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.

Photodynamic inactivation of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents.

There is an increasing need to discover effective methods for treating municipal wastewater and addressing the threat of multidrug-resistant (MDR) strains of bacteria spreading into the environment and drinking water. Photodynamic inactivation (PDI) that combines a photosensitiser and light in the presence of oxygen to generate singlet oxygen and other reactive species, which in turn react with a range of biomolecules, including the oxidation of bacterial genetic material, may be a way to stop the spread of antibiotic-resistant genes. The effect of 5,10,15,20-(pyridinium-3-yl)porphyrin tetrachloride (TMPyP3) without light, and after activation with violet-blue light (VBL) (394 nm; 20 mW/cm2), on MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and K. pneumoniae OXA-48 in tap water and municipal wastewater was investigated. High toxicity (~2 µM) of TMPyP3 was shown in the dark on both strains of K. pneumoniae in tap water, while on P. aeruginosa toxicity in the dark was low (50 µM) and the PDI effect was significant (1.562 µM). However, in wastewater, the toxicity of TMPyP3 without photoactivation was much lower (12.5-100 µM), and the PDI effect was significant for all three bacterial strains, already after 10 min of irradiation with VBL (1.562-6.25 µM). In the same concentrations, or even lower, an anti-adhesion effect was shown, suggesting the possibility of application in biofilm control. By studying the kinetics of photoinactivation, it was found that with 1,562 µM of TMPyP3 it is possible to achieve the complete destruction of all three bacteria after 60 min of irradiation with VBL. This study confirmed the importance of studying the impact of water constituents on the properties and PDI effect of the applied photosensitiser, as well as checking the sensitivity of targeted bacteria to light of a certain wavelength, in conditions as close as possible to those in the intended application, to adjust all parameters and perfect the method.

Periodontal Pathogens and Their Links to Neuroinflammation and Neurodegeneration.

Pathogens that play a role in the development and progression of periodontitis have gained significant attention due to their implications in the onset of various systemic diseases. Periodontitis is characterized as an inflammatory disease of the gingival tissue that is mainly caused by bacterial pathogens. Among them, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia are regarded as the main periodontal pathogens. These pathogens elicit the release of cytokines, which in combination with their virulence factors induce chronic systemic inflammation and subsequently impact neural function while also altering the permeability of the blood-brain barrier. The primary objective of this review is to summarize the existing information regarding periodontal pathogens, their virulence factors, and their potential association with neuroinflammation and neurodegenerative diseases. We systematically reviewed longitudinal studies that investigated the association between periodontal disease and the onset of neurodegenerative disorders. Out of the 24 studies examined, 20 showed some degree of positive correlation between periodontal disease and neurodegenerative disorders, with studies focusing on cognitive function demonstrating the most robust effects. Therefore, periodontal pathogens might represent an exciting new approach to develop novel preventive treatments for neurodegenerative diseases.

Temporal dynamics of adhesion of oral bacteria to orthodontic appliances.

Adhesion of the most common dental biofilm bacteria to alloys used in orthodontics in relation to surface characteristics was analyzed. Streptococcus mutans (S. mutans), Streptococcus oralis (S. oralis), Veillonella parvula (V. parvula), and Aggregatibacter actinomycetemcomitans (A. actynomicetemcomitans) were incubated for 4 h with nickel-titanium (NiTi) and stainless-steel (SS) wires. The surface roughness and free energy of the alloys, as well as the hydrophobicity of the alloys and bacteria, were assessed. NiTi had higher surface free energy and rougher (p<0.001) and more hydrophilic surfaces than SS (p<0.001). The hydrophobic properties of the bacteria decreased in the following order: V. parvula>S. oralis>S. mutans>A. actynomicetemcomitans. Bacterial adhesion generally increased over time, though this pattern was influenced by the type of alloy and the bacteria present (p<0.001). In a multiple linear regression, the principal predictor of adhesion was bacterial hydrophobicity (p<0.001), followed by time (p<0.001); alloy surface characteristics had a low influence.

Streptococcus salivarius as an Important Factor in Dental Biofilm Homeostasis: Influence on Streptococcus mutans and Aggregatibacter actinomycetemcomitans in Mixed Biofilm.

A disturbed balance within the dental biofilm can result in the dominance of cariogenic and periodontopathogenic species and disease development. Due to the failure of pharmacological treatment of biofilm infection, a preventive approach to promoting healthy oral microbiota is necessary. This study analyzed the influence of Streptococcus salivarius K12 on the development of a multispecies biofilm composed of Streptococcus mutans, S. oralis and Aggregatibacter actinomycetemcomitans. Four different materials were used: hydroxyapatite, dentin and two dense polytetrafluoroethylene (d-PTFE) membranes. Total bacteria, individual species and their proportions in the mixed biofilm were quantified. A qualitative analysis of the mixed biofilm was performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results showed that in the presence of S. salivarius K 12 in the initial stage of biofilm development, the proportion of S. mutans was reduced, which resulted in the inhibition of microcolony development and the complex three-dimensional structure of the biofilm. In the mature biofilm, a significantly lower proportion of the periodontopathogenic species A. actinomycetemcomitans was found in the salivarius biofilm. Our results show that S. salivarius K 12 can inhibit the growth of pathogens in the dental biofilm and help maintain the physiological balance in the oral microbiome.

Chelation in Antibacterial Drugs: From Nitroxoline to Cefiderocol and Beyond.

In the era of escalating antimicrobial resistance, the need for antibacterial drugs with novel or improved modes of action (MOAs) is a health concern of utmost importance. Adding or improving the chelating abilities of existing drugs or finding new, nature-inspired chelating agents seems to be one of the major ways to ensure progress. This review article provides insight into the modes of action of antibacterial agents, class by class, through the perspective of chelation. We covered a wide scope of antibacterials, from a century-old quintessential chelating agent nitroxoline, currently unearthed due to its newly discovered anticancer and antibiofilm activities, over the commonly used antibacterial classes, to new cephalosporin cefiderocol and a potential future class of tetramates. We show the impressive spectrum of roles that chelation plays in antibacterial MOAs. This, by itself, demonstrates the importance of understanding the fundamental chemistry behind such complex processes.

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.

The effect of nickel ions on the susceptibility of bacteria to ciprofloxacin and ampicillin.

To explore the interaction effects of ciprofloxacin and ampicillin with nickel on the growth of bacteria, Staphylococcus aureus strain ATCC 29213, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922 were used. Minimum inhibitory concentrations (MICs) were determined for nickel, ciprofloxacin and ampicillin, and the checkerboard method was used to assess their cumulative effects on bacterial growth. The interactions between the metal and antibiotics were assessed by the fractional inhibitory concentration (FIC). The MICs for ciprofloxacin and ampicillin were 0.31 and 1 mg/L for E. faecalis, 0.62 and 1 mg/L for S. aureus and 0.005 and 2.5 for E. coli, respectively. The MIC for nickel was 1000 mg/L for all bacteria. The FIC results for ciprofloxacin and nickel demonstrated an antagonistic effect of the two agents on the growth of E. coli and E. faecalis and an additive effect on S. aureus. The FICs for ampicillin and nickel demonstrated a synergistic effect on the growth of E. faecalis and E. coli. Different interactions of metals and antibiotics were observed depending on the bacteria and the type of antibiotic.

Adhesion of Oral Bacteria to Commercial d-PTFE Membranes: Polymer Microstructure Makes a Difference.

Bacterial contamination of the membranes used during guided bone regeneration directly influences the outcome of this procedure. In this study, we analyzed the early stages of bacterial adhesion on two commercial dense polytetrafluoroethylene (d-PTFE) membranes in order to identify microstructural features that led to different adhesion strengths. The microstructure was investigated by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR). The surface properties were analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), and surface free energy (SFE) measurements. Bacterial properties were determined using the microbial adhesion to solvents (MATS) assay, and bacterial surface free energy (SFE) was measured spectrophotometrically. The adhesion of four species of oral bacteria (Streptococcus mutans, Streptococcus oralis, Aggregatibacter actinomycetemcomitas, and Veilonella parvula) was studied on surfaces with or without the artificial saliva coating. The results indicated that the degree of crystallinity (78.6% vs. 34.2%, with average crystallite size 50.54 nm vs. 32.86 nm) is the principal feature promoting the adhesion strength, through lower nanoscale roughness and possibly higher surface stiffness. The spherical crystallites ("warts"), observed on the surface of the highly crystalline sample, were also identified as a contributor. All bacterial species adhered better to a highly crystalline membrane (around 1 log10CFU/mL difference), both with and without artificial saliva coating. Our results show that the changes in polymer microstructure result in different antimicrobial properties even for chemically identical PTFE membranes.

Combined Inhibitory Effect of Fir (Abies alba Mill.) Honeydew Honey and Probiotic Bacteria Lactiplantibacillus plantarum on the Growth of Salmonella enterica Serotype Typhimurium.

Honey is a natural food consisting mainly of sugars, enzymes, amino acids, organic acids, vitamins, minerals and aromatic substances. In addition to specific organoleptic properties, honey also has other components that contribute to its nutritional and health value. Proteins, vitamins, minerals, organic acids and phenolic compounds, the most variable components of honey, are predominantly responsible for its strong bioactive effect. Honeydew honey is a less known type of honey with outstanding antimicrobial and antioxidant properties that also demonstrates prebiotic effects and can promote the growth of probiotic bacteria. Foodborne illnesses can be prevented by using probiotic strains in combination with prebiotics. The aim of this study was for the first time to determine potential synergistic antimicrobial effect of fir (Abies alba Mill.) honeydew honey (HS) and probiotic bacteria Lactiplantibacillus plantarum on Salmonella enterica serotype Typhimurium, a primary cause of foodborne illnesses. The effect of three different samples of fir honeydew honey on the growth of L. plantarum in de Man, Rogosa and Sharpe (MRS) medium and the potential synergistic effect of HSs and L. plantarum on the growth of S. Typhimurium in the Brain Heart Infusion (BHI) medium were examined. The results indicate that concentrations of 1 and 5% of all three HS samples stimulate the growth and metabolic activity of L. plantarum, while a concentration of 10% inhibits the growth of L. plantarum. The concentration of 5% of all three HS and L. plantarum combined inhibits the growth of S. Typhimurium in BHI broth. Fir honeydew honey showed potential prebiotic properties and antimicrobial activity, both of which can synergistically enhance the probiotic efficacy of L. plantarum against S. Typhimurium To conclude, the combination of fir honeydew honey and L. plantarum represents a successful combination against S. Typhimurium and additional experiments are necessary regarding the mechanisms of their combined effect.

Effects of Long-Term Lead Exposure on Antioxidant Enzyme Defense System in Organs of Brown Hare (Lepus europaeus Pallas) as a Bioindicator of Environmental Pollution in Croatia.

In Croatia, Podravina is a well-known lead-polluted region due to the intensive exploitation of natural gas, a highly developed agricultural industry, and a traffic hub with several heavily traveled roads. It represents a natural environment with a great variety of wildlife, especially hares (Lepus europaeus Pallas), which may serve as an indicator for environmental quality assessment. This study was conducted to estimate the bioaccumulation of lead in hare liver, kidney, muscle, and brain during long-term exposure and its impact on the oxidative status of the organism and to investigate a possible lead exchange ionic mechanism in the brain. In the organs of two hare groups (experimental from polluted area and control from the island of Krk), Ca, Fe, Mg, Na, lead concentrations, and antioxidant enzyme defense system were analyzed. The accumulation of lead was highest in the brain (3.7-fold higher compared to the control group) and lowest in the liver (1.6-fold higher compared to the control group). Kendall-Tau and multiple regression analysis showed that the increased lead content caused a stronger exchange of Ca and Na ions in the brain. We proposed that lead either mobilizes intracellular cation stores or causes competitive displacement of Ca from the binding site in intoxicated cells. A linear predictive model for cell intoxication by lead was proposed, where GPx and SOD were predominantly influenced by long-term lead exposure. The presented results showed that long-term lead exposure in hares negatively affected their oxidative status and caused the strongest toxicity in the brain and muscles, making their survival and/or population vulnerable.

Juniperus communis essential oil limit the biofilm formation of Mycobacterium avium and Mycobacterium intracellulare on polystyrene in a temperature-dependent manner.

Formation of biofilms allows bacterial cells to survive in adverse environments. Nontuberculous mycobacteria are ubiquitous in aqueous environments, where they adhere to surfaces and create a biofilm. This has led to the emergence of healthcare-associated infections and the use of biomaterials in medicine. Essential oils (EO) are substances of natural origin whose effect on microorganisms has been the subject of numerous studies. Here, we investigated the effect of Juniperus communis EO on nontuberculous mycobacteria and their early and mature biofilm formation in sterilised tap water. The combination of Juniperus communis EO and increasing ambient temperature showed a synergistic effect on the reduction of biofilm formation of Mycobacterium avium and Mycobacterium intracellulare on a polystyrene surface. A significant antibiofilm effect of Juniperus communis EO was also found at subinhibitory concentrations, suggesting a potential role for it as an alternative disinfectant of natural water.

Ozone disinfection efficiency against airborne microorganisms in hospital environment: a case study.

Even though ozone has shown its potential for air disinfection in hospital environment, its more frequent use has earned attention only with the COVID-19 pandemic due to its proven antimicrobial effect and low cost of production. The aim of this study was to determine its antimicrobial efficiency against the most common bacterial species in a real-life setting, that is, in the air of one postoperative room of the General Hospital Dr Ivo Pedisic (Sisak, Croatia). Air was sampled for aiborne bacteria before and after treatment with the ozone concentration of 15.71 mg/m3 for one hour. The most dominant Gram-positive bacteria of the genera Micrococcus, Staphylococcus, and Bacillus were reduced by 33 %, 58 %, and 61 %, respectively. The genus Micrococcus proved to be the most resistant. Considering our findings, we recommend longer air treatment with higher ozone concentrations in combination with mechanical cleaning and frequent ventilation.

How can probiotic improve irritable bowel syndrome symptoms?

The onset and manifestations of irritable bowel syndrome (IBS) is associated with several factors, and the pathophysiology involves various central and peripheral mechanisms. Most studies indicate that the management of gut microbiota could significantly affect the improvement of subjective disorders in patients with IBS. Numerous clinical trials have assessed the efficacy of probiotics for IBS with controversial conclusions. Several clinical trials have suggested that probiotics can improve global IBS symptoms, while others only improve individual IBS symptoms, such as bloating scores and abdominal pain scores. Only a few clinical trials have found no apparent effect of probiotics on IBS symptoms. Generally, probiotics appear to be safe for patients with IBS. However, the question of which probiotics should be used for certain IBS subtypes remains unresolved. In everyday practice, the dose of the recommended probiotic remains questionable, as well as how long the probiotic should be used in therapy. The use of probiotics in the M subtype and non-classified IBS is particularly problematic, in which combination therapy should be recommended due to the change in symptoms. Therefore, new approaches are needed in the design of clinical studies that should address certain subtypes of IBS.

Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness.

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs' stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.

Bacterial Exposure to Nickel: Influence on Adhesion and Biofilm Formation on Orthodontic Archwires and Sensitivity to Antimicrobial Agents.

The presence of nickel could modify bacterial behavior and susceptibility to antimicrobial agents. Adhesion and biofilm formation on orthodontic archwires can be a source of bacterial colonization and possible health hazards. Staphylococcus aureus was subjected to exposure and adaptation to various sub-inhibitory concentrations of nickel. Five strains of bacteria adapted to nickel in concentrations of 62.5-1000 µg/mL were tested for adhesion and biofilm formation on nickel-titanium archwires. Archwires were previously incubated in artificial saliva. Bacteria were incubated with orthodontic wire with stirring for 4 h (adhesion) and 24 h (biofilm formation). The number of adherent bacteria was determined after sonication and cultivation on the Muller-Hinton agar. Disk diffusion method was performed on all bacteria to assess the differences in antimicrobial susceptibility. Bacteria adapted to lower concentrations of nickel adhered better to nickel-titanium than strains adapted to higher concentrations of nickel (p < 0.05). Biofilm formation was highest in strains adapted to 250 and 500 µg/mL of nickel (p < 0.05). The highest biofilm biomass was measured for strains adapted to 250 µg/mL, followed by those adapted to 1000 µg/mL. Bacteria adapted to lower concentrations of nickel demonstrated lower inhibition zone diameters in the disk diffusion method (p < 0.05), indicating increased antimicrobial resistance. In conclusion, bacteria adapted to 250 µg/mL of nickel ions adhered better, demonstrated higher biofilm formation and often had higher antimicrobial resistance than other adapted and non-adapted strains.

Sea water whirlpool spa as a source of Legionella infection.

Bacterial pneumonia caused by the inhalation of aerosols contaminated with Legionella spp. is also known as Legionnaires' disease. In this study, we report a case of pneumonia caused by Legionella pneumophila sg.1 in a 58-year-old man who visited a sea water-filled whirlpool within a hotel and spa complex. The patient's Legionella urine antigen test was positive for L. pneumophila sg.1. During the field study, samples were taken from both the outdoor and indoor sea water-filled pools. Samples from the whirlpool were culture positive for L. pneumophila sg.1. Typing results indicated sea water isolate belonged to Sequence type ST82 and Allentown/France MAb subgroup. In vitro experiments showed that L. pneumophila strains are able to survive within sea water up to 7 days, and survival time is prolonged with sea water dilution. Also, our results indicate that L. pneumophila Allentown strain was the most resistant to adverse conditions in sea water with the highest values of DT50 (420 min) and DT90 (1,396 min). The possible source of infection was adding potable water for filling up the whirlpool. The survival of the L. pneumophila in additionally conditioned sea water should be considered in a further study.

Assessment of the Biological Activity and Phenolic Composition of Ethanol Extracts of Pomegranate (Punica granatum L.) Peels.

Pomegranate (Punica granatum L.) is a rich source of constituents with confirmed strong biological activities. However, pomegranate peel, which encompasses approximately 30-40% of its weight, is treated as a biological waste. The aim of this paper was to evaluate the potential of pomegranate peel extracts and to propose its functional properties that can be used for development of functional products. Eight ethanol extracts of pomegranate peels (PPEs) were characterized by use of direct infusion quadrupole-time of flight (Q-TOF), and afterwards tested on their antioxidant, antibacterial and antiproliferative activities. Mass spectrometry analysis revealed that the most prevalent compounds in pomegranate peels were punicalagin, granatin and their derivatives. Analysed extracts had high total phenolic contents that ranged from 5766.44 to 10599.43 mg GAE/100 g, and strong antioxidant activity (7551.31-7875.42 and 100.25-176.60 µmol TE/100 g for DPPH and FRAP assays, respectively). The results of biological activity assays showed that all PPEs possessed antibacterial activity, and that S. aureus was the most sensitive specie with minimum inhibitory concentration and minimum bactericidal concentrations ranging from 0.8 to 6.4 mg/mL. Additionally, the analysis of antiproliferative activity revealed high potency of PPEs, as the IC50 values ranged from 0.132 mg/mL to 0.396 mg/mL. Multivariate analysis pointed out the most discriminative metabolites for antioxidant or antiproliferative activity. Overall, the pomegranate peel confirmed to be a highly valuable source of bioactive compounds that could be used to improve the food functional characteristics.

Juniper and immortelle essential oils synergistically inhibit adhesion of nontuberculous mycobacteria to Acanthamoeba castellanii.

Acanthamoeba is an opportunistic protozoon, widespread in the aquatic environment, where it can be in endosymbiosis with over 30 pathogenic bacteria, including nontuberculous mycobacteria (NTM). Protozoa play a crucial role in mycobacterial pathogenesis and serve as a reservoir of infection. Since the first step in bacteria making contact with amoebae is adhesion, we were interested in investigating whether essential oils (EOs) can affect it. To that end we investigated the effects of juniper (Juniperus communis) and immortelle (Helichrysum italicum) EOs against Mycobacterium avium, M. intracellulare, and M. gordonae in tap water and against their adhesion to Acanthamoeba castellanii by combining them in synergistic EO concentrations. M. avium and M. intracellulare adhered to A. castellanii to a greater extent than M. gordonae. The adhesion of all NTMs was prevented by the subinhibitory concentrations of EOs. When comparing the effect of synergistic combinations of EOs and the effect of a single concentration from a combination, a higher percentage of adhesion inhibition in all synergistic combinations observed, except against M. gordonae. Neither oil was cytotoxic to A. castellanii. Our findings suggest that the EOs or their components weaken the contact of environmental NTMs and free-living amoebae and indirectly diminish their pathogenic potential, which could be of value in developing strategies for maintenance of water supply systems.

Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila.

Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires' disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0-108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.