This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 103–109 c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 109, 108, 107 and 106 c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 105 c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 104 c.f.u. caused sporadic death of mice. All animals that received 103 c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48–72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.

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.

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.

Legionella is an opportunistic premise plumbing pathogen that can be present in municipal and other water supplies. Building water systems may provide conditions (such as low flow, water hardness, low disinfectant residual levels and optimal temperature) that accelerate Legionella growth to levels that may result in an increased risk to public health. The standard disinfection of water systems (periodic overheating of water and chlorination) in the interest of prevention of Legionnaires' disease have often proved to be inefficient. It is therefore necessary to develop new approaches for removing Legionella from water systems. One of the new methods is antimicrobial photodynamic therapy (aPDT), which includes the combined activity of a photosensitizer (PS), molecular oxygen and visible light of appropriate wavelength to create singlet oxygen (1O2) and other oxygen reactive species (ROS) leading to the oxidation of numerous cellular components and cell death. In this study, a newly synthesized cationic, amphiphilic porphyrin TMPyP3-C17H35, was tested against Legionella in tap water. The minimal effective concentration (MEC) of PS photoinactivation test and PS uptake assay in sterile tap water were explored to determine the anti-Legionella activity. The complete inactivation of Legionella in sterile tap water was achieved with 0.024 μM of the PS. Also, the tested PS was found to be very effective in reducing Legionella growth in the sterile tap water and photoinactivation was dose-dependent. The tested PS binds well to the bacterial cell, after only 10 minutes of incubation in the dark. In conclusion, these studies indicate that TMPyP3-C17H35 is highly efficient in aPDT which leads to reducing Legionella growth in sterile tap water, and these results suggest that cationic amphiphilic photosensitizers may have a broader application in the photoinactivation of bacterial cells implicated in water disinfection.

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.

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.

Supplementary Table S1. FIC indices and the type of interaction of the strawberry tree (STE) (MIC 1.25 and 2.5 mg ml -1 ) and bearberry tea (BTE) (MIC 5.0 mg ml -1 ) extracts. *synergistic effect (S) -FICi ≤ 0.5; additive effect (Ad) -FICi = 0.5 -1; indifferent effect (I) -FICi = 1 -2 and antagonistic effect (An) -FICi ≥ 2. S2. FIC indices and type of interaction of the strawberry tree (STE) (MIC 2.5 mg ml -1 ) and bearberry (BE) (MIC 2.5 and 5.0 mg ml -1 ) extracts. *synergistic effect (S) -FICi ≤ 0.5; additive effect (Ad) -FICi = 0.5 -1; indifferent effect (I) -FICi = 1 -2 and antagonistic effect (An) -FICi ≥ 2. S3. FIC indices and the type of interaction of strawberry tree (STE) (MIC 1.25 mg ml -1 ) and bearberry tea (BTE) or bearberry (BE) (MIC 2.5 mg ml -1 ) extracts. *synergistic effect (S) -FICi ≤ 0.5; additive effect (Ad) -FICi = 0.5 -1; indifferent effect (I) -FICi = 1 -2 and antagonistic effect (An) -FICi ≥ 2. Supplementary Table Supplementary Table Supplementary Table S4. Evaluation of initial adhesiveness and biofilm formation ofEnterococcus isolates after 24 and 48 h. *ODc -cut-off OD value (three standard deviations above the average mean OD of the negative control); Supplementary Table S5. Evaluation of the initial adhesiveness and biofilm formation of E43 and E94 isolates in pretreatment with plant extracts after 24 and 48 h. *Category 0 (-); Category 1 (+); Category 2 (++); Category 3 (+++). ODc -cut-off OD value (three standard deviations above the average mean OD of the negative control). Van -Vancomycin; STE -strawberry tree extract; BE -bearberry extract; BTE -bearberry tea extract. S6. Evaluation of initial adhesiveness and biofilm formation of E61 and E89 isolates in pretreatment with plant extracts after 24 and 48 h. *Category 0 (-); Category 1 (+); Category 2 (++); Category 3 (+++). ODc -cut-off OD value (three standard deviations above the average mean OD of the negative control). Van -Vancomycin; STE -strawberry tree extract; BE -bearberry extract; BTE -bearberry tea extract. Supplementary Table

Pseudomonas fluorescens is one of the first colonizers of bacterial biofilm in water systems and a member of opportunistic premise plumbing pathogens (OPPPs). The aim of this study was to examine the effect of UV light and sodium hypochlorite on the formation and destruction of mature P. fluorescens biofilm on ceramic tiles. Planktonic bacteria or bacteria in mature biofilm were exposed to UV light (254 nm) for 5, 20 s. and to 0.4 mg/L sodium hypochlorite for 1 min. Mature biofilm was also exposed to increased concentration of sodium hypochlorite of 2 mg/L for 0.5, 1 and 2 h and combined with UV. Prolonged action of sodium hypochlorite and an increase in its concentration in combination with UV gave the best results in the inhibition of biofilm formation after the pre-treatment and destruction of mature biofilm. The effect of hyperchlorination in combination with UV radiation shows better results after a long exposure time, although even after 120 min there was no completely destroyed biofilm. Furthermore, the mechanism of the effect of combined methods should be explored as well as the importance of mechanical cleaning that is crucial in combating bacterial biofilm in swimming pools.