Reduction of the microbiological load on hospital surfaces through probiotic-based cleaning procedures: A new strategy to control nosocomial infections
Introduction
Healthcare-Associated Infections (HAIs) are the most frequent complications occurring in healthcare facilities. It has been estimated that about 5% to 15% of all hospitalized patients develop a HAI during hospitalization. 1 A three point-prevalence survey conducted in Italy showed a frequency of 6.7% HAI, 2 with the most frequent HAI represented by lower respiratory tract infections, followed by urinary tract infections. In 2012, the European Centre for Disease Prevention and Control (ECDC) published European report on HAI frequency, 3 stating that every year over 3 million patients contract a HAI in the European Union. The high incidence of this severe complication is responsible for a huge cost on the society and makes the abatement of HAIs a priority in healthcare industry. A great number of studies and surveys based on the screening of patients have been reported, but the actual sources of contamination have been poorly characterized. In this view, the contribution of environment and surfaces to HAI development is still a matter of discussion. Nevertheless, it is well known that surfaces act as reservoirs for microorganisms, 4 and represent a potential risk of cross-contamination for patients, caused by direct or indirect contact of patients with such potentially contaminated surfaces. To reduce these risks, in healthcare facilities sanitation procedures are applied to each surface that directly or indirectly may come in contact with people and patients. Despite the existence of experimental evidence suggesting that a reasonable, strategic use of disinfectants is recommended, the routine use of these agents remains still a matter of debate. 5-7 Notwithstanding, a proper surface disinfection is highly recommended by all international guidelines as a primary procedure for preventing infections. 8-10 However, the widespread application of chemical disinfectants may account for several risks both for the environment and for the safety of users. Since microorganisms can adapt to a variety of environmental physical and chemical conditions, it is therefore not surprising that acquired resistance to extensively used antiseptics and disinfectants, together with the ever increasing antimicrobial resistance, has been reported in vitro. 11-14 For these reasons, there is increasing interest about the improvement of efficient, though sustainable, sanitation methodologies capable of containing or limiting the proliferation of pathogenic microorganisms. The importance of this issue is also outlined by recent research aimed to evaluate the antibacterial efficacy of natural detergents/biodetergents as a potential alternative to conventional disinfectants. 15,16 A very promising strategy, as previously suggested by Falagas & Makris, 17 is represented by the use of non pathogenic microbiological products to colonize surfaces in order to counteract the proliferation of other bacterial species, in accordance with the competitive exclusion principle (Gause’s law). 18 This approach is based on the well-established adoption of probiotics as food supplements, referred as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. The problem of hospital hygiene is thus completely reversed, since the aim of the procedure is no more represented by a general disinfection, which minimizes the presence of any kind of microorganisms on hospital surfaces, but to counteract the development of potentially pathogenic strains, instead tolerating the presence of microorganisms that are not harmful to humans.
The broadening of the probiotic concept towards the environment has been designated as biocontrol when the application is antagonistic towards a certain pathogen. 19 This strategy has been already successfully applied for the abatement of Legionella in water systems, 20 but there is no evidence about its effective application on surfaces by means of cleaning. Accordingly, such bio-stabilization through cleaning procedures may introduce a new conception of cleaning systems targeted to the establishment of a controlled and less harmful microbiota instead of a generic removal of microorganisms from the environment. The aim of this study was to provide an experimental evaluation of the efficacy of a probiotic-based solution containing spore forms of Bacillus spp in comparison with traditional chlorine-based chemical disinfectants. As a study-model environment we chose the Sant’Anna University Hospital of Ferrara (Italy). The microbiological screening was focused on the most common HAI-related microorganisms known to reside on surfaces, namely Staphylococcus aureus, Coliforms, Pseudomonas spp. and Candida spp.. The outcome should provide whether the strategy of bio-stabilization with a probiotic-based product could act as an effective and sustainable alternative to chemical disinfectants for treating inanimate surfaces, in particular those located in a nosocomial context.
Materials and methods
Sanitation solutions
The probiotic-based solution used contains 1% spores (30 x 106 CFU/ml) of probiotic bacteria (Bacillus subtilis, Bacillus pumilus and Bacillus megaterium), added with ionic surfactants (0.6%), anionic surfactants (0.8%) and enzymes (amylase, 0.02%). The probiotic-based cleaning procedure was defined as PCHS (Probiotic Cleaning Hygiene System). This product is manufactured by Chrisal (Lommel, Belgium). The exact composition of the probiotic-based preparation is restricted. A common chemical chlorine-based solution (CBS) containing 0.65% sodium hypochlorite and 0.02% surfactants was used as control (Actichlor, Diversey S.p.A., Italy).
Bacterial strains and culture media
For the in vitro tests, Escherichia coli ATCC 10536 (Chrisope Technologies, Lake Charles, LA, USA), Staphylococcus aureus ATCC 25923 (Chrisope Technologies) and Pseudomonas aeruginosa ATCC 9027 (PBI International, Milan, Italy) strains were cultivated on MacConkey Agar (Merck Millipore, Darmstadt, Germany), Baird- Parker Agar (Merck Millipore) and Cetrimide agar (BD Diagnostic Systems, Franklin Lakes, New Jersey, USA) media, respectively. The same culture media were used to detect the presence of correspondent species of microorganisms in the samples. For the in situ trial, the Tryptic Soy Contact Agar (TSA, Merck Millipore) was used for the Total Microbial Count (TMC) together with the above reported media. The growth of all bacterial strains was obtained by incubation at 37°C for 18-24 hours followed by 48 hours incubation at room temperature. The presence of Candida spp. was determined by the Sabouraud Dextrose Contact Agar added with chloramphenicol (Merck Millipore), by incubation at 25°C for 72-90 hours, followed by 48 hours incubation at room temperature.
Identification of the pathogenic strains
The identification of pathogenic strains, initially identified through the Gram staining, was assessed by using the API 20 E (bioMérieux, Inc, Durham, NC, USA) or BBL Enterotube II (BD Diagnostic Systems) for Coliforms including Escherichia coli, the API Staph (20500 bioMérieux, Inc) for Staphylococcus aureus, the BBL Oxi/ Ferm Tube II (BD Diagnostic Systems) for Pseudomonas spp., and the API AUX C for Candida spp.
Quantification of the microbial load
Counting was performed by preparing 10 -6 and 10 -7 dilutions of the test suspension in diluents buffer. A sample of 1 ml for each dilution was inoculated in duplicate using the pour plate technique. 21 Each 1 ml sample was transferred into separate Petri dishes and a volume of 15ml of melted TSA, previously cooled to 45°C, was added. The number of Colony-forming Units (CFU) was determined by colony counting after incubation of TSA plates at 37°C for 20-24 h. A further incubation of plates for 20-24 hours was necessary. The numbers of CFU/ml in the bacterial stock suspension were finally calculated.
Sanitation procedures
Sanitation procedures were performed using microfiber mops, cleaned after each use according to the manufacturer’s instructions and colour-coded according to the type of target surface. A dry dust mopping phase was followed by a wet cleaning phase fresh prepared just before each use, with aqueous solutions of either the probiotic- based or the chemical-based solutions. They were the test and control preparations, respectively. The microfiber cloths were soaked into the solution and stored inside clean containers until use. Mopping phases were performed always by the same trained operator, in order to exclude or minimize the introduction of potential variables in the implementation of the procedures. Floors were treated with 700 ppm/ m2 active chlorine. Hand/body-touched surfaces such as doorknobs, bed frames, tables and chairs or sink, toilet and other bathroom fixtures, were treated with 140 ppm/m 2 active chlorine. These concentrations were used according to the manufacturer’s specifications. Following the manufacturer’s instructions (Chrisal), a preparation of 1.5 x 106 spores/m2 of the probiotic-based solution was used for the treatment of the same surfaces describe above. During the testing, the environments were used in the usual way, either by personal care, and patients and visitors.
Contamination-controlled experiments (in-vitro tests)
In-vitro experiments were conducted by treating samples of the hospital’s areas current materials (i.e. ceramic, PVC, rubber, vitreous- china) with the probiotic-based solution. A solution containing a known concentration (30 x 10 6 cells/ml, 15 ml/m 2 ) of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains was used for the contamination of the sampling surfaces. The bacterial load was measured by the determination of the colony count on RODAC plates (BD), containing TSA medium added with lecithin, histidine and Tween-20, in order to neutralize the action of disinfectants. The number of colonies was determined as total microbial count (TMC), or as specific colony count, by exploiting strain-specific medium. Control plates for sterility were used to guarantee sterility.