Planktonic Cultures Research Articles

Membrane vesicles in Acidithiobacillia class extreme acidophiles: influence on collective behaviors of 'Fervidacidithiobacillus caldus'.

Membrane vesicles (MVs) are envelope-derived extracellular sacs that perform a broad diversity of physiological functions in bacteria. While considerably studied in pathogenic microorganisms, the roles, relevance, and biotechnological potential of MVs from environmental bacteria are less well established. Acidithiobacillaceae family bacteria are active players in the sulfur and iron biogeochemical cycles in extremely acidic environments and drivers of the leaching of mineral ores contributing to acid rock/mine drainage (ARD/AMD) and industrial bioleaching. One key aspect of such a role is the ability of these bacteria to tightly interact with the mineral surfaces and extract electrons and nutrients to support their chemolithotrophic metabolism. Despite recent advances in the characterization of acidithiobacilli biofilms and extracellular matrix (ECM) components, our understanding of its architectural and mechanistic aspects remains scant. Using different microscopy techniques and nano-tracking analysis we show that vesiculation is a common phenomenon in distant members of the Acidithiobacillaceae family, and further explore the role of MVs in multicellular colonization behaviors using 'Fervidacidithiobacillus caldus' as a bacterial model. Production of MVs in 'F. caldus' occurred in both planktonic cultures and biofilms formed on sulfur surfaces, where MVs appeared individually or in chains resembling tube-shaped membranous structures (TSMSs) important for microbial communication. Liquid chromatography-mass spectrometry data and bioinformatic analysis of the MV-associated proteome revealed that 'F. caldus' MVs were enriched in proteins involved in cell-cell and cell-surface processes and largely typified the MVs as outer MVs (OMVs). Finally, microbiological assays showed that amendment of 'F. caldus' MVs to cells and/or biofilms affects collective colonizing behaviors relevant to the ecophysiology and applications of these acidophiles, providing grounds for their exploitation in biomining.

The Study of Antistaphylococcal Potential of Omiganan and Retro-Omiganan Under Flow Conditions.

Staphylococcus aureus is considered one of the leading pathogens responsible for infections in humans and animals. The heterogeneous nature of diseases caused by these bacteria is due to the occurrence of multiple strains, differentiated by several mechanisms of antibiotic resistance and virulence factors. One of these is the ability to form biofilm. Biofilm-associated bacteria exhibit a different phenotype that protects them from external factors such as the activity of immune system or antimicrobial substances. Moreover, it has been shown that the majority of persistent and recurrent infections are associated with the presence of the biofilm. Omiganan, an analog of indolicidin - antimicrobial peptide (AMP) derived from bovine neutrophil granules, was found to exhibit high antistaphylococcal and antibiofilm potential. Furthermore, its analog with a reversed sequence (retro-omiganan) was found to display enhanced activity against a variety of pathogens. Based on experience of our group, we found out that counterion exchange can improve the antistaphylococcal activity of AMPs. The aim of this study was to investigate the activity of both compounds against S. aureus biofilm under flow conditions. The advantage of this approach was that it offered the opportunity to form and characterize the biofilm under more controlled conditions. To do this, unique flow cells made of polydimethylsiloxane (PDMS) were developed. The activity against pre-formed biofilm as well as AMPs-treated bacteria was measured. Also, the incorporation of omiganan and retro-omiganan into the channels was conducted to learn whether or not it would inhibit the development of biofilm. The results of the microbiological tests ultimately confirmed the high potential of the omiganan and its retro-analog as well as the importance of counterion exchange in terms of antimicrobial examination. We found out that retro-omiganan trifluoroacetate had the highest biofilm inhibitory properties, however, acetates of both compounds exhibited the highest activity against planktonic and biofilm cultures. Moreover, the developed methodology of investigation under flow conditions allows the implementation of the studies under flow conditions to other compounds.

The effect of light intensity on microalgae biofilm structures and physiology under continuous illumination

The interest by biofilm-based microalgae technologies has increased lately due to productivity improvement, energy consumption reduction and easy harvesting. However, the effect of light, one key factor for system’s operation, received less attention than for planktonic cultures. This work assessed the impact of Photon Flux Density (PFD) on Chlorella vulgaris biofilm dynamics (structure, physiology, activity). Microalgae biofilms were cultivated in a flow-cell system with PFD from 100 to 500 μmolm-2s-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upmu {\ extrm{mol}} \\, \ extrm{m}^{-2} \\, \ extrm{s}^{-1}}$$\\end{document}. In the first stage of biofilm development, uniform cell distribution was observed on the substratum exposed to 100 μmolm-2s-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upmu \ extrm{mol} \\, \ extrm{m}^{-2} \\, \ extrm{s}^{-1}}$$\\end{document} while cell clusters were formed under 500 μmolm-2s-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upmu \ extrm{mol} \\, \ extrm{m}^{-2} \\, \ extrm{s}^{-1}}$$\\end{document}. Though similar specific growth rate in exponential phase (ca. 0.3 d-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extrm{d}^{-1}}$$\\end{document}) was obtained under all light intensities, biofilm cells at 500 μmolm-2s-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upmu \ extrm{mol} \\, \ extrm{m}^{-2} \\, \ extrm{s}^{-1}}$$\\end{document} seem to be ultimately photoinhibited (lower final cell density). Data confirm that Chlorella vulgaris showed a remarkable capability to cope with high light. This was marked for sessile cells at 300 μmolm-2s-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upmu \ extrm{mol} \\, \ extrm{m}^{-2} \\, \ extrm{s}^{-1}}$$\\end{document}, which reduce very rapidly (in 2 days) their chlorophyll-a content, most probably to reduce photodamage, while maintaining a high final cell density. Besides cellular physiological adjustments, our data demonstrate that cellular spatial organization is light-dependent.

Evaluation of approaches to increase the effectiveness of various disinfectants against biofilm communities of different ages

Disinfectants are used as the main agents against microorganisms circulating on the surfaces of food enterprises. However, the adaptive ability of microorganisms to form biofilms complicates the process of surface cleaning and reduces the effectiveness of disinfectants. Modern disinfectants act against freely circulating microflora, but it is known that they are not always effective against biofilms. The purpose of this study was to investigate effective disinfectant compositions with bactericidal effect on binary bacterial biofilms of different ages. The article describes the effects of disinfectants based on chlorine, peracetic acid and quaternary ammonium compounds with enzymes in concentrations recommended by the manufacturer and increased several times on Salmonella 38, Brochothrix thermosphacta 2726 and Staphylococcus equorum 2736 planktonic cultures and binary biofilms. Binary biofilms of different ages (2 and 9 days old) were exposed to disinfectants with various active ingredients in combination with adjuvants, i. e. hydrogen peroxide 6% and various concentrations of isopropyl alcohol (30%). All products in concentrations recommended by the manufacturer did not have a disinfectant effect against the studied biofilm cultures. As a result of the work, it was found that the most effective disinfectants against multispecies biofilms were quaternary ammonium compounds in combination with enzymes and chlorine in combination with isopropyl alcohol (30%). The results obtained allow to expand knowledge about effective methods for controlling biofilms.

Linking methanotroph phenotypes to genotypes using a simple spatially resolved model ecosystem.

Connecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental context in laboratory settings. Laboratory-based model ecosystems offer a means to better account for environmental conditions compared with standard planktonic cultures and can help link genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs) within the methane-oxygen counter gradient typically found in the natural environment of these organisms. Culturing the methanotroph Methylomonas sp. strain LW13 in this system resulted in the formation of a distinct horizontal band at the intersection of the counter gradient, which we discovered was not due to increased numbers of bacteria at this location but instead to an increased amount of polysaccharides. We also discovered that different methanotrophic taxa form polysaccharide bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this band, we identified genes upregulated within the band and validated their involvement in growth and band formation within the model ecosystem using knockout strains. Notably, deletion of these genes did not negatively affect growth using standard planktonic culturing methods. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover bacterial phenotypes missing from standard laboratory conditions, and to link these phenotypes with their genetic determinants.

Exploring microbial adaptation of immobilised acidophilic cultures to improve microbial oxidation rates and copper tolerance in e-waste bioleaching

The rate of ferric iron regeneration by microbial oxidation is the rate determining step in the bioleaching of printed circuit boards (PCBs). Therefore, improvement of the volumetric rate of ferric iron formation is key for the overall bioleaching efficiency. The volumetric rate is impacted by both the specific rate and the biomass concentration. Limiting the exposure of the bioleaching microorganisms to accumulated toxic metal ions in solution, as well as the presence of inhibiting material components in PCBs through biofilm formation and immobilisation, as well as their adaptation to these inhibitors, is proposed to mitigate against decrease in specific rate while biomass retention by immobilisation is expected to positively impact volumetric rate. In this study, a mixed mesophilic culture, consisting of Leptospirillum (L.) ferriphilum, Acidithiobacillus (At.) caldus, Acidiplasma (Ap.) cupricumulans as dominant species was immobilised on polyurethane foam (PUF) and adapted to 6.0 g/L Cu2+. The rate of microbial ferrous iron oxidation by the immobilised culture was compared to that of planktonic cultures. Further, the performance of both the planktonic and immobilised Cu-adapted cultures (6.0 g/L of Cu2+) were compared with non-adapted planktonic and immobilised cells in the presence of increasing Cu2+ concentrations (0–50 g/L Cu2+).Both the Cu-adapted planktonic cells and the non-adapted immobilised cells demonstrated improved tolerance to Cu2+ stress relative to non-adapted planktonic cells. The Cu-adapted planktonic cells showed higher volumetric ferrous iron oxidation rates (0.0359 ± 0.0023 g Fe2+.L−1.h−1 at 22 g/L Cu2+) than the non-adapted immobilised cells (0.0331 ± 0.0021 g Fe2+.L−1.h−1) in the presence of Cu2+ concentrations exceeding 6 g/L. The Cu-adapted immobilised cells exhibited higher tolerance to Cu2+ stress and higher oxidation kinetics (0.0622 ± 0.0064 g Fe2+.L−1.h−1) than the Cu-adapted planktonic and non-adapted immobilised cells. This study provides a novel approach for improving culture performance and minimising the inhibitory effect of Cu2+ ion on L. ferriphilum dominated cultures. High microbial activity and metal tolerance of Cu-adapted immobilised cells presents an opportunity for immobilised microbial systems to be used both in bioleaching of e-waste streams, such as PCBs, and in bioleaching of mineral concentrates, ores and wastes with Cu2+-rich leachates.

Antibiofilm Activity and Biocompatibility of Temporin-SHa: A Promising Antimicrobial Peptide for Control of Fluconazole-Resistant Candida albicans.

The aim of the study was to investigate the effect of antimicrobial peptides (AMPs) Hylin-a1, KR-12-a5, and Temporin-SHa in Candida albicans as well as the biocompatibility of keratinocytes spontaneously immortalized (NOK-si) and human gingival fibroblasts (FGH) cells. Initially, the susceptible (CaS-ATCC 90028) and fluconazole-resistant (CaR-ATCC 96901) C. albicans strains were grown to evaluate the effect of each AMP in planktonic culture, biofilm, and biocompatibility on oral cells. Among the AMPs evaluated, temporin-SHa showed the most promising results. After 24 h of Temporin-SHa exposure, the survival curve results showed that CaS and CaR suspensions reduced 72% and 70% of cell viability compared to the control group. The minimum inhibitory/fungicide concentrations (MIC and MFC) showed that Temporin-SHa was able to reduce ≥50% at ≥256 µg/mL for both strains. The inhibition of biofilm formation, efficacy against biofilm formation, and total biomass assays were performed until 48 h of biofilm maturation, and Temporin-SHa was able to reduce ≥50% of CaS and CaR growth. Furthermore, Temporin-SHa (512 µg/mL) was classified as non-cytotoxic and slightly cytotoxic for NOK-si and FGH, respectively. Temporin-SHa demonstrated an anti-biofilm effect against CaS and CaR and was biocompatible with NOK-si and FGH oral cells in monolayer.

A bacterial pigment provides cross-species protection from H2O2- and neutrophil-mediated killing.

Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.

Engineering the Soil Bacterium Pseudomonas synxantha 2-79 into a Ratiometric Bioreporter for Phosphorus Limitation.

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25-100 μM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8-8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter's performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts.

Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

Exposure to blue light reduces antimicrobial resistant Pseudomonas aeruginosa isolated from dog ear infections.

Pseudomonas aeruginosa is a leading cause of canine otitis externa. Enrofloxacin is often applied topically to treat this condition, although recalcitrant and recurring infections are common. There is evidence that exposure to blue light (400-470 nm) has a bactericidal effect on P. aeruginosa and other microorganisms. In the present study, we tested the biocidal effect of blue light (375-450 nm), alone or in combination with enrofloxacin, against six isolates of P. aeruginosa from dogs with otitis externa (5 of which were resistant to enrofloxacin). Treatment of planktonic cell cultures with blue light resulted in significant (p < 0.5) reductions in Colony Forming Units (CFU) for all seven strains tested, in some cases below the limit of detection. The greatest bactericidal effect was observed following exposure to light at 405 nm wavelength (p < 0.05). Exposure to blue light for 20 min usually resulted in a greater reduction in Pseudomonas aeruginosa than enrofloxacin treatment, and combination treatment typically resulted in the largest reductions in CFU. Analysis of the genome sequences of these strains established that enrofloxacin resistance was likely the result of a S466F substitution in GyrB. However, there was no clear association between genotype and susceptibility to blue light treatment. These results suggest that blue light treatment, particularly at 405 nm wavelength, and especially in combination with enrofloxacin therapy, could be an effective treatment for otherwise recalcitrant canine otitis externa caused by Pseudomonas aeruginosa. It may also provide a way of extending the usefulness of enrofloxacin therapy which would otherwise be ineffective as a sole therapeutic agent.

Photoinactivation Effects of Curcumin, Nano-curcumin, and Erythrosine on Planktonic and Biofilm Cultures of Streptococcus mutans.

Introduction: This in vitro study was conducted to assess the phototoxic effects of curcumin, nano-curcumin, and erythrosine on the viability of Streptococcus mutans (S. mutans) in suspension and biofilm forms. Methods: Various concentrations of curcumin (1.5 g/L, 3 g/L), nano-curcumin (3 g/L), and erythrosine (100 μM/L, 250 μM/L) were examined for their impact on planktonic and biofilm cultures of S. mutans, either individually or in conjunction with light irradiation (photodynamic therapy or PDT). A blue light-emitting diode (LED) with a central wavelength of 450 nm served as the light source. The results were compared to 0.12% chlorhexidine digluconate (CHX) as the positive control, and a solution containing neither a photosensitizer (PS) nor a light source as the negative control group. The dependent variable was the number of viable microorganisms per experiment (CFU/mL). Results: Antimicrobial PDT caused a significant reduction in the viability of S. mutans in both planktonic and biofilm forms, compared to the negative control group (P<0.05). The highest cell killing was observed in PDT groups with curcumin 3 g/L or erythrosine 250 μmol/L, although the difference with PDT groups using curcumin 1.5 g/L or erythrosine 100 μmol/L was not significant (P>0.05). Antimicrobial treatments were more effective against planktonic S. mutans than the biofilm form. Conclusion: PDT with either curcumin 1.5 g/L or erythrosine 100 μmol/L may be suggested as an alternative to CHX to inactivate the bacteria in dental plaque or deep cavities. Nano-curcumin, at the selected concentration, exhibited lower efficacy in killing S. mutans compared to Curcumin or erythrosine.

Biofilms: A developmental niche for vancomycin-intermediate Staphylococcus aureus

Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 μg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

Acid adaptation alters Streptococcus mutans drug susceptibility profile

The ability of Streptococcus mutans, the primary causative agent of dental caries, to generate and tolerate an acidic environment is a leading factor in its capacity to outcompete other organisms within the oral cavity. The acid tolerance response (ATR) involves coordination of multiple pathways to upregulate macromolecule repair, maintenance of cytosolic pH, and restructuring the membrane. In this study, we asked if the changes associated with adaptation to a low pH environment would result in detectable changes to drug susceptibility. Accordingly, we screened using the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward planktonic cultures of S. mutans or disrupt biofilm formation in growth medium titrated to pH 5 or in the presence of hydrogen peroxide. Results revealed that a total of 182 compounds displayed antimicrobial properties toward the test strain in at least one screening condition. Our findings provide evidence that the S. mutans ATR is characterized by a concerted shift in drug susceptibility. The presence of an intact ATR, associated with growth in continuous culture at pH 5, leads to increased sensitivity to 22 compounds and decreased sensitivity to a separate set of 20 compounds. Disruption of the ATR in a mutant lacking the fabM gene resulted in a susceptibility profile that was distinct from the result of our results. This suggests that loss of unsaturated fatty acids produces a physiological change that is not only measurable, but perhaps more significant than the process of ATR. Additional tests revealed that S. mutans exhibits a distinct drug susceptibility profile compared to a closely related, non-cariogenic bacterium, S. gordonii. Overall, shifts in S. mutans susceptibility patterns based on pH of growth medium or type of culture (batch vs. continuous culture), indicate that the ATR can be tracked using changes in drug susceptibility profile. Furthermore, our results provide clues that compounds could be useful in treatment of dental caries as well as providing molecular tools to study shifts in physiology of disease-associated bacteria.

Phage-Host Interaction Analysis Using Flow Cytometry.

Phage-host interactions are commonly evaluated by culture-based methods. However, these techniques are very laborious and time-consuming. Therefore, other time-efficient, not labor-intensive, and cost-effective methods have been developed.This chapter describes the methodology used to assess the susceptibility of planktonic cultures of bacteria to phage infection and to study their interactions over time by flow cytometry.

The influence of the experimental composition of disinfection preparations based on polyhexamethyleneguanidine and nanoaquachelates of metals on test cultures of microorganisms

When developing a new and effective disinfectant, the existing microbial composition and the ability of microorganisms to form biofilms should be considered. Bacteria in the environment on various surfaces exist in two forms: in the form of planktonic cultures and the form of a formed microbial biofilm. Microorganisms in biofilm are more resistant to factors existing in nature and to the action of various antibacterial compounds. Therefore, when developing disinfectants, it is essential to study the bactericidal effect of newly created agents on bacteria that have formed biofilms. The purpose of the work was to investigate the effect of an experimental composition of a disinfectant based on polyhexamethyleneguanidine (PHMG) and nanoaquachelates of metals on the biofilm-forming properties of microorganisms and to determine the antimicrobial effect of the agent applied to test materials (stainless and galvanized steel, wood, tile). Microbiological studies on the determination of the bactericidal effect of experimental variants of the disinfectant with PHMG and nanoaquachelates of metals were carried out following generally accepted methods. Research on the effect of different disinfectant concentrations on microorganisms in biofilms was carried out according to the methodological recommendations “Determining the bactericidal activity of disinfectants on bacteria in biofilms”. All studies were carried out in triplicate, and the obtained data were subjected to statistical processing. The use of a 2 % solution of the developed disinfectant based on nanoaquachelates of metals and PHMG with an exposure of 40 minutes provides an entirely bactericidal effect on the test cultures of S. aureus, E. coli, and C. albicans in the biofilm. When studying the bactericidal activity of the research composition of the developed disinfectant against museum strains of microorganisms applied to the test materials, it was established that: when using the museum strain of S. aureus applied to galvanized and stainless steel to ensure the bactericidal effect, a concentration of 0.0625 % and 0.0312 % is required for exposures of 20 and 40 minutes. When using wooden and tiled surfaces, a higher concentration of disinfectant is needed, namely 0.125 % and 0.0625 %, for exposures of 20 and 40 minutes; for contamination by the museum strain of E. coli of galvanized and stainless steel, the bactericidal effect was observed at a concentration of 0.0312 % for both 20 min and 40 min. To achieve the bactericidal effect of the agent on a wooden surface within 20 minutes of action, the required concentration of the agent was at least 0.125 %, and after exposure for 40 minutes – 0.0625 %. The bactericidal effect on the tile surface was provided at concentrations of 0.0625 % and 0.0312 % for 20 and 40 min of exposure, respectively; when using the museum strain of C. albicans applied to metal surfaces, the bactericidal effect was observed at disinfectant concentrations of 0.0625 % and 0.0312 % and exposure for 20 and 40 minutes. The tiled surface was disinfected from C. albicans at a composition concentration of 0.0625 % within 20 minutes of exposure, and a wooden surface was disinfected at a composition concentration of 0.125 %.

In Vitro Eradication of Planktonic, Saliva and Biofilm Bacteria Using Lingonberry Extract as a Photosensitizer for Visible Light Plus Water-Filtered Infrared-A Irradiation

Antimicrobial photodynamic treatment (aPDT) with visible light plus water-filtered infrared-A irradiation (VIS-wIRA) and natural single- or multi-component photosensitizers (PSs) was shown to have potent antimicrobial activity. The aim of this study was to obtain information on the antimicrobial effects of aPDT-VIS-wIRA with lingonberry extract (LE) against bacteria that play a role in oral health. Planktonic bacterial cultures of the Gram-positive E. faecalis T9, S. mutans DSM20523, S. oralis ATCC 35037 and S. sobrinus PSM 203513, the Gram-negative N. oralis 14F2 FG-15-7B, F. nucleatum ATCC 25586, and V. parvula DSM, the anaerobic F. nucleatum ATCC 25586 and V. parvula DSM 2008, and the total mixed bacteria from pooled saliva and supra- and subgingival plaques of volunteers were all treated and compared. aPDT-VIS-wIRA with LE as PS significantly (p &lt; 0.008) reduced the growth of all tested Gram-positive, Gram-negative, as well as aerobic and anaerobic bacterial strains, whereas without irradiation no reductions were seen (p &lt; 0.0001). NaCl, with or without irradiation, was ineffective. After treatment with CHX 0.2%, the highest killing rate (100%) was observed, and no bacteria (0 log10 CFU) were cultivable. The method also significantly reduced all of the bacteria present in saliva and in the gingival biofilms. Three-dimensional visualization of viable and non-viable microorganisms revealed that LE penetrated deeper into the cell wall layers than CHX 0.2%. LE was an appropriate PS for eradicating microorganisms with VIS-wIRA, either in their planktonic form or in saliva and gingival plaque biofilms. These results encourage further investigation in order to determine which LE compounds contribute to the photosensitizing effect and to evaluate the size of the effect on maintaining oral health.

Biofilm formation and desiccation survival of Listeria monocytogenes with microbiota on mushroom processing surfaces and the effect of cleaning and disinfection

Microbial multispecies communities consisting of background microbiota and Listeria monocytogenes could be established on materials used in food processing environments. The presence, abundance and diversity of the strains within these microbial multispecies communities may be affected by mutual interactions and differences in resistance towards regular cleaning and disinfection (C&D) procedures. Therefore, this study aimed to characterize the growth and diversity of a L. monocytogenes strain cocktail (n = 6) during biofilm formation on polyvinyl chloride (PVC) and stainless steel (SS) without and with the presence of a diverse set of background microbiota (n = 18). L. monocytogenes and background microbiota strains were isolated from mushroom processing environments and experiments were conducted in simulated mushroom processing environmental conditions using mushroom extract as growth medium and ambient temperature (20 °C) as culturing temperature. The L. monocytogenes strains applied during monospecies biofilm incubation formed biofilms on both PVC and SS coupons, and four cycles of C&D treatment were applied with a chlorinated alkaline cleaning agent and a disinfection agent based on peracetic acid and hydrogen peroxide. After each C&D treatment, the coupons were re-incubated for two days during an incubation period for 8 days in total, and C&D resulted in effective removal of biofilms from SS (reduction of 4.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), while C&D treatments on biofilms formed on PVC resulted in limited reductions (reductions between 1.2 and 2.4 log CFU/cm2, which equals a reduction of 93.7 % and 99.6 %, respectively). Incubation of the L. monocytogenes strains with the microbiota during multispecies biofilm incubation led to the establishment of L. monocytogenes in the biofilm after 48 h incubation with corresponding high L. monocytogenes strain diversity in the multispecies biofilm on SS and PVC. C&D treatments removed L. monocytogenes from multispecies biofilm communities on SS (reduction of 3.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), with varying dominance of microbiota species during different C&D cycles. However, C&D treatments of multispecies biofilm on PVC resulted in lower reductions of L. monocytogenes (between 0.2 and 2.4 log CFU/cm2) compared to single species biofilm, and subsequent regrowth of L. monocytogenes and stable dominance of Enterobacteriaceae and Pseudomonas. In addition, planktonic cultures of L. monocytogenes were deposited and desiccated on dry surfaces without and with the presence of planktonic background microbiota cultures. The observed decline of desiccated cell counts over time was faster on SS compared to PVC. However, the application of C&D resulted in counts below the detection limit of 1.7 log CFU/coupon on both surfaces (reduction of 5.9 log CFU/coupon or less). This study shows that L. monocytogenes is able to form single and multispecies biofilms on PVC with high strain diversity following C&D treatments. This highlights the need to apply more stringent C&D regime treatments for especially PVC and similar surfaces to efficiently remove biofilm cells from food processing surfaces.

Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media

Bacillus Calmette–Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.

The Controversial Effect of Antibiotics on Methicillin-Sensitive S. aureus: A Comparative In Vitro Study.

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include β-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus's ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that β-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that β-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence.