Legionella pneumophila is a Gram-negative bacterial pathogen that colonizes natural and artificial water systems and has the ability to form a biofilm. The biofilm protects L. pneumophila from various environmental factors and makes it more resistant to chlorine-based disinfectants. This study investigated the anti-bacterial properties of tea tree (Melaleuca alternifolia (Maiden and Betche) Cheel) oil and lemon eucalyptus tree (Eucalyptus citriodora Hook) essential oils (EOs) and their synergistic, additive inhibitory and anti-adhesive effects against L. pneumophila biofilm formation on polystyrene. The minimum effective concentration (MEC) for tea tree is 12.8 mg ml(-1) and for lemon eucalyptus tree EO 6.4 mg ml(-1). In the checkerboard assay, different combinations of these two EO show synergistic and additive anti-microbial activity. The minimum anti-adhesive concentration (MAC) for tea tree is 12.8 mg ml(-1) and for lemon eucalyptus tree EO 6.4 mg ml(-1). A combination of 3.2 mg ml(-1) tea tree EO and 0.8 mg ml(-1) lemon eucalyptus tree EO showed the strongest anti-adhesive effect against L. pneumophila on polystyrene. The tested oils and their combination showed intriguing potential to inhibit L. pneumophila biofilm formation.

The study aimed to analyse the potential of Lamiaceae essential oils, extracts, and hydrolats against bacterial biofilms. Bacterial cells Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus cereus were exposed to Thymus vulgaris L. (thyme), Salvia officinalis L. (sage), Mentha x piperita L. (mint) essential oils, extracts, and hydrolats. The result of the minimal inhibitory concentration assessment shows the highest antibacterial potential for essential oils, followed by extracts and hydrolats respectively. The anti-biofouling capacity revealed that thyme essential oil has the highest potential for biofilm prevention for all tested bacteria, reducing up to 91% of biofilm, followed by mint (88%) and sage (87%) essential oil. While the thyme extract (84%), sage extract (83%) and hydrolat (77%) we less effective. The chemical composition of thyme essential oil showed a high percentage of monoterpene hydrocarbons and oxygenated monoterpenes, among which p-cymene and thymol were the most predominant. The bacterial cell membrane integrity assessment shows a significant increase in dead cells by increasing the concentration of thyme essential oil. The findings of our research indicate that the choice of herbal preparation significantly affects the active components, thereby influencing both antibacterial and anti-biofouling capabilities. Lamiaceae essential oils show great potential for biofilm management and represent a good candidate for antibacterial application in pharmacy, medicine, and industry.

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 log10 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.

This study aimed to analyze the potential of phytochemicals linalool and citronellal against E. coli and S. aureus biofilms and to compare the results to sodium hypochlorite. We tested the minimal inhibitory concentration, bacterial cell reduction, respiratory chain dehydrogenase activity, cell membrane integrity, and biomass reduction. The results show the lowest inhibitory concentration for both E. coli and S. aureus for sodium hypochlorite, followed by a combination of linalool and citronellal, the sole use of linalool, and the sole use of citronellal, respectively. Furthermore, we found that linalool was effective in biofilm cell reduction, cell respiratory inhibition, membrane integrity, and biomass reduction, while citronellal was less effective. Overall, this indicates that linalool has some benefits in biofilm management, especially with a focus on reducing toxic sodium hypochlorite consumption.

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.

Phosphate-based drinking water softeners are commonly used to prevent scale formation in drinking water distribution infrastructure. The main reason for drinking water softening is primarily economic (protection of pipes and extension of equipment life), while the health aspect of such treatment is usually neglected. The aim of this work is to investigate the effects of phosphate-based drinking water softeners on growth stimulation of Legionella pneumophila. Bacterial growth was observed at two different phosphate concentrations. On average, an increase in growth of 1.19-1.28 log CFU/mL was observed in selected samples with added phosphates compared with the control. The results of the in vitro experiment confirmed that the added phosphates stimulate the growth of L. pneumophila; growth stimulation could therefore be expected in drinking water distribution systems (DWDS) when phosphates are used as well. The availability of phosphorus in DWDS may be a crucial limiting factor for biofouling control. Consequently, phosphate-based chemicals for drinking water should be avoided or used with prudence, especially in drinking water with high concentrations of other nutrients.

This study was carried out with the aim of establishing how the outcomes of polyelectrolyte multilayer formation can be predicted on the basis of the results of complexation studies in solution and vice versa. For this purpose, the correlation between the processes of complex and multilayer formation involving three pairs of vinylic polyions in solutions of binary 1 : 1 sodium salts (NaX; X = F, Cl, Br, I, NO3, ClO4) was explored by means of dynamic and electrophoretic light scattering, potentiometry, microcalorimetry, spectrophotometry and quartz crystal microbalance. The gradual reactant mixing in solution at lower salt concentrations resulted in a Fuoss-Sadek sequence of events (primary complexes → secondary complexes → 1 : 1 flocculate), whereby the obtained nano-complexes could be successively overcharged. At high salt concentration and with excess polycation present, metastable nano-complexes and precipitates containing surplus of positively charged monomers were formed. The amount of extrinsically compensated charge was in accord with the polycation affinities toward counteranions, established by monitoring the electrolyte-induced aggregation of positively charged nano-complexes. Perfect analogy with respect to counteranion influence on the amount of adsorbed polycation was noticed for corresponding multilayers. Aside from providing a deeper understanding of interpolyelectrolyte neutralization, the gained insights can also be used to steer the polyelectrolyte multilayer composition and properties.

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.

Bacterial adhesion on material surfaces can be reduced by different type of coatings. In our study a silica surface on which polyelectrolyte multilayers were formed, with various proteins being the terminating layer, was used as a material surface. We examined three different types of proteins: bovine serum albumin (BSA), lysozyme and glucanase. Adhesion experiments were performed with two bacterial strains: Escherichia coli and Pseudomonas aeruginosa. In order to characterize the surface prior to bacterial adhesion, surface roughness and hydrophobicity were determined by profilometry, atomic force microscopy (AFM) and tensiometry, whereas the surface charge was estimated by zeta potential measurements of silica particles covered with polyelectrolyte multilayers and proteins. The extent of adhered bacteria was examined using scanning electron microscopy (SEM). It was confirmed that in the case where lysozyme was the outermost layer the lysis takes place, whereas in the case of BSA and glucanase specific protein-bacteria interactions are dominant. We showed that the adhesion strongly depends on the protein specificity and the surface physical properties do not play the key role in the adhesion process.

Legionella pneumophila can cause a potentially fatal form of humane pneumonia (Legionnaires’ disease), which is most problematic in immunocompromised and in elderly people. Legionella species is present at low concentrations in soil, natural and artificial aquatic systems and is therefore constantly entering man-made water systems. The environment temperature for it’s ideal growth range is between 32 and 42°C, thus hot water pipes represent ideal environment for spread of Legionella. The bacteria are dormant below 20°C and do not survive above 60°C. The primary method used to control the risk from Legionella is therefore water temperature control. There are several other effective treatments to prevent growth of Legionella in water systems, however current disinfection methods can be applied only intermittently thus allowing Legionella to grow in between treatments. Here we present an alternative disinfection method based on antibacterial coatings with Cu-TiO2 nanotubes deposited on preformed surfaces. In the experiment the microbiocidal efficiency of submicron coatings on polystyrene to the bacterium of the genus Legionella pneumophila with a potential use in a water supply system was tested. The treatment thus constantly prevents growth of Legionella pneumophila in presence of water at room temperature. Here we show that 24-hour illumination with low power UVA light source (15 W/m2 UVA illumination) of copper doped TiO2 nanotube coated surfaces is effective in preventing growth of Legionella pneumophila. Microbiocidal effects of Cu-TiO2 nanotube coatings were dependent on the flow of the medium and the intensity of UV-A light. It was determined that tested submicron coatings have microbiocidal effects specially in a non-flow or low-flow conditions, as in higher flow rates, probably to a greater possibility of Legionella pneumophila sedimentation on the coated polystyrene surfaces, meanwhile no significant differences among bacteria reduction was noted regarding to non or low flow of medium.

The fourth principle of Green Chemistry, a critically important area of sustainability science, identifies that substances should maintain intended functions while possessing limited intrinsic hazards to public health and the environment. In the present study, we empirically determined efficacy of 20 cleaning products against E. coli attachment to surfaces. Subsequently, probabilistic assessments using novel chemical efficacy distributions (CEDs) were conducted. Results for most chemicals indicated bacterial detachment with increasing concentration. Using a threshold concentration of 5th centiles, these cleaning product ingredients were predicted to detach E. coli (Lowest Observable Effect Concentration) at or below 0.7 (0.2, 2.2) mg L-1 for 5% of ingredients. We then employed chemical toxicity distributions (CTDs) to examine acute toxicity, based on currently available information, for common mammalian models. Results demonstrate that probabilities of associated hazards for rat (oral exposure route), mouse (oral exposure route), rabbit (dermal exposure route), and rat (inhalation exposure route) were 43.2%, 36.7%, 38.6% and 75.5%, respectively. A novel joint probability distribution analysis approach was then developed and applied to identify substances with various efficacy (CED) and hazard (CTD) characteristics. Our observations indicate that combining efficacy and toxicity information using joint probability curves may be useful for identifying classes of antimicrobial products, and other chemical classes, to optimize efficacy while minimizing environment and health hazards. (c) 2020 Elsevier Ltd. All rights reserved.

Cleaning the surfaces enables reducing microbial populations and consequently the risk of food contamination. The aim of this research was to study the potential of natural extracts from soap nuts, quillaja bark, and horse chestnuts for the reduction of Escherichia coli on glass surfaces. Analogous to that, we tested the efficacy of standard saponins as the active components in the plants mentioned above. The results show that the numbers of bacteria cells are decreased by increasing concentrations of all cleaning product, except for quillaja standard saponin (p < 0.05). Furthermore, the findings indicate that natural plant extracts have greater efficacy than standard saponins do. In particular, quillaja bark extract shows the potential to reduce bacterial populations by up to 66%. Moreover, the research also demonstrates that natural plant extracts have excellent abilities to reduce water surface tension (22 mN/m for soap nut extract) and emulsification potential (74 for quillaja bark extract). Natural plant extracts are inexpensive, biodegradable, and residues are less toxic than conventional ones. The evidence of this study suggests that natural plant extracts are efficient against attached E. coil cells on the glass surface and are a good candidate for surface hygiene management.

Abstract Preventing bacterial attachment to surfaces is the most efficient approach to controlling biofilm proliferation. The aim of this study was to compare anti-adhesion potentials of 5 and 50 mmol/L polyelectrolyte multilayers of poly(allylamine hydrochloride)/poly(sodium 4–styrenesulfonate), poly(4-vinyl-N-ethylpyridinium bromide)/ poly(sodium 4–styrenesulfonate), and poly(4-vinyl-N-isobutylpyridinium bromide)/poly(sodium 4–styrenesulfonate) against Escherichia coli. Glass surface was covered with five polyelectrolyte layers and exposed to bacterial suspensions. Poly(4-vinyl-N-ethylpyridinium bromide)/poly(sodium 4–styrenesulfonate) was the most effective against bacterial adhesion, having reduced it by 60 %, followed by poly(4-vinyl-N-isobutylpyridinium bromide)/poly(sodium 4– styrenesulfonate) (47 %), and poly(allylamine hydrochloride)/poly(sodium 4–styrenesulfonate) (38 %). Polyelectrolyte multilayers with quaternary amine groups have a significant anti-adhesion potential and could find their place in coatings for food, pharmaceutical, and medical industry.

There is a wide range of factors affecting bacterial adhesion and biofilm formation. However, in both food processing and medical settings, it is very hard to obtain suitably controlled conditions so that the factors that reduce surface colonisation and biofouling can be studied. The aim of this study was to evaluate the effect of glucose concentration, temperature and stainless steel (SS) surface roughness on biofouling by four common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and L. monocytogenes). Among the tested variables, the untreated SS surface (3C) was shown to be fouled more than 3D polished, brushed or electropolished SS surfaces. Although an array of parameters influenced biofouling, the most promising control measure was the influence of low temperature (4 degrees C) that reduced biofouling even in the case of the psychrophilic Listeria monocytogenes. The study findings could significantly contribute to the prevention of SS surface contamination and consequential biofouling by food and healthcare associated pathogens.

Abstract Biofilms on food contact materials represent public health issues because they are resistant to cleaning and disinfection. This study aims to assess the Bacillus cereus biofilm formation capacity on silicone, polyethylene terephthalate, Teflon, and aluminium food contact materials. The biofilm biomass was analysed with the crystal violet assay method. We used the standard strain B. cereus CCM 2010, wild strain B. cereus 100 and spores of those two strains. The results show that both the vegetative form the bacteria and it spores form large amounts of biofilm on silicone, followed by polyethylene terephthalate, Teflon, and aluminium. More detailed analysis has shown that spores form more biomass on all materials in comparison to the vegetative form and that the standard strains form low levels of biofilm in contrast to the wild strains. Selecting proper material with the lowest biofilm formation potential can prevent or reduce food contamination and consequently increase food safety.

In sufficient concentrations, the pathogenic bacteria L. pneumophila can cause a respiratory illness that is known as the "Legionnaires" disease. Moreover, toxic Shiga strains of bacteria E. colt can cause life-threatening hemolytic-uremic syndrome. Because of the recent restrictions imposed on the usage of chlorine, outbreaks of these two bacterial species have become more common. In this study we have developed a novel rotation generator and its effectiveness against bacteria Legionella pneumophila and Escherichia colt was tested for various types of hydrodynamic cavitation (attached steady cavitation, developed unsteady cavitation and supercavitation). The results show that the supercavitation was the only effective form of cavitation. It enabled more than 3 logs reductions for both bacterial species and was also effective against a more persistent Gram positive bacteria, B. subtilis. The deactivation mechanism is at present unknown. It is proposed that when bacterial cells enter a supercavitation cavity, an immediate pressure drop occurs and this results in bursting of the cellular membrane. The new rotation generator that induced supercavitation proved to be economically and microbiologically far more effective than the classical Venturi section (super)cavitation.

The aim of this study was to determine and compare the efficacy of a standard cleaning agent, sodium dodecyl sulphate, and natural extracts from pomegranate peel grape skin and bay laurel leaf against E. coli biofilm. The biofilm was exposed for 10 minutes to three different concentrations of each tested compound. The results show that bay laurel leaf extract is the most efficient with 43% biofilm biomass reduction, followed by pomegranate peel extract (35%); sodium dodecyl sulphate and grape skin extract each have 30% efficacy. Our study demonstrated that natural extracts from selected plants have the same or even better efficacy against E. coli biofilm removal from surfaces than the tested classical cleaning agent do. All this indicates that natural plant extracts, which are acceptable from the health and environment points of view, can be potential substitutes for classical cleaning agents.

The aim of this study was to analyze the impact of hydrodynamic forces on the multiplication of E. coli, and biofilm formation and dispersion. The experiments were provided in a flow chamber simulating a cleaning-in-place system. Biofilm biomass was measured using a crystal violet dye method. The results show that hydrodynamic forces affect not only biofilm formation and dispersion but the multiplication of E. coli in the first place. We found more biofilm biomass on the rough surface than on the smooth one. The results of the biofilm formation test show that laminar flow promotes the biofilm growth over 72 h, meanwhile turbulent flow after 48 h causes decrease in biomass. The results of the biofilm dispersion test, in contrast, show that laminar flow removed less biofilm from both materials that turbulent flow did. Therefore, taking into account these findings in cleaning-in-place technology can substantially reduce E. coli multiplication and biofilm formation.

The aim of this study was to analyse the adhesion of E. coli, P. aeruginosa and S. aureus on food contact materials, such as polyethylene terephthalate, silicone, aluminium, Teflon and glass. Surface roughness, streaming potential and contact angle were measured. Bacterial properties by contact angle and specific charge density were characterised. The bacterial adhesion analysis using staining method and scanning electron microscopy showed the lowest adhesion on smooth aluminium and hydrophobic Teflon for most of the bacteria. However, our study indicates that hydrophobic bacteria with high specific charge density attach to those surfaces more intensively. In food services, safety could be increased by selecting material with low adhesion to prevent cross contamination.

Bakery food contact surfaces generally include conveyor belts where the foodstuff is in contact with surfaces for long periods. The aim of the study was to analyse biofilm levels of B. cereus by initially inoculated vegetative cells and spores on different polyurethane conveyor belts. The overall biomass of the biofilm was measured using the optical density of absorbed crystal violet dye in cells. Moreover, the efficacy of cleaning agents, ultrasound, and a combined method was tested. The results show biofilm inoculated from spores attaches to surfaces the most firmly, followed by wild and standard strains. We did not prove that material roughness or contact angle impact the overall biomass of the biofilm. Furthermore, an efficacy assessment shows that strongly attached biofilm from spores is more difficult to eliminate in comparison to biofilm from vegetative cells. Moreover, we determined that using either the cleaning agent or ultrasound had limited capacity for biofilm removal in comparison with the combined method. Therefore, prevention of food contamination should also include hygiene management of food contact materials. The bakery industry should plan to change cleaning procedures to those implementing hurdle technologies. (C) 2017 Elsevier Ltd. All rights reserved.