Klebsiella pneumoniae is an emerging multidrug-resistant pathogen that can contaminate hospital surfaces in the form of a biofilm which is hard to remove with standard disinfectants. Because of biofilm resistance to conservative disinfectants, the application of new disinfection technologies is becoming more frequent. Ozone gas has antimicrobial activity but there is lack of data on its action against K. pneumoniae biofilm. The aim of this study was to investigate the effects and mechanisms of action of gaseous ozone on the OXA-48-procuding K. pneumoniae biofilm. A 24 h biofilm of K. pneumoniae formed on ceramic tiles was subsequently exposed to different concentrations of ozone during one and two hours to determine the optimal ozone concentration. Afterwards, the total bacteria count, total biomass and oxidative stress levels were monitored. A total of 25 ppm of gaseous ozone was determined to be optimal ozone concentration and caused reduction in total bacteria number in all strains of K. pneumoniae for 2.0 log₁₀ CFU/cm², followed by reduction in total biomass up to 88.15%. Reactive oxygen species levels significantly increased after the ozone treatment at 182% for the representative K. pneumoniae NCTC 13442 strain. Ozone gas in the concentration of 25 ppm caused significant biofilm reduction but did not completely eradicate the K. pneumoniae biofilm formed on ceramics. In conclusion, ozone gas has great potential to be used as an additional hygiene measure in joint combat against biofilm in hospital environments.

Acinetobacter baumannii is a prominent emerging pathogen responsible for a variety of hospital-acquired infections. It can contaminate inanimate surfaces and survive in harsh environmental conditions for prolonged periods of time in the form of biofilm. Biofilm is difficult to remove with only one method of disinfection, so combined disinfection methods and biocidal active substances are needed for biofilm eradication. Additionally, having in mind ecological demands, legislators are more prone using fewer toxic substances for disinfection that produce less solid waste and hazardous disinfection byproducts. Gaseous ozone and citric acid are natural biocidal compounds, and the purpose of this study was to determine their combined biocidal effects on A. baumannii biofilm formed on ceramics and polystyrene. Twenty-four-hour A. baumannii biofilm formed on ceramic tiles and polystyrene was exposed to different combinations of disinfection protocols with 25 ppm of gaseous ozone for 1 h exposure time and 15% citric acid for 10 min exposure. The total number of bacteria was counted afterwards and expressed as CFU/cm². The determined disinfection protocols of A. baumannii biofilm with combined citric acid and gaseous ozone caused reduction of 2.8 to 5.89 log₁₀ CFU (99.99% inhibition rate) of total viable bacteria for each method, with the citric acid–ozone–citric acid disinfection protocol being most successful in eradication of viable bacteria on both ceramics and polystyrene. In conclusion, gaseous ozone and citric acid showed good combined biocidal effects on A. baumannii biofilm and successfully reduced early A. baumannii biofilm from ceramic and polystyrene surfaces. The given combination of active substances can be a good option for eco-friendly disinfection of hospital inanimate surfaces from A. baumannii biofilm contamination with prior mechanical cleaning.

Benzalkonium chloride (BAC) is a chlorine-based chemical compound with proven antimicrobial properties against bacteria, viruses, and fungi, depending on the length of the alkyl chain. It can be used as a biocide, as a cationic surfactant, and as a phase transfer agent. The aim of this study was to investigate the effectiveness of commercial cleaning agents for sanitary areas Bis duo Active (BDA) with BAC as an active compound in working concentrations of 5% and 20% on the destruction of bacterial biofilm formed on ceramic tiles. A biofilm of Staphylococcus aureus, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were grown on ceramic tiles with dimensions of 2.5 × 2.5 cm over 24 h. These plates were then treated with working concentrations of BAC for 10 min. After washing, ATP bioluminescence was measured with a luminometer, CFU/cm² was determined and the total biomass reduction was measured after crystal-violet staining. Both working concentrations of benzalkonium chloride proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Both treatments caused a reduction in total bacteria number of up to 3.12 log₁₀CFU/cm², followed by a reduction in total biomass up to circa (ca.) 64%. Inhibition rates varied from ca. 28% to ca. 99%, depending on the method and concentration used. Mechanical cleaning prior or after treatment with BAC is essential to improve biofilm destruction. All methods used in this study are effective for the determination of the anti-biofilm activity of BAC. Further analyses are needed.

Ozone is a triatomic allotropic modification of oxygen with very high oxidation potential and strong antimicrobial properties, and can be used as a disinfecting agent. The aim of this work was to investigate the effectiveness of gaseous ozone in reducing the number of bacteria and the total biomass of E. coli biofilm using different methods of quantification and detection. Biofilm of all tested clinical isolates and standard strain was grown on ceramic tiles with dimensions of 1.0 × 1.0 cm over 24 h. These plates were then treated with gaseous ozone for 1 h. After washing, CFU/cm² was determined, ATP bioluminescence was measured with a luminometer, and the total biomass reduction was measured after crystal-violet staining. Gaseous ozone proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Treatment caused a reduction in total bacteria number of up to 2.00 log₁₀CFU/cm², followed by a reduction in total biomass of up to 61.40%. Inhibition rates varied from 35.80% to 99.41%, depending on the method of quantification used. All methods used in this study are effective in determining the anti-biofilm activity of gaseous ozone, but more research is needed.

Environmental contamination with Klebsiella pneumoniae biofilm can be a source of healthcare-associated infections. Disinfection with various biocidal active substances is usually the method of choice to remove contamination with biofilm. In this study we tested 13 different disinfection protocols using gaseous ozone, citric acid, and three working concentrations of benzalkonium chloride-based professional disinfecting products on 24-hour-old biofilms formed by two K. pneumoniae strains on ceramic tiles. All tested protocols significantly reduced total bacterial counts compared to control, varying from a log ₁₀ CFU reduction factor of 1.4 to 5.6. Disinfection combining two or more biocidal active substances resulted in significantly better anti-biofilm efficacy than disinfection with single substances, and the most effective combination for both strains was that of citric acid, gaseous ozone, and benzalkonium chloride. This follow up study is limited to K. pneumoniae alone, and to overcome this limitation, future studies should include more bacterial species, both Gram-positive and Gramnegative, and more samples for us to find optimal disinfection protocols, applicable in real hospital settings.

Sažetak Zaštita prirode i okoliša danas čini sastavni dio gospodarskog i šireg društvenog razvoja. Podizanjem razine svijesti, proaktivnim razmišljanjem i djelovanjem, možemo računati na 10.održivi razvoj nadolazećih generacija. Ipak, u toj namjer...