collection contamination. In taps, contamination can arise from DWDS or retrograde routes; components like solenoid valves and flow straighteners can harbor higher biofilm loads; PVC tends to support less biofilm than stainless steel or cast iron. Remediation has included component removal, proprietary cleaners, and antimicrobial fittings; severe outcomes have been linked to tap biofilms (e.g., P. aeruginosa outbreaks). For storage containers, studies show increases in turbidity, heterotrophs, and indicators in stored versus source water; qPCR detection of pathogens (e.g., V. cholerae) in container water and dislodged biofilms; rapid biofilm development on polyethylene and galvanized steel; and variable biofilm loads not consistently associated with container type or cleaning practices. Modeling suggests biofilms significantly impact water quality and childhood diarrhea. Microscopy confirms biofilm formation on plastics within 24–48 h. Prior recommendations for routine washing exist, but rigorous assessments of practical, locally available cleaning agents and methods for biofilm removal in household contexts are limited.

Two laboratory experiments assessed efficacy of locally-available cleaning agents and methods in removing E. coli biofilms from taps and storage container surfaces. Agents: bleach (≈0.5–0.53% sodium hypochlorite), recently boiled water (~80 °C), soapy water (20 g/L grated local bar soap; pH ~11.5; taps only), and vinegar (5% acetic acid; pH ~2.4). Methods for taps: flowing agent through tap for 60 s; soaking assembled 60 s; soaking assembled 5 min; soaking disassembled 60 s; scrubbing with bottle brush 5 times assembled; scrubbing 5 times disassembled; scrubbing then soaking disassembled 5 min. Methods for container coupons: soaking 5 min; wiping with sponge; wiping with cloth. Controls received no cleaning. Tap biofilm growth system: 24 Tomlinson HFSLT taps mounted on three 10 L buckets circulated E. coli-spiked LB broth under laminar conditions (20 psi; ~1.5 L/min through each tap) for 5 days. The system was sterilized with 5% NaOCl and rinsed to zero chlorine residual. E. coli ATCC 11229 cultures were prepared to spike target 10^2–10^3 CFU/mL into sterile LB. Culture samples were enumerated 15 min after spiking and after 22.5 h. Cooling controls maintained temperature. After growth, taps were cleaned per assigned agent/method. Swabbing of vertical and horizontal internal surfaces used hygiene swabs with standard membrane filtration and mColiBlue24 enumeration; results expressed in CFU/cm^2. For imaging, coupons were cut from the seat cup (SC) and tap tube (TT), stained with DAPI, and imaged via epifluorescence/confocal microscopy. Image stacks were processed in FIJI/ImageJ (thresholding, filtering, particle analysis); quantitative estimates (e.g., thickness, density) and qualitative ratings (no growth to dense structure) were recorded. Storage container coupons: 45 coupons (4 cm^2) were cut from polypropylene containers. Surface roughness was profiled; outliers removed, leaving 30 coupons. Coupons were sterilized and submerged individually in 25 mL E. coli-spiked LB, incubated 21 days at 35 °C with gentle shaking; media refreshed every 48 h. After growth, exterior surfaces were sterilized, and interior biofilms were cleaned with assigned agent/method. For soaking, coupons were immersed in 25 mL agent for 5 min; for wiping, sterile sponge or cloth soaked in agent wiped five times in four directions. Two of three replicates per condition were sonicated in PBS (with neutralizers as needed) after vortexing to enumerate dislodged E. coli via membrane filtration; one replicate was stained and imaged as for taps (z-step 0.5 µm). Data were analyzed in Excel and R. Outcome measures: E. coli CFU/cm^2 on surfaces (taps via swabbing; coupons via sonication) and biofilm structure via imaging (thickness, qualitative scoring). Log reduction values (LRVs) were calculated versus controls.

Biofilm growth was confirmed on taps and coupons by culture increases and microscopy. In tap cultures, E. coli increased from ~10^2–10^3 CFU/mL at 15 min to ~3.9×10^5–8.25×10^8 CFU/mL at 22.5 h; control tap swabs showed 93.5–178,060 CFU/cm^2 (horizontal) and 944–543,217 CFU/cm^2 (vertical). Imaging confirmed dense biofilms; calculated densities were within one order of magnitude across sites. Tap cleaning efficacy: - Bleach (0.5%) and vinegar (5%) reduced surface E. coli to ≤1 CFU/cm^2 (non-detect by method) across all methods (>2.3 LRV for bleach; >5.3 LRV for vinegar, relative to controls), though imaging indicated remaining thin structures in some bleach cases. - Boiled water reduced E. coli variably (-0.84 to 3.42 LRV), achieving non-detect only when methods included soaking (assembled or disassembled); flowing or scrubbing without soaking was insufficient. - Soapy water reduced E. coli (1.05–6.04 LRV) but generally did not reach non-detect except when scrubbing and soaking disassembled for 5 min. - The scrubbing and soaking disassembled 5 min method achieved ≤1 CFU/cm^2 with all agents. Imaging of taps: - Boiled water (most methods) and bleach (all methods) often left thin residual biofilms (0–41 µm and 0–46 µm, respectively). Vinegar with scrubbing or disassembled soaking produced the thinnest structures (0–26 µm). Soapy water left thicker biofilms (9.8–150 µm). Qualitative analysis showed no biofilm or only isolated cells in select conditions: soaking assembled in boiled water, scrubbing assembled or scrubbing+soaking 5 min in vinegar, and soaking assembled/disassembled or scrubbing+soaking 5 min in bleach. Scrubbing aligned biofilm structures; soapy water sometimes left live cells in bubble outlines; bleach soaking left elongated dead cells in structures. Storage container coupons: - Only soaking for 5 min reduced surface E. coli to <1 CFU/cm^2 for all three tested agents. LRVs versus controls were >7.95 for bleach and boiled water, and 6.78 for vinegar. - Wiping with sponge or cloth yielded plates too numerous to count at all dilutions (>12,500 CFU/cm^2); conservative LRVs were <3.25. - Imaging of coupons was inconsistent; quantitative image metrics were unreliable. Recommendation (considering efficacy and practicality): Soak assembled taps in recently boiled water (<76.7 °C rating, ~80 °C at use) for 5 minutes. Bleach (all methods) and vinegar (scrubbing only) are technically effective alternatives, but disassembly and specialized brushes are impractical. No practical cleaning recommendation for storage containers emerged because soaking large containers is impractical.

The study directly addressed the question of which locally available agents and methods best remove biofilms from taps and container surfaces. Findings show that soaking is critical for effective biofilm removal: bleach and vinegar achieved non-detectable E. coli across tap methods, but imaging suggests EPS removal can require contact time; boiled water required soaking to be effective, likely due to heat-mediated disruption of EPS. Soapy water was largely ineffective, consistent with expectations that surfactants alone cannot penetrate or extract organisms embedded in EPS. Scrubbing without soaking tended to smear or align biofilm rather than remove it, and wiping container surfaces was ineffective, likely due to limited diffusion-penetration into thick, mature biofilms. The results align with literature indicating that disinfectant efficacy against biofilms depends on concentration and contact time, with acids often requiring longer exposures and alkaline chlorine penetrating EPS more effectively. Practically, disassembling taps and using specialized brushes is burdensome and risks damage or contamination; preparing dilutions can be error-prone; boiled water avoids chemical residues and may be more effective against complex biofilms including protozoa, supporting its recommendation. For containers, while soaking was technically effective, the volume and logistics make it impractical in households, underscoring a gap in feasible cleaning strategies. The work highlights the need for realistic, user-friendly protocols to mitigate recontamination from biofilm reservoirs in domestic water systems.

This study established laboratory models of E. coli biofilms on household taps and storage container surfaces and evaluated locally available cleaning agents and household-feasible methods. Key contributions include: (1) demonstration that soaking is essential for effective removal of biofilms from taps, with recently boiled water for 5 minutes recommended as an effective and practical method; (2) confirmation that bleach and vinegar can be effective under specific method constraints (e.g., soaking for bleach; scrubbing for vinegar), though practicality may limit uptake; and (3) identification that only soaking effectively removed biofilms from container coupons, but that soaking entire containers is impractical, leaving no actionable recommendation for container cleaning at present. Future research should assess acceptability and taste impacts, determine optimal cleaning frequency and regrowth dynamics, evaluate antimicrobial materials for taps and containers, study complex mixed-species biofilms, and develop feasible container cleaning approaches that provide sufficient contact time without full-volume soaking (e.g., sprays, abrasive scrubbing with disinfectants). A systematic review of safe water storage outcomes is also recommended.

- Biofilms were grown from high-concentration E. coli cultures, potentially forming faster and denser than in field conditions. - Only E. coli ATCC 11229 (moderate adherence) was used; real-world biofilms are multispecies and may respond differently to agents. - A single concentration per cleaning agent was tested; varying concentrations/contact times may alter efficacy. - Agents like bleach and vinegar were tested against E. coli-only biofilms; efficacy against other organisms or mixed biofilms may differ. - Imaging for storage container coupons was inconsistent and not relied upon; sonication-based counts were used instead. - New, clean taps and containers were used; aged or roughened surfaces may affect biofilm adhesion and cleaning efficacy. - Taps could not be sonicated; swabbing is less efficient than sonication, making coupon E. coli results more reliable than tap results. - Live/dead staining to differentiate viable cells in biofilms was not performed, limiting insight into viability post-cleaning.