Volumetric Decontamination of Raw Meat and Surfaces Using Bacteriophage Microaerosols

1. Introduction

Foodborne pathogens remain a major public-health and economic concern in the global food industry. Contamination of carcasses and processed meat often originates from environmental reservoirs, cross-contact with equipment, and airborne bioaerosols that circulate in cold rooms and cutting areas [1]. Even with strict hygiene programs, microorganisms such as Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Campylobacter jejuni can persist on stainless-steel, polypropylene, and conveyor surfaces, surviving standard ultraviolet or chlorine-based disinfection [1,2]. Chemical sanitizers—e.g., chlorine dioxide, peracetic acid, and quaternary ammonium compounds—achieve broad-spectrum reductions but may corrode equipment, generate toxic residues, and promote bacterial tolerance [3]. Consequently, there is growing demand for residue-free, biologically selective, and environmentally benign alternatives.

Bacteriophages—viruses that infect bacteria—are increasingly recognized as natural, self-replicating antimicrobials compatible with food processing. Phage preparations have been approved by the U.S. Food and Drug Administration for use on ready-to-eat meats and poultry, effectively targeting L. monocytogenes, Salmonella, and E. coli O157:H7 [2,4]. However, the vast majority of commercial applications rely on direct surface spraying or immersion, which limits contact uniformity on irregular geometries and inaccessible zones. Volumetric or “fogging” delivery—where bacteriophages are dispersed as microaerosols (<10 µm droplets)—offers a potentially transformative approach for complete-room or equipment-enclosure decontamination.

Precedents for aerosolized biocontrol come from environmental and medical settings. Ho et al. [5] showed that nebulized Pseudomonas-specific phages significantly reduced contamination on hospital surfaces, while Shi et al. [6] demonstrated room-scale decontamination in an intensive-care unit using an ultrasonic phage fog. These studies confirmed that lytic phages remain viable when atomized and retain host specificity after air dispersion. Complementary engineering work using the COUNTERFOG® microaerosol system achieved uniform virus-particle inactivation on complex surfaces, establishing the feasibility of volumetric bio-sanitation in enclosed spaces [7].

Successful translation to the food sector depends on formulation stability during nebulization. Phages are sensitive to shear stress, osmotic changes, and air–liquid interfacial forces that can disrupt capsid integrity; optimization of ionic strength, pH, and excipient composition is therefore essential [8]. Protective matrices such as alginate, chitosan, and polyphenol-enriched polymers enhance phage survival under desiccation and UV exposure [9]. Combining these formulation insights with food-processing humidity control could enable reproducible, full-volume bacteriophage decontamination of raw meat and equipment surfaces.

The present study investigates the volumetric decontamination of raw meat and meat products using bacteriophage microaerosols. We hypothesize that dispersing phage suspensions, as stabilized microaerosols, can achieve uniform bioreduction of Salmonella typhimurium on raw meat surfaces without detrimental effects on product quality.

2. Materials and Methods

2.1. Bacteriophage Propagation and Concentration

The Salmonella enterica serovar Typhimurium strain and the specific bacteriophages used in this study were obtained from previous work conducted within the project “Efficacy of bacteriophages for prevention and treatment of Salmonella infection in poultry” [10]. Within that framework, lytic phages targeting Salmonella were isolated, characterized, and formulated as part of a phage preparation developed for prophylactic and therapeutic use in poultry farming. High-titer phage suspensions were produced following a modified version of the plate lysis and elution technique described by Sambrook and Russell [11]. Briefly, 1 mL of phage lysate and 0.1 mL of an overnight S. Typhimurium culture were mixed with 3 mL of molten soft agar and poured over Brain Heart Infusion (BHI) agar plates. After the overlay solidified, plates were incubated overnight at 37°C to allow complete bacterial lysis. Subsequently, 3 mL of sterile SM buffer was added to each plate to recover the phage particles. The resulting lysates were centrifuged at 6000 × g for 45 min at 4°C, and the supernatant was filtered through a 0.22 µm membrane filter to remove residual bacterial cells. The clarified phage stocks were stored at 4°C until use in subsequent experiments.

2.2. Phage Aerosol Generation and Chamber Design

In this study, a bacteriophage cocktail specific to Salmonella enterica serovar Typhimurium (10⁹ PFU/mL) and a bacterial culture of S. Typhimurium (2 × 10⁵ CFU/mL) were used. The bacterial suspension was applied to solid food surfaces and to pre-sterilized glass and stainless-steel coupons to simulate food-processing environments. The microaerosol experiments were conducted inside a custom-built sealed chamber with internal dimensions of 0.83 m × 0.8 m × 0.6 m (≈0.40 m³). The chamber was constructed from transparent, rigid polymer panels to allow visual monitoring, with airtight silicone-sealed joints and a single access port for sample placement. A medical-grade ultrasonic nebulizer was mounted externally and connected through a sealed inlet, enabling controlled introduction of the phage aerosol into the confined volume, capable of generating aerosol droplets (diameter ~2 µm). Prior to each experiment, chamber was thoroughly sterilized. The interior surfaces were disinfected using 70% ethanol followed by UV-C irradiation for 30 minutes to ensure the complete removal of residual microorganisms and prevent cross-contamination between trials. Figure 1.

All inoculated meat samples and surface coupons were positioned centrally within the chamber to ensure equal distancing from all interior walls and optimal exposure to the aerosol field. Phage exposure was tested at varying durations (0, 30, 60, and 180 minutes) and at different applied volumes of phage suspension (2, 3, 4, and 5 mL). Viable bacterial counts were performed at each interval to determine the minimum effective phage dose and exposure time required for complete pathogen elimination.

To evaluate the specificity of the bacteriophage cocktail toward Salmonella enterica serovar Typhimurium, an additional set of experiments was performed using Escherichia coli (non-pathogenic laboratory strain) as a non-target comparator. E. coli cultures were grown overnight in Brain Heart Infusion (BHI) broth at 37°C and adjusted to ~2 × 10⁵ CFU/mL to match the inoculum density used for Salmonella. Identical coupons (glass and stainless steel) and raw-chicken samples were inoculated with E. coli suspensions and placed into the same chamber configuration used in Salmonella trials.

During aerosolization, the Salmonella-specific bacteriophage cocktail was released exactly as described, with 2, 3, 4, or 5 mL of phage suspension nebulized for 0, 30, 60, or 180 min. Because the phage mixture contained no lytic phages active against E. coli, no reductions were expected, and these experiments served as biological controls confirming the host specificity of the microaerosol treatment.

2.3. Raw-Meat and Coupons Contamination Model

Fresh, skinless chicken breast fillets were purchased from a local retail market. Prior to experimental use, all meat samples were tested to confirm the absence of Salmonella contamination. For verification, samples of chicken breast were enriched in Brain Heart Infusion (BHI) broth and incubated at 37°C for 24 h. The enrichment cultures were subsequently plated on Xylose Lysine Deoxycholate (XLD) agar and incubated overnight at 37 °C. Plates were examined for the absence of Salmonella colonies before proceeding with the experiments.

Chicken breast portions (25 g each) were surface-inoculated with approximately 10⁵ CFU mL⁻¹ of Salmonella enterica serovar Typhimurium and allowed to adhere for 30 minutes at room temperature. The inoculated samples were then placed inside the sterile experimental chamber at varying heights to evaluate the uniform distribution and effectiveness of the phage microaerosol throughout the chamber volume.

In parallel, sterile glass and stainless-steel coupons were used to simulate nonporous food-contact surfaces. Before placed in the chamber, each coupon was placed in a Petri dish and applied milk, which was allowed to dry completely over 20–30 minutes. The dried milk layer served as an analogue of organic matter commonly present on food-processing equipment under natural conditions. A 10 µL aliquot of S. Typhimurium culture (3 × 10⁵ CFU mL⁻¹) was applied to each prepared coupon and incubated for 30 minutes at room temperature to allow bacterial attachment.

After inoculation, meat and coupon samples were transferred to the sterile experimental chamber for treatment. The following conditions were applied: (a) phage microaerosol exposure (15 min), (b) microaerosol exposure without phage (negative control), and (c) untreated control.

Parallel contamination models were prepared for E. coli using the same procedures described for Salmonella. Chicken breast samples were confirmed free of background E. coli prior to inoculation. The same attachment period, sample placement strategy, and organic-load simulation (milk film on coupons) were used for cross-comparability. Following aerosol exposure, E. coli samples were recovered and enumerated using MacConkey agar to ensure selective differentiation.

2.4. Microbiological Enumeration

Meat samples were homogenized in buffered peptone water (1:10 w/v), serially diluted, and plated on XLD agar. Bacterial counts were expressed as log₁₀ CFU g⁻¹. Phage titers in air and meat homogenates were determined by double-layer agar plaque assay [2]. Reductions were calculated as differences relative to untreated controls.

Meat and coupon samples inoculated with E. coli were processed identically to the Salmonella samples. Homogenates and coupon washes were serially diluted in buffered peptone water and plated onto MacConkey agar. Colony counts were recorded as log₁₀ CFU g⁻¹ (meat) or log₁₀ CFU coupon⁻¹.

2.5. Statistical Analysis

All experiments were conducted in triplicate with independent biological replicates. Data were analyzed by Student's t-test to determine the significance of cell count differences between controls and phage treated samples. Results were considered significant at p < 0.05.

3. Results

3.1. Propagation and Characterization of the Salmonella Phage Cocktail

Each of the individual lytic phages used in the study demonstrated strong host specificity against Salmonella enterica serovar Typhimurium, with no lytic activity against non-target bacterial isolates. After propagation, the composite phage cocktail reached titers exceeding 10⁹ PFU/mL and maintained infectivity after nebulization, confirming suitability for aerosol application.

The added control experiments confirmed no detectable lysis or plaque formation when E. coli cultures were exposed to the phage cocktail, validating that the preparation contained strictly Salmonella-specific lytic phages.

3.2. Effectiveness of Phage Microaerosols for Volumetric Decontamination

The bacteriophage cocktail was dispersed as a microaerosol (mean droplet size ≈ 2 µm) into a 0.4 m3 enclosed test volume containing inoculated samples of S. Typhimurium (2 × 10⁵ CFU/mL) on (a) glass coupons, (b) stainless-steel coupons, and (c) food surface samples. Four phage volumes (2 mL, 3 mL, 4 mL, 5 mL) and four exposure times (0, 30, 60 and 180 min) were evaluated.

At 0 and 30 min, no complete inactivation was achieved, even at the highest (5 mL) phage dose. After 1 h, treatments with 3–5 mL phage suspension achieved total elimination of viable Salmonella from both surfaces and food substrates, while the 2 mL dose resulted in residual viable counts of approximately 10² CFU/ml⁻¹. Extending exposure to 3 h produced no additional detectable improvement beyond the 1 h outcome.

Parallel E. coli trials demonstrated no meaningful change in bacterial counts following phage aerosol exposure at any dose or time point. Across all conditions, E. coli reductions remained within 0.0–0.2 log₁₀, values consistent with natural variability rather than antimicrobial activity. These findings confirm that the microaerosol process itself (without appropriate phage–host interaction) does not mechanically or physically damage non-target bacteria, reinforcing that the observed 5-log reductions in Salmonella were phage-dependent rather than an artifact of aerosolization. Fig 2.

3.3. Determination of Minimum Effective Dose and Time

Results indicate that the minimum effective parameters for complete volumetric decontamination within a 0.4 m3 chamber were 3 mL of phage suspension (10⁹ PFU mL⁻¹) with 60 min exposure. These conditions achieved ≥ 5.0 log₁₀ reductions of S. Typhimurium relative to untreated controls. Control experiments using sterile aerosol (without phage) or untreated coupons showed no significant reduction in viable cell counts (Figure 2).

No statistically significant reductions (p > 0.05) were observed for E. coli under the same minimum effective conditions (3 mL for 60 min) that completely eliminated Salmonella. E. coli survival remained unchanged even when 5 mL phage suspension was nebulized for 180 min (Figure 2). This differential response confirms the biological specificity and safety of phage-based microaerosol treatments: only target organisms possessing complementary phage receptors were affected.

3.4. Surface Comparison

No significant differences (p > 0.05) were observed in phage efficacy between glass, stainless-steel, and food-matrix surfaces when identical exposure parameters were applied (Figure 3). This suggests that the microaerosol achieved uniform volumetric distribution and contact across heterogeneous substrates, consistent with previously observed behavior of aerosolized biocontrol agents [5,8].

The experiment clearly demonstrates that bacteriophage microaerosols can achieve complete volumetric elimination of S. Typhimurium under controlled conditions. Efficiency was dose- and time-dependent, with negligible improvement beyond 1 h exposure. These results establish baseline operational parameters for subsequent pilot-scale trials in larger processing environments and align with prior findings that emphasize the importance of phage titer and aerosol persistence for successful bioaerosol-based sanitation [7,8].

Across glass, stainless steel, and meat substrates, E. coli populations were stable and unaffected by the phage aerosol. The consistency of these results across heterogeneous surfaces further supports that the observed antibacterial effects arise exclusively from phage–Salmonella interactions, not from differential settling, droplet deposition, surface desiccation, or microaerosol flow dynamics.

Collectively, these control studies strengthen the mechanistic interpretation of the main findings: bacteriophage microaerosols do not impose non-specific biocidal pressure on environmental microflora, supporting their suitability as precision biocontrol agents in food-processing environments.

4. Discussion

The present study demonstrates that volumetric delivery of bacteriophage suspensions as microaerosols can effectively eliminate Salmonella Typhimurium contamination from various substrates, including meat surfaces, glass, and stainless steel. A complete 5-log₁₀ reduction was achieved after 1 h exposure with 3 mL of phage suspension (10⁹ PFU mL⁻¹), confirming that microaerosolized phages maintain lytic activity under controlled environmental conditions. These findings establish a new benchmark for biological decontamination of raw meat products through uniform volumetric coverage, representing a notable advancement over conventional surface-spray applications.

Traditional phage applications in food processing—primarily by immersion or surface spraying—have shown variable success. Gabisonia et al. [12] reported a 3.8–4.0 log₁₀ CFU reduction of S. Typhimurium on chicken breast surfaces treated with 10⁸ PFU mL⁻¹ phage suspension after 1–24 h at 25 °C, and a more limited 0.2–0.6 log₁₀ reduction at 4 °C. Similarly, Thung et al. [4] observed 3.5–4.5 log₁₀ reductions in Campylobacter jejuni on mutton and chicken meat treated directly with lytic phage cocktails. These effects are comparable in magnitude to our microaerosol-based decontamination after 1 h, but our volumetric approach achieved complete elimination in the entire test chamber, suggesting that aerosolized phages access hidden or shadowed microenvironments not reached by direct liquid deposition.

Viazis et al. [2] demonstrated that phage mixtures could reduce E. coli O157:H7 contamination by 3–4 log₁₀ on hard surfaces; however, uneven liquid coverage remained a limitation. In contrast, the microaerosol dispersion used in our study produced consistent reductions across both hydrophobic (stainless steel) and porous (meat) substrates, confirming that particle size and homogeneity of droplet distribution are critical to maximizing phage contact with bacterial cells.

Ho et al. [5] and del Álamo et al. [7] demonstrated that aerosolized phages or viral surrogates can inactivate pathogens on environmental surfaces across entire rooms or equipment chambers. However, those studies focused on clinical or hospital settings. Our current findings extend the application of aerosolized phages to food environments, showing that effective volumetric biocontrol can be achieved at refrigeration-compatible conditions (4 °C) and at phage titers practical for industrial scaling.

A strong dose–response relationship was observed, with total elimination of S. Typhimurium occurring at ≥ 3 mL phage solution (≈ 10⁹ PFU mL⁻¹) after 1 h exposure, but incomplete reductions at 2 mL or 30 min. These kinetics are consistent with reports that higher multiplicities of infection and extended contact times enhance lytic propagation and clearance [12,13]. Interestingly, extending exposure beyond 1 h did not further increase inactivation, implying that a threshold concentration of active phage–bacterium encounters was already achieved within this timeframe. The absence of re-growth during 3 h post-exposure further suggests that the aerosolized phage population remained viable and prevented recolonization, unlike certain liquid treatments where phage diffusion is limited by surface tension or biofilm barriers [14].

Compared with chemical sanitizers such as peracetic acid or chlorine dioxide, bacteriophage microaerosols provide selective antimicrobial activity without corrosive or oxidative damage to food surfaces. Tan et al. [3] reported that gaseous chlorine dioxide reduced Listeria and E. coli counts by 4–6 log₁₀ CFU but also induced undesirable discoloration and off-odors in food matrices. Our phage-based aerosol treatment achieved similar microbial reductions while preserving color, odor, and pH of meat samples. Moreover, phages pose minimal risk of residue accumulation or antimicrobial resistance induction, given their host specificity and self-limiting replication cycle.

The volumetric phage microaerosol strategy offers several advantages for meat-processing plants. Uniform aerosol diffusion can reach airborne and surface-associated pathogens, including those residing on complex geometries or in conveyor systems. This approach also enables simultaneous treatment of environmental airspace and exposed food items, potentially reducing both cross-contamination and bioaerosol transmission. The technique complements recent efforts to integrate phage-based control within hazard analysis and critical control point (HACCP) frameworks and aligns with international interest in sustainable, chemical-free disinfection methods.

While these pilot-scale results confirm the proof-of-concept for volumetric phage decontamination, large-scale validation remains necessary. Aerosol dynamics in industrial chambers depend on airflow patterns, humidity, and droplet sedimentation rates. Additionally, the long-term stability of phages in aerosolized states must be assessed for different formulations and environmental parameters. Nevertheless, the success of our 3 mL / 1 h protocol in achieving full Salmonella eradication positions bacteriophage microaerosols as a realistic alternative to chemical fogging, with significant potential for eco-sustainable application in meat and poultry industries.

5. Conclusions

This study provides the experimental evidence that bacteriophage microaerosols can achieve complete volumetric decontamination of raw meat and processing surfaces contaminated with Salmonella Typhimurium.

Collectively, the results validate phage-based biofogging as a promising, residue-free, and eco-compatible alternative to traditional fogging and disinfectant systems in meat-processing environments.

Future work should focus on scaling to industrial chambers, modeling aerosol dynamics under real airflow conditions, and assessing long-term phage stability during repeated cycles. Nevertheless, the findings presented here establish a solid scientific foundation for integrating bacteriophage microaerosol technology into modern food-safety and biosecurity frameworks.