Potential Use of a Combined Bacteriophage-Probiotic Sanitation System to Control Microbial Contamination and AMR in Healthcare Settings: A Pre-Post Intervention Study

Abstract

Microbial contamination in the hospital environment is a major concern for public health, since it significantly contributes to the onset of healthcare-associated infections (HAIs), which are further complicated by the alarming level of antimicrobial resistance (AMR) of HAI-associated pathogens. Chemical disinfection to control bioburden has a temporary effect and can favor the selection of resistant pathogens, as observed during the COVID-19 pandemic. Instead, probiotic-based sanitation (probiotic cleaning hygiene system, PCHS) was reported to stably abate pathogens, AMR, and HAIs. PCHS action is not rapid nor specific, being based on competitive exclusion, but the addition of lytic bacteriophages that quickly and specifically kill selected bacteria was shown to improve PCHS effectiveness. This study aimed to investigate the effect of such combined probiotic-phage sanitation (PCHSφ) in two Italian hospitals, targeting staphylococcal contamination. The results showed that PCHSφ could provide a significantly higher removal of staphylococci, including resistant strains, compared with disinfectants (-76%, p < 0.05) and PCHS alone (-50%, p < 0.05). Extraordinary sporadic chlorine disinfection appeared compatible with PCHSφ, while frequent routine chlorine usage inactivated the probiotic/phage components, preventing PCHSφ action. The collected data highlight the potential of a biological sanitation for better control of the infectious risk in healthcare facilities, without worsening pollution and AMR concerns.

Keywords: AMR; HAI; bacteriophages; bioburden; probiotics; sanitation.

Figure 1

Study design. The study period (14 weeks) was subdivided in three phases: the pre-PCHS T0 period (red; four weeks), during which wards received conventional chemical sanitation; the PCHS T1 period (yellow; four weeks), during which PCHS replaced chemical sanitation; and the PCHSφ T2 period (blue; six weeks), during which phages were added to PCHS sanitation in the bathrooms of 12/24 randomly selected rooms (treatment group), while the remaining rooms received only PCHS without phages (control group). Phage applications (green arrows) and samplings (red arrows) are indicated.

Figure 2

Phage susceptibility test of MRSA isolated from hospital surfaces in the pre-PCHS period. Each isolate was assessed for Sb-1 phage susceptibility by the spot test assay. CTR-, negative control (TSB); Sb-1, phage suspension. The results shown are for five representative MRSA isolates.

Figure 3

Total and staphylococcal contamination in the enrolled hospital wards at T0 (pre-PCHS period). Total contamination is expressed as the sum of Staphylococcus spp., Enterobacteriaceae spp., Pseudomonas spp., Clostridium spp., Aspergillus spp., and Candida spp. CFU counts. Samplings were performed in rooms (floor, bed footboard) and bathrooms (floor, sink, shower plate). The results are expressed as CFU number per m²; median values (lower part of the box) and Q3 values (upper part of the box, representing the 75% percentile values) are shown, together with min. and max. values. (A) HS-1. (B) HS-2.

Figure 4

Total pathogen contamination in enrolled hospitals. The sanitation types applied in the different study periods are identified by colors: red (chemical-based sanitation), yellow (PCHS) and blue (PCHSφ). (A) Rooms of HS-1. (B) Bathrooms of HS-1. (C) Rooms of HS-2. (D) Bathrooms of HS-2. All the results are expressed as CFU number per m²; Median values (lower part of the box) and Q3 values (upper part of the box, representing the 75% percentile values) are shown, together with min. and max. values. Sampling times: T0 (chemical-based sanitation in all enrolled bathrooms), T1 (PCHS in all enrolled bathrooms), and T2 (PCHS and PCHSφ in control and treated bathrooms, respectively). *, p < 0.05.

Figure 5

Staphylococcal contamination in enrolled hospitals. The sanitation types applied in the different study periods are identified by colors: red (chemical-based sanitation), yellow (PCHS) and blue (PCHSφ). (A) Rooms of HS-1. (B) Bathrooms of HS-1. (C) Rooms of HS-2. (D) Bathrooms of HS-2. All the results are expressed as CFU number per m²; Median values (lower part of the box) and Q3 values (upper part of the box, representing the 75% percentile values) are shown, together with min. and max. values. Sampling times: T0 (chemical-based sanitation in all enrolled bathrooms), T1 (PCHS in all enrolled bathrooms), and T2 (PCHS and PCHSφ in control and treated bathrooms, respectively). *, p < 0.05.

Figure 6

Amounts of PCHS-Bacillus in the enrolled hospital wards. (A) Rooms of HS-1. (B) Bathrooms of HS-1. (C) Rooms of HS-2. (D) Bathrooms of HS-2. All the results are expressed as mean CFU number per m² ± S.D. values. Sampling times: T0 (chemical-based sanitation in all enrolled bathrooms), T1–T2 (PCHS in all enrolled bathrooms), and T3–T6 (PCHS and PCHSφ in control and treated bathrooms, respectively).

Figure 7

Anti-Staphylococcus Sb-1 phage DNA quantification in treated areas. (A) HS-1 setting. (B) HS-2 setting. Results are expressed as mean genome copy number per surface sample (corresponding to 100 cm²) ± S.D. values. Mean values obtained in positive samples are also shown in labels.

Figure 8

Resistome characterization of hospital surface microbiome. Left panels, HS-1. Right panels, HS-2. T0, R genes detected at basal level during the pre-PCHS period. T1, R genes detected during the PCHS period (T1₂ sampling). T2, R genes detected during the PCHSφ period (T2₂ sampling). Results are expressed as mean values of Log₁₀ fold change compared with the respective controls, represented by negative controls (NTC) at T0, T0 values for T1 results, and T1 values for T2 results. Results refer to duplicate samples in two independent assays.