Decontamination of microbiologically contaminated abiotic porous surfaces in an oral surgery clinic using vaporised hydrogen peroxide (VHP)

Abstract

Purpose

The aims of the study were to identify microorganisms, including those in the VBNC state, inhabiting porous surfaces in oral surgery offices and to assess the biocidal effectiveness and impact of 300 ppm vaporised hydrogen peroxide (VHP) for 20 min on decontaminated materials.

Methods

From the surfaces of textured armrests of dental chairs, pinewood doors and window frames and cotton medical aprons, 30 swabs were taken with moistened sponges. The identification of isolated microorganisms was performed using molecular methods with MALDI-TOF MS, DNA Sanger sequencer and Illumina MiSeq. To evaluate the impact of VHP decontamination (independent variable) on the number of microorganisms (response variable) ANOVA and LSD tests were used. After application of 10 processes of VHP decontamination, changes in the properties of the materials were assessed using FTIR spectroscopy, SEM microscopy and XPS spectrometry.

Results

The concentration of microorganisms was 10¹–10⁴ CFU/100 cm² on the tested surfaces and 10² CFU/m³ in the air. Twenty species of bacteria, one yeast and 16 filamentous fungi were identified, with the predominance of Bacillus, Staphylococcus, Alternaria, Aspergillus and Penicillium. Moreover, Janthinobacterium, Acremonium, Aureobasidium, Coprinellus and Cosmospora in the VBNC state were metagenomically detected. VHP decontamination resulted in a reduction in the majority of tested microbial strains by a minimum of 3 log, and all tested mixed cultures inhabiting porous surfaces were above 98% and in the air, 100%. VHP decontamination did not affect the structural and morphological properties of cotton fibres, wood or stainless steel.

Conclusions

VHP decontamination at a concentration of 300 ppm for 20 min can be used for the holistic disinfection of air, surfaces and equipment in oral surgery offices.

Introduction

Oral surgery offices as well as other outpatient healthcare facilities require constant maintenance of microbiological cleanliness to ensure the safety of patients and staff. This is a problem with such a high rotation of people who contaminate the air and surfaces [1]. Microorganisms transmitted by people can survive on dry abiotic surfaces for a few months. It is particularly difficult to maintain microbiological cleanliness on very porous surfaces. Therefore, it is very important to choose decontamination methods that have the ability to penetrate hard-to-reach places, including the free spaces of porous surfaces [2].

Smooth washable surfaces predominate in healthcare facilities, but there are also porous surfaces, such as textured elements of equipment, wooden finishes and fabrics. Porosity may take on several forms depending on the pore size: microporosity refers to pores smaller than 2 nm in diameter; mesoporosity, between 2 nm and 50 nm; and macroporosity, greater than 50 nm, which are perceptible to the naked eye. Smooth surfaces roughen over time as a result of cleansing with disinfectants, which increases the specific surface area. Both degraded smooth surfaces and porous surfaces are easily inhabited by microorganisms due to the large amount of free spaces connected to the external surface [3]. Biofilms, which are difficult to remove, are often created in these free spaces. Biofilm-forming microorganisms are characterised by increased invasiveness and the ability to cause dangerous infections, especially in hospitals. The severity of infections depends on the characteristics of the microorganisms and prevalence of resistant pathogens in the hospital environment [4]. In addition, biofilm-forming microorganisms are more resistant to disinfectants than planktonic microorganisms. Therefore, disinfection methods should be adjusted in such a way that they do not negatively affect the surfaces, i.e., they do not cause an increase in the porosity.

The microbiological cleanliness of surfaces is closely related to the composition of bioaerosol in the air. Air is not a favourable environment for the growth of microorganisms, but it is a main environment of microbial transmission between living organisms and abiotic matter. To provide a safe environment in outpatient healthcare facilities, decontamination should cover both surfaces and air. Many methods of chemical and physical decontamination of surfaces and indoor air are currently available. Some teams of experts regularly describe the standards of cleaning and disinfection in order to unify this procedures in healthcare units [5–7]. In outpatient healthcare facilities, the following chemical methods for decontaminating surfaces are most commonly used: alcohols, halogens and quaternary ammonium salts [8–12]. The air is usually decontaminated with ultraviolet irradiation, but this physical method only allows for local decontamination of a small volume of air. Simultaneous decontamination of both the surfaces and air in the entire volume of a room, along with the equipment, is possible using fumigation methods, for example, with hydrogen peroxide (H₂O₂).

The biocidal properties of H₂O₂ in liquid form consist of the oxidation of cell components such as lipids, nucleic acids and proteins by free hydroxyl radicals and have been known for many years. For the antimicrobial effects of liquid H₂O₂, a high concentration and a long time of interaction are required. In the gaseous phase, the effectiveness of H₂O₂ is much greater at lower concentrations, e.g., the effectiveness of gaseous H₂O₂ at a concentration of 1 mg/l is the same as that of liquid H₂O₂ at a concentration of 400 mg/l. H₂O₂ in a gaseous form is safe for the environment because it decomposes into water vapour and oxygen [13]. A stabilised solution of 35% H₂O₂, which is used to generate gaseous H₂O₂, is registered by the Environmental Protection Agency (EPA) as a sterilising agent [14]. Currently, gaseous H₂O₂ in the form of vapour (hydrogen peroxide vapour, HPV) is sometimes used for decontamination [15–17]. In this paper, the patented vaporised H₂O₂ (VHP – vaporised hydrogen peroxide) was tested. Both techniques differ primarily in the conditions of the process. The VHP system does not allow for the condensation of disinfectant due to the dehumidification of the air both before and during the process. Thus, VHP interacts primarily with microorganisms and with the decontaminated surface to a lesser extent. In contrast, the HPV technique uses the phenomenon of micro-condensation, so it might react more intensively with decontaminated surfaces [18].

VHP shows a broad spectrum of viricidal, bactericidal, fungicidal and sporicidal activities [19, 20]. Most of the studies assessing the bactericidal properties of VHP have focused on selected pathogenic species, such as Acinetobacter baumannii, Clostridium difficile, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, for which this process has been highly effective. Yeast belonging to the genera Candida, Saccharomyces and Rhodotorula, as well as filamentous fungi belonging to the genera Aspergillus, Chaetomium, Fusarium, Penicillium, Stachybotrys and Trichophyton, demonstrate a higher sensitivity to VHP decontamination than bacteria [21, 22].

To date, studies on the effectiveness of VHP and its impact on decontaminated surfaces have been carried out on non-porous materials using high doses of VHP. The aims of the study were to identify microorganisms, including those in a viable but non-culturable (VBNC) state, inhabiting porous surfaces in the oral surgery office and to assess the biocidal effectiveness of 300 ppm VHP for 20 min in the air and on porous surfaces while simultaneously assessing its impact on the decontaminated materials to a depth of 2–5 nm. The research was carried out to find a decontamination method that is effective and safe for decontaminated surfaces (especially porous) for use in oral surgery offices. The scope of the study included the following: 1. a quantitative assessment and identification of microorganisms, including those in the VBNC state, inhabiting the environment of the oral surgery office; 2. evaluation of the effectiveness of VHP decontamination performed on a porous material inoculated with the isolated microorganisms (model tests); 3. evaluation of the effectiveness of VHP decontamination performed under real conditions; and 4. assessment of the impact of VHP on the morphology, structure and elemental composition and types of functional groups on the superficial layer with a depth of 2–5 nm of selected materials.

Materials and methods

Porous surfaces and air in an oral surgery office

Abiotic porous surfaces located in an oral surgery office in the clinic were tested for microbiological contamination and isolation of microbial strains for the model study. Samples (N = 30) were taken from surfaces with high porosity, which are the most difficult to disinfect: rough elements of dental units equipment (i.e., armrests of dental chairs, N = 10), pine wooden elements (doors and windows frames, N = 10) and textile medical aprons made of cotton (N = 10). Microbiological contamination of the air in the oral surgery office was also determined. Samples of air (N = 20) were taken at a height of 1.5 m in 2 representative locations (at the door, N = 10, and in the middle of the room between the dental chairs, N = 10). All the samples were taken from the same places after an entire working day, both before and after VHP decontamination.

Assessment of microbiological contamination

Swabs were taken with sponges (sterile, moistened with sterile saline (0.85% NaCl), biocide-free, dimensions: 3.5 × 7.5 cm) from the tested porous surfaces with dimensions of 5.0 × 5.0 cm using disposable templates according to the ISO 18593:2018 standard [23]. The sponges were placed in sterile bottles containing 100 ml of sterile saline and shaken. Serial decimal dilutions were made from the initial suspension. For the culturing of bacteria and fungi, tryptic soy agar (TSA) with nystatin (0.2%) and Sabouraud agar with chloramphenicol (0.1%) media were used, respectively. Microbiological contamination of the air was determined using a SAS Super IAQ air sampler (International PBI S.p.A., Italy) according to the EN 13098:2000 standard [24]. Air samples of 0.05 m³ and 0.1 m³ were collected using the same media as used for the isolation of microorganisms from porous surfaces. All plates were incubated at 30 ± 2 °C for 2 days (bacteria) and 25 ± 2 °C for 5 days (fungi). After incubation, the colonies were counted, and the total number of bacteria or fungi was presented as colony forming units (CFU) per 100 cm² of the surface or 1 m³ of the air.

Identification of microorganisms

The identification of bacteria isolated from the porous surfaces was performed using a molecular method based on the nucleotide sequence of the 16S rRNA gene. Genomic DNA was isolated using the Bead-Beat Micro Gravity (A&A Biotechnology, Poland) kit and was used as a template for amplification of the 16S rRNA gene. The polymerase chain reaction (PCR) was carried out in a 50 μl volume containing 5 μl (~50 ng) of genomic DNA, 25 μl of 2x PCR Master Mix Plus High GC, 0.2 μl of each primer (B-all forward, GAG TTT GAT CCT GGC TCA G; reverse, ACG GCT ACC TTA CGA CTT) at a concentration of 100 μM and 19.6 μl of sterile water. The prepared mixtures were subjected to an amplification reaction under the following conditions: initial denaturation at 94 °C for 2 min; 30 cycles of denaturation at 94 °C for 30 s; annealing at 58 °C for 45 s; and elongation at 72 °C for 90 s; and final elongation at 72 °C for 5 min. The amplified DNA fragments were purified using the Clean-Up AX (A&A Biotechnology, Poland) kit. The PCR products were suspended in 10 mM Tris-HCl buffer (pH 8.0), diluted to a concentration of 100 ng/μl and sequenced using the ABI3730XL DNA Sequencer (Thermo Fisher Scientific, USA). The obtained nucleotide sequences of 16S rRNA genes were analysed and compared with the sequences published in the National Center for Biotechnology Information (NCBI) database using the BLASTN 2.2.32+ program and Vector NTI Express software (Thermo Fisher Scientific, USA).

The identification of yeast isolated from the porous surfaces was performed using MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) mass spectrometry. A small amount of yeast culture was applied as a thin layer directly to the plate. The sample was extracted with 70% formic acid and then ionised with an IVD HCCA matrix (Bruker, USA). The measurements and data analysis were performed on a Microflex LT system (Bruker Daltonics, Germany) with the presence of a BTS (Bacterial Test Standard) control. The identification score value (SV) criteria were used according to the manufacturer’s protocol: > 2.3 indicates highly probable species identification, 2.299–2.0 indicates a secure genus identification and probable species identification, 1.999–1.7 indicates a probable genus identification and < 1.7 indicates unreliable identification.

Filamentous fungi isolated from porous surfaces were identified based on macroscopic and microscopic observations on malt extract agar (MEA) and Czapek yeast agar (CYA) media. The characteristics of the growth of the tested strains were determined macroscopically by considering the size, shape, colour and structure of the mycelium, the presence of a secretion and the presence of a pigment diffusing into the substrate. Based on microscopic slides, the construction of hyphae, conidia and spores was determined. The identification was carried out using a diagnostic key [25].

Metagenomic analysis

To determine the diversity of the microbial community, including bacteria and fungi in the VBNC state, inhabiting porous surfaces in the oral surgery office, metagenomic analysis was performed using next-generation sequencing (NGS) of the V3-V4 region of the 16S rRNA gene and the ITS1 region. The test sample was a mixture of all initial suspensions of the microorganisms in sterile saline. Metagenomic analysis included the following stages: isolation of the total genetic material of the sample using the Sherlock AX (A&A Biotechnology, Poland) kit; control of the presence of DNA by real-time PCR in a Mx3000P thermocycler (Stratagene, USA) using the fluorescent double-stranded DNA-binding dye SYBR Green; fluorimetric assessment of genomic DNA concentration using the Quant-iT PicoGreen dsDNA Assay (Life Technologies, USA) kit; preparation of libraries for sequencing covering the V3-V4 regions of the 16S rRNA gene and ITS1 based on the ‘16S Metagenomic Sequencing Library Preparation. Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System’ (Illumina, USA) protocol using sequences of the specific primers 341F and 785R (for 16S analysis) as well as ITS1FI2 and 5.8S (for ITS1 analysis); NGS sequencing using the MiSeq (Illumina, USA) instrument and paired-end (PE) technology using 2 × 250 bases and the MiSeq Reagent Kit v2 reagents; and bioinformatic analysis with the QIIME software package to determine the species of bacteria and fungi based on the reference sequences databases GreenGenes v13_8 (bacteria) and UNITE v7 (fungi). Analyses were performed by assigning pyrosequencing reads as an operational taxonomic units (OTUs) with sequence identity ≥97%. The classifications with less than 2% of abundance were gathered into the category ‘other’.

Materials samples

In model microbiological studies, cotton fabric (SDC Enterprises Limited, UK) samples (N = 56) were used. The fabric did not contain any optical lighteners, dyes or additives. Samples were sterilised before the decontamination effectiveness test (121 °C, 20 min).

To evaluate the impact of VHP decontamination on the properties of the materials, pinewood and stainless steel samples were used in addition to the cotton fabric.

Cultures of microorganisms in model studies

For studies of decontamination effectiveness, microorganisms isolated from the tested surfaces at the highest isolation frequency (>15%) were used: Bacillus licheniformis, Lysinibacillus fusiformis, Micrococcus luteus, Psychrobacillus psychrodurans, Staphylococcus epidermidis, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides, Epicoccum nigrum, Penicillium chrysogenum and Rhizopus nigricans. In addition, two strains of microorganisms from approved Pure Culture Collections, which show significant resistance to decontamination, were selected for the VHP decontamination effectiveness tests: Bacillus subtilis WDCM 00003 and Aspergillus niger WDCM 00053 [26, 27].

Pure cultures of isolates were activated before experiments using the same media and incubation conditions as used for the isolation of microorganisms. For the preparation of fabric samples inoculated with isolates, the requirements of the AATCC Test Method 100–2012 created by the American Association of Textile Chemists and Colorists were used [28]. Inocula were suspensions of bacterial cells or fungal conidia in sterile saline. The concentrations of the inocula were established using the plating method and were 10⁸ CFU/ml for bacteria and 10⁷ conidia/ml for fungi. Standardised inoculum (0.2 ml) was transferred onto a sterile cotton fabric sample placed on M₀ medium (MgSO₄ × 7H₂O, 5 g; (NH₄)₂SO₄, 3 g; KH₂PO₄, 1 g; glucose, 20 g; agar, 15 g in 1000 ml of H₂O) and incubated for 21 days in a climatic chamber (air temperature 28 ± 2 °C, air relative humidity RH 80%). The experiment was performed in duplicate. Next, decontamination with VHP was performed, and the effectiveness was evaluated.

Vaporised hydrogen peroxide (VHP) decontamination

The samples were treated with VHP in an oral surgery office equipped with five dental workstations (capacity of approximately 200 m³) using a mobile VHP decontamination system. Samples for microbiological tests were decontaminated with VHP once, and samples for physico-chemical tests were treated ten times in one week intervals.

The system consisted of 4 elements: a generator in which the vaporisation of H₂O₂ occurred; a gas circulation unit that forced the VHP into the room and enforced air movement; a drying tank that dehumidified the air in the room before and during the process; and a set of sensors that monitored the conditions in the room. The decontamination process consisted of 4 consecutive phases: air drying to a relative humidity of less than 40%; vaporisation of 35% liquid H₂O₂ at a temperature of 120 °C in the generator and its progressive introduction into the room; maintaining the set VHP concentration for a given period of time by continuously supplying air with VHP; stopping the injection of VHP and wicking away air containing VHP to the generator for catalytic reduction of VHP to water vapour and oxygen. The process was performed under a controlled VHP concentration, air temperature, air relative humidity (RH) and pressure so that the actual concentration of VHP in the decontaminated area did not exceed the condensation point. To minimise the negative oxidative effect of H₂O₂ on treated surfaces, the lowest VHP dose that was biocidally effective was used for decontamination. The process conditions were as follows: VHP concentration, 300 ppm; exposure time, 20 min; air temperature, 20–22 °C; air RH, 37–43%; and air pressure, 1 bar.

Decontamination effectiveness tests

The numbers of microorganisms present before and after VHP decontamination were established to assess the effectiveness of VHP decontamination using a culture-dependent method including the same media and incubation conditions as used for the isolation of microorganisms. The reduction (R) in the number of microorganisms (individual species in model studies and mixed cultures on porous surfaces and in the air under real conditions) due to decontamination was calculated using Eq. 1 [28]:

$$\mathrm{R}=\left({\mathrm{N}}_0-\mathrm{N}\right)/{\mathrm{N}}_0·100\%$$ 1

where N₀ is the number of microorganisms in the sample before decontamination and N is the number of microorganisms in the sample after decontamination (CFU/100 cm²).

Morphological analysis of the materials

The surface morphology of cotton fibres in the fabric samples, as well as the pinewood and stainless steel samples before and after 10 decontamination processes, was investigated by scanning electron microscopy (SEM). The samples of materials were deposited onto an aluminium specimen mount with carbon tape and sputter-coated with an Au/Pd alloy using a JFC-1200 evaporator prior to examination. Imaging was performed at a magnification of 5000× for cotton fibres and 100× for wood and steel using a Quanta 3D FEI scanning electron microscope (Thermo Scientific, USA). The tests were performed under a low vacuum for cotton fabric and a high vacuum for wood and steel in topographic contrast mode.

Structural analysis of the materials

To determine changes in the molecular structure of the cotton fabric, pinewood and stainless steel after 10 processes of VHP decontamination, the samples were examined by Fourier transform infrared spectroscopy (FTIR). Analysis of the samples was performed using a Nicolet 8700 FTIR spectrometer (Thermo Scientific, USA) equipped with a Smart Orbit diamond attenuated total reflectance (ATR) accessory and a fast, high sensitivity Mercury Cadmium Telluride (MCT-A) detector cooled with liquid nitrogen. Spectra before and after decontamination were collected from a layer with a thickness of 2–3 μm in the range of 4000–650 cm⁻¹ with a resolving power of 4 cm⁻¹. ATR spectra were subjected to baseline correction, ATR correction and scaled normalisation, resulting in obtained spectra that were equivalent to the transmission spectra. The test results were analysed with the software application OMNIC 3.2 (Thermo Scientific, USA).

Chemical analysis of the materials

Due to the oxidative nature of VHP, the superficial layer of material that is in direct contact with the disinfectant is the most exposed to deterioration. Therefore, X-ray photoelectron spectroscopy (XPS), which collects data from a layer at a depth of 2–5 nm, was used to assess the changes in the elemental composition and chemical condition of a surface of the material.

Samples of cotton fabric before and after 10 decontamination processes were analysed under high vacuum conditions in a multi-chamber UHV (Prevac, Poland) system. The samples of material were mounted on a molybdenum mount and degassed to a constant high vacuum in the charging lock of the UHV system at room temperature. Then, after transferring the mount with the sample to the analysing chamber of the UHV system, proper XPS analysis was performed. The monochromatic radiation from the Al anode with the Al Kα characteristic line and an energy of 1486.7 eV was used as the excitation source. Quantitative determination of the elemental composition of the surface of the investigated material was based on review spectra, and regional spectra were used for chemical analysis (functional groups). The energy bands were subjected to unravelling with a deconvolution method to the component peaks using the asymmetric Gauss/Lorentz function, and then the sum of the components was programmatically adjusted to the envelope of the spectral band. To normalise spectroscopic measurements, the X-axis of the spectra (binding energy, E_B) was calibrated to the C1s aliphatic carbon peak, E_B = 285 eV. The binding energies for individual spectral bands were 284.7 eV for C1s, 531.2–531.7 eV for O1s (review spectra) and 284.7 eV for C1s A, 286.3–286.4 eV for C1s B, 287.7–287.9 eV for C1s C, and 288.9 eV for C1s D (regional spectra).

The quality assurance in research

The validation of the quantitative methods for assessment of microbiological contamination was performed by 1 and 2 analysts by repeating the tests 20 times. Precision in the repeatability conditions (for 1 analyst) of <0.25 log and < 0.33 log in the reproducibility conditions (between 2 analysts) were obtained. For quantitative physico-chemical methods, precision in the conditions of repeatability was determined: 1% for FTIR and 7% for XPS. Moreover, to assure quality in the model microbiological studies, each sample was tested twice. In the tests of effectiveness of VHP decontamination performed under real conditions, the samples were taken in two parallel replicates, and each sample was tested twice.

Statistical analysis

The total numbers of bacteria or fungi on the tested surfaces and in the air and for the number of microorganisms before and after decontamination (individual species in model studies and mixed cultures under real conditions) were expressed as the mean ± standard deviation. To evaluate the impact of VHP decontamination (independent variable) on the number of microorganisms (response variable) one-way analysis of variance (ANOVA) and least significant difference (LSD) tests at a significance level of p < 0.05 were used. All data were analysed using the statistical software STATISTICA 6.0 (Statsoft, USA).

Results

Quantitative assessment and identification of microorganisms

The microbiological contamination of the porous surfaces in the oral surgery office ranged from 10¹ to 10⁴ CFU/100 cm², while in the air, it was approximately 10² CFU/m³. The average concentrations of bacteria on tested surfaces were as follows: 5.5 × 10³ ± 4.6 × 10² CFU/100 cm² (armrests of dental chairs), 2.4 × 10² ± 5.2 × 10¹ CFU/100 cm² (wooden frames of doors and windows) and 6.2 × 10² ± 9.1 × 10¹ CFU/100 cm² (textile medical aprons). Fungi contaminated the surfaces with 2.3 × 10² ± 2.0 × 10² CFU/100 cm² (armrests of dental chairs), 5.3 × 10⁴ ± 8.6 × 10³ CFU/100 cm² (wooden frames of doors and windows) and 7.1 × 10¹ ± 6.6 × 10¹ CFU/100 cm² (textile medical aprons) on average. The average concentration of bacteria in the air was 8.8 × 10² ± 5.8 × 10¹ CFU/m³ at the door and 7.2 × 10² ± 9.4 × 10¹ CFU/m³ in the middle of the room. Fungi contaminated the air with 6.9 × 10² ± 1.2 × 10² CFU/m³ (at the door) and 5.7 × 10² ± 1.1 × 10² CFU/m³ (in the middle of the room) on average. The surfaces of armrests of dental chairs and textile medical aprons as well as air were more highly contaminated with bacteria than fungi. In the case of the surfaces of the wooden elements of doors and windows, an inverse relationship was observed.

Using culture-dependent methods, 20 species of bacteria, 1 species of yeast and 16 species of filamentous fungi were isolated from all tested surfaces (Table 1). Based on the 16S rRNA gene nucleotide sequences, the following species of bacteria were identified: Bacillus (9 species), Enterobacter (1), Lysinibacillus (1), Micrococcus (2), Moraxella (1), Pseudomonas (2), Psychrobacillus (1) and Staphylococcus (3). The degree of similarity in the nucleotide sequence of the 16S rRNA gene was over 99% for all analysed strains. Additionally, 16 species of fungi belonging to the following genera were identified: Alternaria (3), Aspergillus (4), Chaetomium (1), Cladosporium (2), Epicoccum (1), Fusarium (1), Penicillium (2), Rhizopus (1), Trichoderma (1) and one yeast species: Rhodotorula mucilaginosa.

Table 1.

Microorganisms identified on the tested surfaces using culture-dependent methods

N°	Species	Compared sequence	S [%]	Accession number
BACTERIA
1	Bacillus amyloliquefaciens	Bacillus amyloliquefaciens SS-12.6	99	KY780586.1
2	Bacillus cereus	Bacillus cereus ML267	99	KC692161.1
3	Bacillus circulans	Bacillus circulans S4A1	99	MG547704.1
4	Bacillus licheniformis	Bacillus licheniformis WAS3–5	99	JF496512.1
5	Bacillus megaterium	Bacillus megaterium GC61	99	KF158230.1
6	Bacillus mycoides	Bacillus mycoides FJAT-45861	99	KY038716.1
7	Bacillus simplex	Bacillus simplex IHB B 7066	99	KJ721216.1
8	Bacillus subtilis	Bacillus subtilis Van3	99	JX049584.1
9	Bacillus thuringiensis	Bacillus thuringiensis ILBB224	99	KT340483.1
10	Enterobacter sp.	Enterobacter sp. LB37	99	JQ692868.1
11	Lysinibacillus fusiformis	Lysinibacillus fusiformis KNUC423	99	JQ071512.1
12	Micrococcus luteus	Micrococcus luteus 3A	99	KF993658.1
13	Micrococcus yunnanensis	Micrococcus yunnanensis KA-20	99	KX108873.1
14	Moraxella osloensis	Moraxella osloensis TT16	99	CP024185.2
15	Pseudomonas oryzihabitans	Pseudomonas oryzihabitans AA21	99	MG571765.1
16	Pseudomonas stutzeri	Pseudomonas stutzeri TH-31	99	KF783212.1
17	Psychrobacillus psychrodurans	Psychrobacillus psychrodurans fwzy181	99	KF208476.1
18	Staphylococcus cohnii	Staphylococcus cohnii HNS003	99	JN128237.1
19	Staphylococcus epidermidis	Staphylococcus epidermidis Fussel	99	NR036904.1
20	Staphylococcus warneri	Staphylococcus warneri UCCB 146	99	MH198281.1
N°	Species	Mattched Pattern	SV	NCBI Identifier
YEAST
1	Rhodotorula mucilaginosa	Rhodotorula mucilaginosa CBS 316 T	2.15	5537
N°	Species
FILAMENTOUS FUNGI
1	Alternaria alternata
2	Alternaria chlamydospora
3	Alternaria tenuissima
4	Aspergillus flavus
5	Aspergillus niger
6	Aspergillus ochraceus
7	Aspergillus versicolor
8	Chaetomium globosum
9	Cladosporium cladosporioides
10	Cladosporium macrocarpum
11	Epicoccum nigrum
12	Fusarium poae
13	Penicillium chrysogenum
14	Penicillium funiculosum
15	Rhizopus nigricans
16	Trichoderma viride

S - similarity, Accession number – GenBank, NCBI 16S rRNA gene sequence comparison; SV - the level of similarity between an unknown tested specimen and a reference sample is indicated by a s(score); NCBI Identifier - sequence record processed by NCBI

The armrests of dental chairs were inhabited mainly by the bacterium S. epidermidis and fungus A. alternata, wooden frames of doors and windows were inhabited mainly by B. licheniformis and A. niger, and the textile medical aprons were dominated by B. cereus and P. chrysogenum. The microorganisms Bacillus licheniformis, Lysinibacillus fusiformis, Micrococcus luteus, Psychrobacillus psychrodurans, Staphylococcus epidermidis, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides, Epicoccum nigrum, Penicillium chrysogenum and Rhizopus nigricans were isolated from more than 15% of the samples taken from porous surfaces (frequency > 15%).

As a result of the NGS sequencing, 105,144 pairs of raw sequences of V3-V4 16S rRNA gene and 152,823 in the case of ITS1 region, were obtained. After quality control process, a high quality sequences were acquired and matched to kingdom Bacteria (23,359 reads, 99.59%) and Fungi (137,907 reads, 99.86%). Figure 1 shows all classifications with relative abundance ≥2%. The metagenomic analysis allowed the identification of bacteria belonging to 7 families and fungi belonging to 10 families. Among the marked families, some microorganisms were identified to the genus level (Fig. 1). On the tested surfaces bacteria of the genera Pseudomonas and Staphylococcus and fungi of the genus Aspergillus and family Pleosporaceae were predominant. Moreover, the following exemplary operational taxonomic units (OTUs) with relative abundance <2% were also detected: Oerskovia, Propionibacterium, Candida, Scopulariopsis, Trichosporon and Wallemia. Bioinformatic analysis showed that many of the sequences did not match with the cultivable microorganisms. With the use of metagenomics analysis, a much greater variety of fungal species (167 OTUs) than bacterial (27 OTUs) was detected on the tested surfaces.

Fig. 1.

Relative abundance of bacterial (a) and fungal (b) taxa identified on the tested surfaces using the metagenomic analysis

Evaluation of the effectiveness of VHP decontamination performed under model conditions

The numbers of individual strains of microorganisms on cotton fabric before and after VHP decontamination are presented in Table 2. VHP decontamination caused a statistically significant reduction in the numbers of all microbial strains inoculated on the fabric (ANOVA at a significance level of p < 0.05). For the individual bacterial strains, the number was R = 2.63–6.26 log reduced, and for fungi, the reduction ranged between R = 2.84 log and R = 6.88 log. Disinfection resulted in a reduction higher than R = 3.0 log in the case of 12 out of 14 tested microorganisms. A reduction below R = 3.0 log was demonstrated for only 2 species of microorganisms: B. licheniformis and A. niger WDCM 00053.

Table 2.

The effectiveness of VHP decontamination for tested microorganisms inoculated on cotton fabric

Microorganisms	Number of microorganisms [CFU/100 cm2]		Reduction R [%]
	before VHP	after VHP
B. licheniformis	8.3 × 105 ± 4.0 × 105	2.0 × 103 ± 7.6 × 102*	99.76
P. psychrodurans	1.2 × 107 ± 9.8 × 106	2.3 × 103 ± 5.8 × 102*	99.98
L. fusiformis	1.9 × 103 ± 6.4 × 102	0.0 × 100 ± 0.0 × 100*	100.00
S. epidermidis	1.8 × 106 ± 2.2 × 106	0.0 × 100 ± 0.0 × 100*	100.00
M. luteus	7.8 × 105 ± 8.8 × 104	4.0 × 100 ± 5.0 × 100*	99.99
B. subtilis WDCM 00003	4.0 × 106 ± 5.4 × 106	4.4 × 101 ± 3.6 × 101*	99.99
A. niger	4.0 × 108 ± 2.4 × 108	1.9 × 105 ± 1.4 × 105*	99.95
R. nigricans	1.5 × 106 ± 5.8 × 105	0.0 × 100 ± 0.0 × 100*	100.00
P. chrysogenum	7.8 × 106 ± 6.1 × 106	0.0 × 100 ± 0.0 × 100*	100.00
A. flavus	2.5 × 107 ± 2.4 × 107	9.0 × 100 ± 1.1 × 101*	99.99
E. nigrum	3.4 × 104 ± 3.2 × 104	0.0 × 100 ± 0.0 × 100*	100.00
C. cladosporioides	1.7 × 104 ± 1.2 × 104	0.0 × 100 ± 0.0 × 100*	100.00
A. alternata	2.2 × 104 ± 8.6 × 103	0.0 × 100 ± 0.0 × 100*	100.00
A. niger WDCM 00053	9.7 × 107 ± 1.2 × 108	1.4 × 105 ± 1.1 × 105*	99.86

Mean ± standard deviation; *statistically significant difference between samples before and after VHP decontamination, ANOVA at a significance level of p < 0.05

The highest resistance to VHP decontamination was found for B. licheniformis (R = 99.76%). The microorganisms most sensitive to VHP decontamination were L. fusiformis, S. epidermidis, M. luteus, B. subtilis WDCM 00003, R. nigricans, P. chrysogenum, A. flavus, E. nigrum, C. cladosporioides and A. alternata, for which a reduction at the level equal to or greater than 99.99% was demonstrated.

Evaluation of the effectiveness of VHP decontamination performed under real conditions

The total numbers of bacteria and fungi on the tested porous surfaces and in the air in the oral surgery office before and after VHP decontamination are presented in Table 3. VHP decontamination resulted in a statistically significant reduction in the number of microorganisms of all tested mixed cultures (ANOVA at a significance level of p < 0.05). For the total number of bacteria on the surfaces, the reduction was R = 98.33–99.19%, and for fungi, it ranged between R = 99.94% and R = 100.00%. Microorganisms in the air were completely eliminated after VHP decontamination (R = 100.00%).

Table 3.

The effectiveness of VHP decontamination in the environment of the oral surgery office

Place of sampling	Total number of bacteria		Reduction R [%]	Total number of fungi		Reduction R [%]
	before VHP	after VHP		before VHP	after VHP
Surfaces:	[CFU/100 cm2]			[CFU/100 cm2]
Armrest of dental chairs	5.5 × 103 ± 4.6 × 102	8.5 × 101 ± 6.1 × 100*	98.45	2.3 × 102 ± 2.0 × 102	0.0 × 100 ± 0.0 × 100*	100.00
Wooden frames	2.4 × 102 ± 5.2 × 101	4.0 × 100 ± 2.0 × 100*	98.33	5.3 × 104 ± 8.6 × 103	3.3 × 101 ± 8.6 × 100*	99.94
Medical aprons	6.2 × 102 ± 9.1 × 101	5.0 × 100 ± 2.4 × 100*	99.19	7.1 × 101 ± 6.6 × 101	0.0 × 100 ± 0.0 × 100*	100.00
Air:	[CFU/1 m3]			[CFU/1 m3]
At the door	8.8 × 102 ± 5.8 × 101	0.0 × 100 ± 0.0 × 100*	100.00	6.9 × 102 ± 1.2 × 102	0.0 × 100 ± 0.0 × 100*	100.00
In the middle of the room	7.2 × 102 ± 9.4 × 101	0.0 × 100 ± 0.0 × 100*	100.00	5.7 × 102 ± 1.1 × 102	0.0 × 100 ± 0.0 × 100*	100.00

Mean ± standard deviation; *statistically significant difference between samples before and after VHP decontamination, ANOVA at a significance level of p < 0.05

Satisfactory results of the reduction in the number of microorganisms have been reported for VHP decontamination: all air samples and 4 out of 6 surface samples were reduced to R > 99%. A reduction below R = 99% was demonstrated only for mixed bacterial cultures inhabiting the surfaces of the armrests of dental chairs and wooden frames of doors and windows. Moreover, mixed fungal cultures were more sensitive to VHP decontamination than mixed bacterial cultures.

Assessment of the impact of VHP on the physico-chemical properties of selected materials

The microscopic images (SEM) of the surfaces of the cotton fabric, pinewood and stainless-steel before and after 10 processes of VHP decontamination are presented in Fig. 2. The images of the surface of all tested materials decontaminated with VHP did not show any changes in surface morphology when compared with materials before decontamination. No additional microcavities, microdamages, defibrations or signs of corrosion in the materials samples after VHP decontamination were observed. Subtle differences in the appearance of cotton fabric, wood and steel samples resulted from the fact that microscopic images were taken from different spots of materials.

Fig. 2.

Microscopic images of the surfaces of the cotton fabric (1a – before VHP, 1b – after VHP), pinewood (2a – before VHP, 2b – after VHP) and stainless steel (3a – before VHP, 3b – after VHP)

Infrared absorption spectra (ATR-FTIR) of the cotton fabric, wood and stainless steel, both before decontamination and after 10 processes of decontamination with VHP, are presented in Fig. 3. In the FTIR spectra of fabric and wood, bands associated with cellulose and lignin structure were predominant. Intense bands in both of the spectra of steel in the region of 2400–1900 cm⁻¹ were derived from the vibrations in a diamond crystal. A comparison of the infrared spectra shows that no significant changes in the spectral profiles of all tested materials after VHP decontamination occurred when compared to the samples between decontamination. In the case of fabric and wood samples, no changes in the intensity of the bands corresponding to the lignin and cellulose structures, indicating that inter- and intramolecular hydrogen bonds (centred at 3406 and 3370 cm⁻¹) and cellulose oxidation products, such as aldehydes and carboxylic acids (centred at 1750 and 1600 cm⁻¹), were observed. There were also no new bands associated with new functional groups generated in the molecular structure of all tested materials during VHP decontamination.

Fig. 3.

Infrared absorption spectra of the cotton fabric (1a – before VHP, 1b – after VHP), pinewood (2a – before VHP, 2b – after VHP) and stainless steel (3a – before VHP, 3b – after VHP)

XPS review and regional spectra of the fabric samples before and after 10 processes of VHP decontamination are presented in Fig. 4. Elementary analysis of the surface composition of the cotton fabric based on the XPS review spectra showed that the ratio of the number of carbon atoms to oxygen atoms (C:O) was 2.1:1 for the sample before decontamination and 2.0:1 for the sample after decontamination. VHP decontamination did not cause a significant change in the C:O ratio and thus did not affect elemental composition of the fabric. Phase analysis of the cotton fabric surface based on regional spectra showed that the ratio of the number of aliphatic carbon atoms (C1s A band: C-C, C-H) to the sum of oxidised forms of carbon (C1s B-D bands: C-O-C, C-OH, C-N<, O-C-O, C=O, N-C=O, O=C-O-) was 1:2.2 for the sample before decontamination and 1:2.9 after decontamination. This indicates only a slight increase in the degree of oxidation of the fabric surface after VHP decontamination, as a result of increase in the number of functional groups named oxidised forms of carbon (C1s B, C1s C and C1s D band).

Fig. 4.

XPS review and regional spectra of the cotton fabric (1a – before VHP, 1b – after VHP)

Discussion

Microorganisms inhabiting the environment of healthcare facilities, including those in the VBNC state

In the current study, the level of contamination of surfaces in the oral surgery office (2.4 × 10²–5.5 × 10³ CFU/100 cm² for bacteria and 7.1 × 10¹–5.3 × 10⁴ CFU/100 cm² for fungi) was slightly higher than that found in previous studies in similar areas. Claro et al. [29] reported a level of contamination of 2.41 × 10²–1.08 × 10³ CFU/100 cm² for high-touch surfaces in medical and surgical wards. Additionally, Garcia-Cruz et al. [30] found 0–1.6 × 10³ CFU/100 cm² bacteria and 0–5.0 × 10² CFU/100 cm² fungi on the surfaces in the stomatology ward of the hospital in Xalapa City, Mexico.

The average concentration of bacteria in the air in the oral surgery office was 8.0 × 10² CFU/m³, and for fungi, it was 6.3 × 10² CFU/m³. A level of microbiological contamination of air similar to what was observed in this work was identified by Barlean et al. [31], who showed that the average total number of mesophilic germs and fungi was 4.3 × 10² CFU/m³ and 2.4 × 10² CFU/m³, respectively, after four hours of clinical activity in dental practices in Iasi, Romania. Additionally, Azari et al. [32] found approximately 1.2 × 10² CFU/m³ bacteria in the air in dental surgeries and fewer fungi than in this study (1.0 × 10¹ CFU/m³).

The most often isolated microorganisms from the tested porous surfaces were bacteria belonging to the genus Bacillus, which have the ability to form spores resistant to many disinfecting agents [33]. Most of the microorganisms isolated in these studies have been previously identified in other healthcare facilities. Bacillus sp., Micrococcus sp., Staphylococcus cohnii, Staphylococcus warneri and filamentous fungi were identified on the surfaces of an operating theatre in a hospital in Sosnowiec [34]. Bacillus sp., Bacillus subtilis, Bacillus cereus, Enterobacter sp., Micrococcus luteus, Staphylococcus epidermidis, Alternaria sp., Aspergillus sp. Penicillium sp. and Rhizopus sp. were present in the air at the University of Benin City Teaching Hospital in Nigeria, as well as in hospitals in Zarqa city in Jordan [35, 36].

In the current study, 18 genera of bacteria and fungi were identified by culture-dependent methods and indirectly using the NGS method. The use of culture-dependent methods allowed the identification of all microorganisms with accuracy to the species level, in contrast to the NGS method. For most of the microorganisms that were identified based on the nucleotide sequence of the 16S rRNA gene or MALDI-TOF MS, it was possible to confirm their presence with accuracy to the genera level by NGS method, while in the case of bacteria belonging to 3 genera and fungi belonging to 3 genera, identification was only possible to the family level. Additionally, metagenomic analysis allowed the identification of 5 other genera, which were not detected using culture-dependent methods, i.e., Janthinobacterium, Acremonium, Aureobasidium, Coprinellus and Cosmospora, including those in the VBNC state. Thus, to fully analyse the biodiversity of microorganisms inhabiting the tested surfaces, these two methods should be used simultaneously. Similar conclusions were presented by Adamiak et al. [37] in studies of halophilic microorganisms inhabiting building objects.

Disinfecting activities in healthcare facilities should focus primarily on the elimination of disease-causing microorganisms. Some of the bacteria isolated from the tested surfaces may pose a danger to human health, such as Enterobacter sp. (health risk group 2 according to Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 and the regulation of the Polish Minister of Health of April 22, 2005) as well as B. subtilis and Bacillus thuringiensis (group 2 according to the regulation of the Polish Minister of Health of April 22, 2005). Among the fungi isolated in the present study, some genera known for their allergenic or toxic properties, such as Alternaria, Aspergillus, Cladosporium and Penicillium, were detected [38]. Furthermore, A. flavus isolated from the tested surfaces is a potential producer of aflatoxins, which are one of the most toxic and potent hepatocarcinogenic natural compounds ever characterised [39].

Effectiveness of VHP decontamination

Decontamination using the VHP method at a concentration of 300 ppm for 20 min resulted in a statistically significant reduction in the number of all tested microorganisms in the model studies and under real conditions. A minimum 3-log reduction in the number of microorganisms is considered an effective disinfection according to the standards EN 1650:2008 and EN 1276:2009 [40, 41] and was achieved for most of the microbial strains tested in model studies. It is impossible to adequately relate the results of the VHP efficiency tests performed under real conditions to the logarithmic reduction because the levels of contamination of the air and surfaces in the oral surgery office before disinfection were in most cases too low. For these studies, it is better to refer to the percentage reduction in the total number of bacteria and fungi, which was satisfactory and reached values between 98.33% and 100%.

The total elimination of fungi from the surfaces of the armrest of dental chairs and medical aprons, on which mainly A. alternata and P. chrysogenum were detected, is analogous to their 100% reduction in model studies. A reduction in the total number of bacteria and fungi lower than 100% was obtained for wooden frames, which were inhabited mainly by B. licheniformis and A. niger, corresponding to a reduction of 99.76% and 99.95% for these strains in model studies, respectively. Only in the case of the armrests of dental chairs was the reduction in the total number of bacteria on their surface lower than the dominant strain (S. epidermidis, R = 100%), which can be explained by the presence of bacterial species less susceptible to VHP in the analysed samples. The effectiveness of VHP disinfection has been previously tested using different doses than those applied in this study. Levels of VHP decontamination effectiveness for individual strains similar to what was observed in this work were achieved by Sójka-Ledakowicz et al. [42], who showed complete elimination of bacteria of the genus Staphylococcus and spores of bacterium B. subtilis after the application of VHP at a dose of 200 ppm for 20 min. However, this dose did not eliminate A. niger, similarly to the dose tested in the present study (300 ppm for 20 min). Complete elimination of A. niger was achieved after increasing the concentration of VHP to 500 ppm, as observed by Sójka-Ledakowicz et al. [42], and applying hydrogen peroxide in gas form (GHP) in doses of 250 ppm for 90 min and 400 ppm for 30 min, as observed by Harmata et al. [19]. However, such high concentrations of VHP and decontamination times were not used in this work because they could have a negative impact on the surface properties.

Most of the current research focuses on the application of HPV due to its relatively low cost [22, 43]. Lemmen et al. [44] tested HPV efficacy (3 cycles of 500–600 ppm for 20 min) against S. aureus, Enterococcus sp. and A. baumannii dried on stainless steel disks and cotton and showed their complete reduction regardless of surface material. The effects achieved by were better than those described in this study because we applied only 1 decontamination process and they have used 3 processes. On the other hand, there are publications describing the use of HPV under real conditions, in which less efficient results were obtained than those presented in this paper. Błażejewski et al. [45] performed tests under real conditions and indicated that HPV (30 min at a dew point) caused only a 33% reduction in the contamination of rooms inhabited by S. aureus, A. baumannii and gram-negative rods. Similarly, Horn and Otter [46] reported a 47% reduction in the rate of S. aureus, Enterococcus sp., C. difficile and gram-negative rods over the 2-year study period, including both the implementation of HPV for terminal disinfection and improvements in hand hygiene.

Only a few recent literature reports refer to VHP, which is more expensive than HPV but has an advantage in terms of safety for the equipment. It was showed that VHP was successful in controlling the nocosomal acquisition of A. baumannii in hospitals after an 8-h cycle, including decontamination at a dose of 250 ppm for 90 min and in one case followed by 400 ppm for 30 min [47, 48]. Moreover, Galvin et al. [21] tested the VHP efficiency on hard surfaces and showed a 6–7 log reduction for all tested pathogens (Aspergillus fumigatus, E. coli, E. faecalis, P. aeruginosa, S. aureus and C. difficile) using a 4-h decontamination process. These reductions are higher than those obtained in this study, probably because they relate to efficacy tests performed using hard materials as opposed to our testing of porous materials.

Impact of VHP on decontaminated materials

VHP decontamination under the conditions of the performed tests caused no marked changes in the morphology and structure of cotton fibres, wood or stainless steel after 10 decontamination processes. The producers of the VHP decontamination system have defined a list of materials that are compatible with this disinfectant, i.e., causing no changes in properties of the materials (strength, flexibility and chemical structure) after application of VHP. The list includes metals, plastics, elastomers, some paints, claddings, ceiling tiles and electronic equipment, but there are no textiles or wood mentioned, which were tested in the present study. There are only a few literature reports describing the impact of VHP on similar materials as used in our study, but higher doses of VHP than those applied in this study were used, and the results were diverse. A slight reduction in the tensile strength of cotton gauze after application of VHP at a dose used for textile bleaching (800 ppm for 4 h) was shown by Sójka-Ledakowicz et al. [42]. Similarly, the Federal Aviation Administration showed that VHP at a concentration of 450 ppm for 4.8 h causes slight or moderate degradation of the tensile strength of textiles after 10 cycles of decontamination [49]. However, in the report of the US EPA [50], it was shown that 250 ppm VHP and exposure times of 1 h and 4 h did not cause physical changes or corrosion of various types of steel after a 12-month observation period. Additionally, Harmata et al. [19] showed that after the use of GHP at doses of 250 ppm for 90 min and 400 ppm for 30 min, there was no significant corrosion of elements made of aluminium, and no changes in the strength parameters of the fabric were observed. It has been indicated that the liquid form of H₂O₂ acts more aggressively than the VHP decontamination system tested in this study. Since the first application of H₂O₂ at 30%, black spots on stainless steel and galvanised steel were perceptible with SEM characterisation, and few principles of corrosion were evidenced with XPS analysis. Moreover, ten times the application of H₂O₂ at 30% significantly changed the FTIR spectra of metallic surfaces, unlike in our study [51]. However, to comprehensively analyse the impact of decontamination methods on surfaces, SEM, FTIR and XPS methods should be used at the same time. Similar conclusions were presented by Jo et al. [52], who showed that some chemical damage of materials after exposure to disinfectants was detected using XPS, whereas more conventional FTIR spectroscopy did not detect significant chemical changes.

Conclusions

The microbiological contamination of the porous abiotic surfaces in the oral surgery office ranged from 10¹ to 10⁴ CFU/100 cm², while in the air, it was approximately 10² CFU/1 m³. Tested surfaces were inhabited by 20 species of bacteria and 17 species of fungi, as well as microorganisms in the VBNC state. The predominant bacteria belonged to the genus Bacillus. The use of both culture-dependent and metagenomic methods in microbiological analysis allowed to obtain complete information on the biodiversity of microorganisms inhabiting the porous surfaces in the oral surgery office. Among the identified microorganisms, bacteria and fungi that pose a threat to human health were detected, what emphasizes the necessity of regular decontamination of the oral surgery office to protect staff and patients.

VHP decontamination at a concentration of 300 ppm for 20 min resulted in a reduction in the majority of the tested microbial strains by a minimum of 3 log. The reduction in microorganisms on the armrest of dental chairs, wooden frames and medical aprons obtained under real conditions correlates with the reduction in the dominant strains on the respective surfaces tested in model studies. VHP decontamination was more efficient against microorganisms in the air, which is a main environment of transmission of microorganisms, than those inhabiting porous surfaces.

VHP decontamination under the conditions of the performed tests did not affect the structural and morphological properties of the cotton fabric, wood or stainless steel after 10 processes of decontamination. VHP decontamination did not cause a significant change in the elemental composition of cotton fabric, but resulted in a slight increase in the degree of oxidation of its surface.

VHP decontamination at a concentration of 300 ppm for 20 min can be used for the holistic disinfection of air, surfaces and equipment in oral surgery offices in clinics with efficacy greater than 98%, which indicates a satisfactory disinfecting effect. Each outpatient healthcare facility should individually determine the frequency of VHP decontamination processes to systematically improve the microbiological cleanliness of the environment. Further research may focus on the evaluation of the effect of several dozen processes of VHP decontamination used for a much longer period of time on the level of microbiological contamination of air and porous surfaces and the properties of decontaminated materials.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.