A synergistic anti-bacterial and anti-adhesion activity of tea tree (Melaleuca alternifolia) and lemon eucalyptus tree (Eucalyptus citriodora Hook) essential oils on Legionella pneumophila.

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.

Phosphate drinking water softeners promote Legionella growth.

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.

Disinfecting Action of Gaseous Ozone on OXA-48-Producing Klebsiella pneumoniae Biofilm In Vitro.

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.

Predicting the outcomes of interpolyelectrolyte neutralization at surfaces on the basis of complexation experiments and vice versa.

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.

Soft nanotechnology: the potential of polyelectrolyte multilayers against E. coli adhesion to surfaces.

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.

Photocatalytic biocidal effect of copper doped TiO2 nanotube coated surfaces under laminar flow, illuminated with UVA light on Legionella pneumophila.

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.

Biofouling of stainless steel surfaces by four common pathogens: the effects of glucose concentration, temperature and surface roughness.

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 °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.

Efficacy of sodium dodecyl sulphate and natural extracts against E. coli biofilm.

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.

Removal of bacteria Legionella pneumophila, Escherichia coli, and Bacillus subtilis by (super)cavitation.

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. coli 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 coli 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.

Escherichia coli biofilm formation and dispersion under hydrodynamic conditions on metal surfaces.

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 influence of shear stress on the adhesion capacity of Legionella pneumophila.

Bacterial adhesion is a complex process influenced by many factors, including hydrodynamic conditions. They affect the transfer of oxygen, nutrients, and bacterial cells in a water supply and cooling systems. The aim of this study was to identify hydrodynamic effects on bacterial adhesion to and detachment from stainless steel surfaces. For this purpose we observed the behaviour of bacterium L. pneumophila in no-flow and laminar and turbulent flow conditions simulated in a fluid flow chamber. The bacterial growth in no-flow and laminar flow conditions was almost identical in the first 24 h, while at 48 and 72 h of incubation, the laminar flow stimulated bacterial growth. In the second part of this study we found that laminar flow accelerated bacterial adhesion in the first 48 h, but after 72 h the amount of bacterial cells exposed to the flow dropped, probably due to detachment. In the third part we found that the turbulent flow detached more bacterial cells than the laminar, which indicates that the strength of shear forces determines the rate of bacterial removal.

Bacterial adhesion capacity on food service contact surfaces.

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.

Use of hydrodynamic cavitation in (waste)water treatment.

The use of acoustic cavitation for water and wastewater treatment (cleaning) is a well known procedure. Yet, the use of hydrodynamic cavitation as a sole technique or in combination with other techniques such as ultrasound has only recently been suggested and employed. In the first part of this paper a general overview of techniques that employ hydrodynamic cavitation for cleaning of water and wastewater is presented. In the second part of the paper the focus is on our own most recent work using hydrodynamic cavitation for removal of pharmaceuticals (clofibric acid, ibuprofen, ketoprofen, naproxen, diclofenac, carbamazepine), toxic cyanobacteria (Microcystis aeruginosa), green microalgae (Chlorella vulgaris), bacteria (Legionella pneumophila) and viruses (Rotavirus) from water and wastewater. As will be shown, hydrodynamic cavitation, like acoustic, can manifest itself in many different forms each having its own distinctive properties and mechanisms. This was until now neglected, which eventually led to poor performance of the technique. We will show that a different type of hydrodynamic cavitation (different removal mechanism) is required for successful removal of different pollutants. The path to use hydrodynamic cavitation as a routine water cleaning method is still long, but recent results have already shown great potential for optimisation, which could lead to a low energy tool for water and wastewater cleaning.

Microbial adhesion capacity. Influence of shear and temperature stress.

Environmental parameters dictate the conditions for both biofilm formation and deconstruction. The aim of this study is to analyse the impact of hydrodynamic and thermodynamic effects on bacterial detachment. Escherichia coli grown on two stainless steel metal surfaces with different roughness (brushed with roughness of 0.05 µm and electropolished with roughness of 0.29 µm) are exposed to laminar and turbulent (shower) flows of phosphate buffered saline media at temperatures of 8, 20 and 37 °C. Results show that the turbulent flow removes significantly more bacterial cells than laminar flow (p <0.05) on both materials. This indicates that the shear force determines the rate of detached bacteria. It is also observed that detachment of cells is more efficient on brushed than on electropolished contact surfaces because on the latter surface, fewer cells were attached before exposure. Moreover, we demonstrate that the temperature of the washing agent has an impact on bacterial detachment. At the same flow conditions, the exposure to higher temperature results in greater detachment rate.

The impact of material surface roughness and temperature on the adhesion of Legionella pneumophila to contact surfaces.

The adhesion of bacterial cells to various surfaces is based on physical and chemical interactions between the micro-organisms and the surfaces. The main purpose of this research is to determine the effect of material roughness and incubation temperature on the adhesion of bacteria. To determine the adhesion of the bacterial strain of Legionella pneumophila ATCC 33153 to the glass coupons, a spectrophotometric method of measuring the optical density of crystal violet dye that is released from pre-stained bacterial cells attached to the test surface was used. The intensity of adhesion is in positive correlation to the increase in surface roughness (p < 0.05). The adhesion is the greatest at an optimal temperature of 36 °C, whereas the temperature of 15 °C has a bacteriostatic effect and the temperature of 55 °C a bactericidal effect.