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This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in different media to induce Brucella abortus biofilm formation and planktonic cells, followed by metabolomics and proteomics analyses for these two samples. Significant differential metabolites were identified, followed by KEGG pathway analysis. Differentially expressed proteins were identified, followed by subcellular localization, GO annotation, and KEGG pathway enrichment. Additionally, a correlation analysis of metabolomics and proteomics was performed. Metabolomics analysis showed 7682 positive and 4433 negative metabolites, including 188 positive and 117 negative significant differential metabolites. These differential metabolites were enriched in fatty acid/unsaturated fatty acid biosynthesis and linoleic acid metabolism. Proteomics analysis revealed 1759 proteins, including 486 differentially expressed proteins, which were enriched in various metabolic and degradation-related pathways. Subcellular localization showed that 74.3% of the differential proteins were cytoplasmic proteins. Correlation analysis showed that 1-palmitoyl-2-oleoyl-phosphatidylglycerol had the most significant correlations with proteins, followed by cytosine. Both metabolites correlated with the protein Q57EI7 (RbsB-1, ribose ABC transporter). One common pathway, fatty acid biosynthesis, was identified by both proteomics and metabolomics analyses that involved the metabolites, oleic acid, and protein Q57DK3 (biotin carboxylase). There were metabolomic and proteomic differences between Brucella abortus biofilm and planktonic cells, and these results provide novel insights into the biofilm-forming process of Brucella abortus., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pediococcus pentosaceus, a bacterium recently used in human and animal probiotics, was used in combination with supports made from polylactic acid composite soybean meal was used to study biofilm formation, and it was found that dense biofilms developed by Day 1. Proteomic comparison between planktonic and biofilm cultures of P. pentosaceus showed distinct expression patterns of intracellular and extracellular proteins. Type I glyceraldehyde-3-phosphate dehydrogenase was upregulated in biofilm cultures and mediated cell adhesion and encouraged biofilm production. GMP synthase, which regulates GMP synthesis and acts as an intracellular signal molecule to control cell mechanisms and has been exploited in the development of new therapeutic agents, was also upregulated in the biofilm mode of growth. The present work serves as a basis for future studies examining the complex network of systems that regulate lactic acid bacterial (LAB) biofilm formation and can serve as a framework for studies of production of therapeutic agents from LAB., (© 2020 The Society for Applied Microbiology.)

The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass-function relationships and aid in the development of biofilm control strategies.

Silver nanoparticles were produced with AgF as the starting Ag(I) salt, with pectin as the reductant and protecting agent. While the obtained nanoparticles (pAgNP-F) have the same dimensional and physicochemical properties as those already described by us and obtained from AgNO 3 and pectin (pAgNP-N), the silver nanoparticles from AgF display an increased antibacterial activity against E. coli PHL628 and Staphylococcus epidermidis RP62A ( S. epidermidis RP62A), both as planktonic strains and as their biofilms with respect to pAgNP-N. In particular, a comparison of the antimicrobial and antibiofilm action of pAgNP-F has been carried out with pAgNP-N, pAgNP-N and added NaF, pure AgNO 3 , pure AgF, AgNO 3 and added NaF and pure NaNO 3 and NaF salts. By also measuring the concentration of the Ag + cation released by pAgNP-F and pAgNP-N, we were able to unravel the separate contributions of each potential antibacterial agent, observing an evident synergy between p-AgNP and the F - anion: the F - anion increases the antibacterial power of the p-AgNP solutions even when F - is just 10 µM, a concentration at which F - alone (i.e., as its Na + salt) is completely ineffective.

Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminolevulinic acid (ALA) - named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread infections. The technique combines the action of the porphyrins acting as photosensitisers with light, to produce reactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been reported although variable responses ranging from total eradication to absence of photokilling were found. ALA-PDI conducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteria hinders the optimization of the treatment. The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gram-negatives bacteria growing on planktonic and biofilm cultures and to correlate the degree of the response with the amount and type of porphyrin synthesised. Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and time exposure of ALA, and light dose were set. In both Gram-positive species analysed, a peak of porphyrin synthesis was observed at 1-2 mM ALA in biofilm and planktonic cultures, which fairly correlated with the decrease in the number of CFU after PDI (5 to 7 logs) and porphyrin content was in the same order of magnitude. In addition, ALA-PDI was similarly effective for planktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low light doses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateau of cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longer incubation periods was observed. The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Gram-positive bacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bacteria. Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated high ALA quantities and the amount of porphyrins did not correlate with the degree of photoinactivation. Our microscopy studies show that porphyrins are not located in the envelopes of Gram-negative bacteria, reinforcing the hypothesis that endogenous porphyrins fail to attack these structures., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction. Cryptococcus The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Aim. The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Cryptococcus species. Methodology. Susceptibility testing of darunavir and the common antifungals against 12 members of the Cryptococcus neoformans / Cryptococcus gattii species complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively. Results. Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. All Cryptococcus species strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation ( P <0.05). The combination of darunavir with antifungals caused a significant reduction in biofilm metabolic activity and biomass when compared to darunavir alone ( P <0.05). Conclusion. species and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms of Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24 h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24 h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4 h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24 h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.

Microbial cells can rapidly form biofilm on endotracheal tubes (ETT) causing ventilator-associated pneumonia, a serious complication in patients receiving mechanical ventilation. A novel polyamide with a good balance of hydrophilic/hydrophobic moieties was used for the embedment of green-reduction silver nanoparticles (AgNPs) for the composite-coated ETT. The films were conformal with a thickness of ∼ 17 ± 3 µm accommodating high loading of 60 ± 35 nm spherical-shaped AgNPs. The coated ETT resulted in a significant difference in reducing both planktonic growth and microbial adhesion of single and mixed-species cultures, compared with uncoated ETT ( p  < 0.05). A time-kill assay demonstrated rapid bactericidal effects of the coating on bacterial growth and cell adhesion to ETT surface. Biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus , commonly encountered pathogens, was inhibited by > 96% after incubation for 72 h. Polyamide/AgNP composite-coated ETT provided a broad-spectrum activity against both Gram-positive and Gram-negative bacteria as well as Candida albicans and prolonged antimicrobial activity.

Multifactorial antibacterial action is an important feature of honey; however, its bactericidal efficacy against biofilm-embedded bacteria is limited. The aim of this study was to investigate the impact of vitamin C (Vit C) on the antibacterial activity of natural honeys against planktonic as well as biofilm-embedded bacterial pathogens. The antibacterial activity of four honey samples supplemented with Vit C was expressed as the minimum inhibitory concentration (MIC). At sub-MICs, Vit C significantly increased the antibacterial activity of the tested honeys against Pseudomonas aeruginosa in planktonic cultures. However, after supplementation, honeydew honey, the most active honey, was ineffective against Staphylococcus aureus . On the other hand, when 100% honeydew honey was supplemented with Vit C (100 mg/g of honey) in a multispecies wound biofilm model, complete eradication of almost all bacterial isolates, including S. aureus , was observed. Furthermore, a mixture of honey and Vit C was partially effective against Enterococcus faecalis , whereas honey alone exhibited no antibacterial activity against this bacterium. Vit C counteracted hydrogen peroxide in honey solution and, thus, eliminated the major antibacterial compound present in honey. It is likely that a combination of honey with Vit C may trigger the intracellular production of reactive oxygen species in bacterial cells, but the exact cellular mechanisms warrant further investigations.

Background: Invasive aspergillosis is a fungal infection that occurs mainly in immunocompromised patients. It is responsible for a high degree of mortality and is invariably unresponsive to conventional antifungal treatments. Histone deacetylase inhibitors can affect the cell cycle, apoptosis and differentiation. The histone deacetylase inhibitor vorinostat (SAHA) has recently received approval for the treatment of cutaneous T cell lymphoma. Here, we investigated the interactions of SAHA and itraconazole, voriconazole, and posaconazole against Aspergillus spp. in vitro using both planktonic cells and biofilms., Results: We investigated 20 clinical strains using broth microdilution checkerboard methods. The results showed synergy between SAHA and itraconazole, voriconazole, and posaconazole against 60, 40, and 25% of tested isolates of planktonic Aspergillus spp., respectively. Similar synergy was also observed against Aspergillus biofilms. The expression of the azole-associated multidrug efflux pumps MDR1, MDR2, MDR3 and MDR4, as well as that of HSP90, was measured by RT-PCR. The results indicated that the molecular mechanism of the observed synergistic effects in Aspergillus fumigatus may be partly associated with dampened expression of the efflux pump genes and, furthermore, that HSP90 suppression may be a major contributor to the observed synergistic effects of the drugs., Conclusions: SAHA has potential as a secondary treatment to enhance the effects of azoles against both biofilm and planktonic cells of Aspergillus spp. in vitro. This effect occurs mostly by inhibition of HSP90 expression.

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7 nm, 26.1 mv) showed time- and concentration-dependent activity against MRSA growth, killing ∼ 90% of planktonic bacterial cells in 3 h at 400 μg ml -1 , and completely inhibiting biofilm formation at 1000 μg ml -1 . Moreover, CNPs at 400 μg ml -1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (∼ 25%) and biofilm formation (∼ 50%). The CNPs-bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.

Studying parallel evolution of similar traits in independent within-species lineages provides an opportunity to address evolutionary predictability of molecular changes underlying adaptation. In this study, we monitored biofilm forming capabilities, motility, and virulence phenotypes of a plethora of phylogenetically diverse clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa . We also recorded biofilm-specific and planktonic transcriptional responses. We found that P. aeruginosa isolates could be stratified based on the production of distinct organismal traits. Three major biofilm phenotypes, which shared motility and virulence phenotypes, were produced repeatedly in several isolates, indicating that the phenotypes evolved via parallel or convergent evolution. Of note, while we found a restricted general response to the biofilm environment, the individual groups of biofilm phenotypes reproduced biofilm transcriptional profiles that included the expression of well-known biofilm features, such as surface adhesive structures and extracellular matrix components. Our results provide insights into distinct ways to make a biofilm and indicate that genetic adaptations can modulate multiple pathways for biofilm development that are followed by several independent clinical isolates. Uncovering core regulatory pathways that drive biofilm-associated growth and tolerance towards environmental stressors promises to give clues to host and environmental interactions and could provide useful targets for new clinical interventions., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Sulfate-reducing bacteria (SRB) are key contributors to microbe-induced corrosion (MIC), which can lead to serious economic and environmental impact. The presence of a biofilm significantly increases the MIC rate. Inhibition of the quorum-sensing (QS) system is a promising alternative approach to prevent biofilm formation in various industrial settings, especially considering the significant ecological impact of conventional chemical-based mitigation strategies. In this study, the effect of the QS stimulation and inhibition on Desulfovibrio vulgaris is described in terms of anaerobic respiration, cell activity, biofilm formation, and biocorrosion of carbon steel. All these traits were repressed when bacteria were in contact with QS inhibitors but enhanced upon exposure to QS signal molecules compared to the control. The difference in the treatments was confirmed by transcriptomic analysis performed at different time points after treatment application. Genes related to lactate and pyruvate metabolism, sulfate reduction, electron transfer, and biofilm formation were downregulated upon QS inhibition. In contrast, QS stimulation led to an upregulation of the above-mentioned genes compared to the control. In summary, these results reveal the impact of QS on the activity of D. vulgaris , paving the way toward the prevention of corrosive SRB biofilm formation via QS inhibition. IMPORTANCE Sulfate-reducing bacteria (SRB) are considered key contributors to biocorrosion, particularly in saline environments. Biocorrosion imposes tremendous economic costs, and common approaches to mitigate this problem involve the use of toxic and hazardous chemicals (e.g., chlorine), which raise health and environmental safety concerns. Quorum-sensing inhibitors (QSIs) can be used as an alternative approach to inhibit biofilm formation and biocorrosion. However, this approach would only be effective if SRB rely on QS for the pathways associated with biocorrosion. These pathways would include biofilm formation, electron transfer, and metabolism. This study demonstrates the role of QS in Desulfovibrio vulgaris on the above-mentioned pathways through both phenotypic measurements and transcriptomic approach. The results of this study suggest that QSIs can be used to mitigate SRB-induced corrosion problems in ecologically sensitive areas., (Copyright © 2019 American Society for Microbiology.)

Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC., Competing Interests: The study employed a plasma source which had been developed and patented by terraplasma GmbH. The prime interest of terraplasma was to determine the safety of the plasma source in an independent study, carried out without undue influence by the company so that it provides a valuable documentation. The scientific value of this work lies in the possibility for advancing plasma treatment and providing the quantitative background for this future application, as well as paving the way for a clinical study. In this sense the interest is complementary – there is no competing interest. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. Here, we analyze the impact of PA5oct jumbo phage treatment on planktonic/cell line associated and sessile P. aeruginosa population. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population correlates with reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa . This phenomenon, combined with the loss or modification of the phage receptor, makes most of the phage-resistant clones significantly less pathogenic in in vivo model. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.

During chronic biofilm infections, Pseudomonas aeruginosa bacteria are exposed to increased oxidative stress as a result of the inflammatory response. As reactive oxygen species (ROS) are mutagenic, the evolution of resistance to ciprofloxacin (CIP) in biofilms under oxidative stress conditions was investigated. We experimentally evolved six replicate populations of P. aeruginosa lacking the major catalase KatA in colony biofilms and stationary-phase cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of CIP or without CIP (eight replicate lineages for controls) under aerobic conditions. In CIP-evolved biofilms, a larger CIP-resistant subpopulation was isolated in the ΔkatA strain than in the wild-type (WT) PAO1 population, suggesting oxidative stress as a promoter of the development of antibiotic resistance. A higher number of mutations identified by population sequencing were observed in evolved ΔkatA biofilm populations (CIP and control) than in WT PAO1 populations evolved under the same conditions. Genes involved in iron assimilation were found to be exclusively mutated in CIP-evolved ΔkatA biofilm populations, probably as a defense mechanism against ROS formation resulting from Fenton reactions. Furthermore, a hypermutable lineage due to mutL inactivation developed in one CIP-evolved ΔkatA biofilm lineage. In CIP-evolved biofilms of both the ΔkatA strain and WT PAO1, mutations in nfxB , the negative regulator of the MexCD-OprJ efflux pump, were observed while in CIP-evolved planktonic cultures of both the ΔkatA strain and WT PAO1, mutations in mexR and nalD , regulators of the MexAB-OprM efflux pump, were repeatedly found. In conclusion, these results emphasize the role of oxidative stress as an environmental factor that might increase the development of antibiotic resistance in in vivo biofilms., (Copyright © 2019 American Society for Microbiology.)

The aim of the present work was to study and compare the effect of growth temperature (20, 30, and 37°C) and surface type (stainless steel and polycarbonate) on the production of virulence factors, such as proteases and siderophores, and the risk of surface contamination associated with Pseudomonas aeruginosa biofilm and planktonic cells. The increase of growth temperature from 20 to 37°C increased (approximately twofold) the electronegative charge and the hydrophobicity of the P. aeruginosa biofilm cell surface. P. aeruginosa biofilm cell adhesion to stainless steel and polycarbonate was 5- and 1.5-fold higher than their planktonic counterparts at 20 and 30°C, respectively. The increase of growth temperature from 20 to 37°C increased the production of proteases (twofold) and siderophores (twofold) and the cytotoxicity (up to 30-fold) against the HeLa cell line in the supernatants of P. aeruginosa planktonic and biofilm cultures. This study also highlighted that biofilm and planktonic P. aeruginosa cells exhibited distinct physiological properties with respect to the production of virulence factors and the cytotoxicity against the Hela cell line. Therefore, effective disinfection procedures should be adapted to inactivate bacteria detached from biofilms.

Most antimicrobials currently used in the clinical practice are tested as growth inhibitors against free-floating microorganisms in a liquid suspension, rather than against sessile cells constituting biofilms. Hence, reliable, fast, and reproducible methods for assessing biofilm susceptibility to antimicrobials are strongly needed. Isothermal microcalorimetry (IMC) is a nondestructive sensitive technique that allows for the real-time monitoring of microbial viability in the presence or absence of antimicrobial compounds. Therefore, the efficacy of specific antimicrobials, alone or in combination, may be promptly validated supporting the development of new drugs and avoiding the administration of ineffective therapies. Furthermore, the susceptibility of both planktonic and biofilm cells to antimicrobials can be conveniently assessed without the need for elaborated staining procedures and under nontoxic working conditions. Quantitative data regarding the antimicrobial effect against different strains might be collected by monitoring the microbial cell replication, and, more importantly, a dose-dependent activity can be efficiently detected by measuring the delay and decrease in the heat flow peak of the treated samples. A limitation of IMC for anti-biofilm susceptibility test is the inability to directly quantify the non-replicating cells in the biofilm or the total biomass. However, as IMC is a nondestructive method, the samples can be also analyzed by using different techniques, acquiring more information complementary to calorimetric data. IMC finds application also for the investigation of antibiotic eluting kinetics from different biomaterials, as well as for studying bacteriophages activity against planktonic and biofilm bacteria. Thus, the wide applicability of this ultra-sensitive and automated technique provides a further advance in the field of clinical microbiology and biomedical sciences.

Background: The global threat of antimicrobial resistance especially due to the bacterial biofilms has engaged researchers in the search of new treatment modalities. Antimicrobial photodynamic inactivation (aPDI) is one of the alternative treatment modalities which kills bacteria by generating endogenous reactive oxygen species (ROS). In this work authors evaluated the antibacterial and antibiofilm efficacy of rose Bengal (RB) conjugated to CNT against E. coli., Methods: The interaction of anionic dye to the CNT was studied using UV-vis spectroscopy, HRTEM, FTIR and spectrofluorometry. Phototoxicity of RBCNT conjugate against E. coli was studied using a green light of 50 mW and radiant exposure of 1674.7 J/ cm 2 for 10 min. The antibiofilm activity and mechanism of action of RBCNT conjugate in presence of light was evaluated., Results: The loading and encapsulation was found to be 15.46 ± 1.05 and 61.85 ± 4.23% respectively. The photodynamic inactivation of planktonic cells of E. coli was found to 5.46 and 3.56 log 10 CFU/ml reduction on treatment with RBCNT and RB respectively. The conjugate also exhibited efficient and enhanced antibiofilm activity against E. coli. The study of mechanism of action has confirmed cell membrane and DNA damage were the two main targets of aPDI., Conclusion: This report has concluded the efficient photodynamic inactivation occurred in Gram negative bacteria E. coli due to the increased production of ROS inside the bacterial cells. Hence, the newly synthesized RBCNT conjugate can be efficiently utilized to control infections caused by E. coli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Despite the high success rate of endodontic treatment, failure may occur in some cases. In this case, Enterococcus faecalis is the most common species in endodontic treatment failure and post-treatment apical periodontitis. Therefore, a new adjunctive strategy is needed for the prevention of endodontic infections due to E. faecalis. The aim of the present study was to compare the antimicrobial and anti-biofilm activities of different common photosensitizers (PSs) for use in antimicrobial photodynamic therapy (aPDT) against E. faecalis., Materials and Methods: E. faecalis strain ATCC 29212 was used as the tested strain and methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG), and curcumin (CUR) were used as PSs. Irradiation was carried out using diode laser and light emitting diode (LED) at wavelengths related to the above PSs. Then, antimicrobial and anti-biofilm activities were measured using the microbial viability assay and crystal violet test, respectively., Results: aPDT with using the above PSs significantly decreased the CFU/mL count of E. faecalis compared to the control group (P < 0.05). The killing percentage of E. faecalis via PS mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0% for CUR, ICG, TBO, and MB, respectively. aPDT using the above PSs significantly decreased the biofilm formation ability of E. faecalis compared to the control group (P < 0.05). The biofilm reduction percentage of the PSs was 68.4%, 62.9%, 59.0%, and 47.6% for CUR, ICG, TBO, and MB, respectively., Conclusion: CUR and ICG mediated aPDT exhibited considerably more antimicrobial activity than other PSs, while TBO and MB demonstrated weaker anti-biofilm effects against E. faecalis compared to other PSs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The role of genes that are essential for development of Staphylococcus aureus biofilm during infection is not fully known. mRNA from two methicillin-resistant S. aureus strains that formed weak and strong biofilm on polystyrene plates were isolated at five time points from cells grown in biofilm and planktonic culture. Quantitative real-time PCR analysis showed that the expression levels of investigated genes under biofilm conditions were significantly higher than under planktonic conditions. The expression levels of the gene encoding elastin binding protein ( ebps ) and laminin binding protein ( eno ) were significantly increased in biofilm at 3 h, both in strongly and weakly adhering strain. The peak expression of fib gene encoding fibrinogen binding protein was found at 6 and 8 h in the case of strongly and weakly adhering strain, respectively. The expression of icaA and icaD genes in both strains was significantly higher under biofilm conditions when comparing to planktonic cells during 12 h. The expression level of the genes encoding binding proteins and the glucosamine polymer polysaccharide intercellular adhesin (PIA) slowly decreased after 24 h. Finally, we found that the expression levels of genes encoding binding factors in weakly adhering strain were significantly lower than in strongly adhering strain.

Objectives: Mupirocin is a useful antibiotic against superficial skin infections. We compared the impact of mupirocin with a cephalosporin, a fluoroquinolone, an aminoglycoside, and a macrolide on planktonic cell growth and biofilm formation of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA)., Materials and Methods: Minimum inhibitory concentration (MIC) of mupirocin was determined against S. aureus strains used in this study. Biofilm formation of S. aureus strains exposed to mupirocin was quantified by crystal violet staining assay. Moreover, biofilm structure and viability of the biofilm cells were visualized by Live/Dead staining assay. Biofilm-related gene expression was investigated by quantitative real-time PCR., Results: MRSA USA300 clone was resistant to mupirocin with MIC of 1,024 mg/L, while MRSA ATCC-43300 and MSSA ATCC-29213 were susceptible with MICs of 0.03 mg/L. Planktonic cell growth of the S. aureus strains was inhibited by mupirocin in a dose-dependent manner. However, some of the low concentrations of mupirocin less than the MICs promoted biofilm formation. Confocal laser scanning microscopy of the biofilm structures and cell viabilities showed established biofilms of slightly higher cell density in the mupirocin treated groups, especially in the MRSA USA300 clone. Gene expression of RNAIII in planktonic cells and biofilms of MRSA USA300 clone showed the highest upregulation after initial exposure to sub-MIC of mupirocin followed by downregulation, whereas the other antibiotics showed various fluctuations., Conclusion: The results showed that subinhibitory concentrations of mupirocin promoted biofilm formation of S. aureus, in particular the MRSA USA300 clone.

Bacterial biofilms can enhance survival in adverse environments and promote infection. However, little is known about biofilm formation by Clostridium perfringens. To better characterize this process, we used SEM to observe the surfaces of C. perfringens biofilms after 12, 24, 48, and 72 h of incubation. Biofilm cells appeared to be encased in a dense matrix material, and the total biomass of the biofilm increased with incubation time. To gain insight into the differentially expressed genes (DEGs) between biofilm and planktonic cells, we carried out comparative transcriptomic analysis using RNA sequencing. In total, 91 genes were significantly differentially expressed, with 40 being up-regulated and 51 down-regulated. In particular, genes encoding sortase, ribosomal proteins, and ATP synthase were up-regulated in biofilms, while genes coding for clostripain and phospholipase C were down-regulated. To validate the RNA sequencing results, qRT-PCR analysis was performed using five randomly selected DEGs. Results showed that all five genes were up-regulated, which was in accordance with the RNA sequencing results. To examine the functional differences, the DEGs were characterized by GO and KEGG pathway enrichment analyses. Results showed that the up-regulated genes were divided into 32 significantly enriched GO terms, with "macromolecular complex" being the most common. Oxidative phosphorylation was the only significantly enriched pathway, suggesting that ATP is required for biofilm stability. This study provides valuable insights into the morphology and transcriptional regulation of C. perfringens during biofilm formation, and will be useful for understanding and developing biofilm-based processes.

Staphylococcus aureus is a leading cause of device-associated biofilm infections, which represent a serious health care concern based on their chronicity and antibiotic resistance. We previously reported that S. aureus biofilms preferentially recruit myeloid-derived suppressor cells (MDSCs), which promote monocyte and macrophage anti-inflammatory properties. This is associated with increased myeloid arginase-1 (Arg-1) expression, which has been linked to anti-inflammatory and profibrotic activities that are observed during S. aureus biofilm infections. To determine whether MDSCs and macrophages utilize Arg-1 to promote biofilm infection, Arg-1 was deleted in myeloid cells by use of Tie-2 Cre mice. Despite Arg-1 expression in biofilm-associated myeloid cells, bacterial burdens and leukocyte infiltrates were similar between wild-type (WT) and Arg-1 fl/fl ; Tie-2 Cre conditional knockout (KO) mice from days 3 to 14 postinfection in both orthopedic implant and catheter-associated biofilm models. However, inducible nitric oxide synthase (iNOS) expression was dramatically elevated in biofilm-associated MDSCs from Arg-1 fl/fl ; Tie-2 Cre animals, suggesting a potential Arg-1-independent compensatory mechanism for MDSC-mediated immunomodulation. Treatment of Arg-1 fl/fl ; Tie-2 Cre mice with the iNOS inhibitor N6-(1-iminoethyl)-l-lysine (l-NIL) had no effect on biofilm burdens or immune infiltrates, whereas treatment of WT mice with the Arg-1/ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) increased bacterial titers, but only in the surrounding soft tissues, which possess attributes of a planktonic environment. A role for myeloid-derived Arg-1 in regulating planktonic infection was confirmed using a subcutaneous abscess model, in which S. aureus burdens were significantly increased in Arg-1 fl/fl ; Tie-2 Cre mice compared to those in WT mice. Collectively, these results indicate that the effects of myeloid Arg-1 are context dependent and are manifest during planktonic but not biofilm infection., (Copyright © 2018 American Society for Microbiology.)

Listeria monocytogenes is a food-borne pathogen that can grow as a biofilm on surfaces. Biofilm formation in food-processing environments is a big concern for food safety, as it can cause product contamination through the food-processing line. Although motile aerobic bacteria have been described to form biofilms at the air-liquid interface of cell cultures, to our knowledge, this type of biofilm has not been described in L. monocytogenes before. In this study we report L. monocytogenes biofilm formation at the air-liquid interface of aerobically grown cultures, and that this phenotype is specifically induced when the media is supplemented with glycerol as a carbon and energy source. Planktonic growth, metabolic activity assays and HPLC measurements of glycerol consumption over time showed that glycerol utilization in L. monocytogenes is restricted to growth under aerobic conditions. Gene expression analysis showed that genes encoding the glycerol transporter GlpF, the glycerol kinase GlpK and the glycerol 3-phosphate dehydrogenase GlpD were upregulated in the presence of oxygen, and downregulated in absence of oxygen. Additionally, motility assays revealed the induction of aerotaxis in the presence of glycerol. Our results demonstrate that the formation of biofilms at the air-liquid interface is dependent on glycerol-induced aerotaxis towards the surface of the culture, where L. monocytogenes has access to higher concentrations of oxygen, and is therefore able to utilize this compound as a carbon source., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.

The use of nitric oxide (NO), a naturally occurring antimicrobial agent, as an alternative strategy to combat bacterial biofilms has recently gained considerable momentum in light of the global threat of emerging antibiotic resistance. While previous NO-based anti-biofilm approaches were aimed at killing bacterial cells within biofilms, NO has also been recently identified as a key mediator of biofilm dispersal, causing the release of cells from the biofilm community. This is of great interest towards the design of more effective anti-biofilm strategies but further studies are warranted to validate this concept. Therefore, in the present study we investigated whether a NO precursor, isosorbide mononitrate (ISMN) or its analogue D-isosorbide can induce bacteria cell dispersal from Staphylococcus aureus (S. aureus) biofilms and explored the potential synergy of ISMN and the antimicrobial compounds mupirocin and ciprofloxacin in biofilm eradication. This study demonstrate that ISMN causes dispersal of S. aureus biofilm bacteria, particularly when exposed to high levels of drug. ISMN at 60mg/mL increased the number of colony forming units (CFU) (~3log 10 and ~5log 10 ) of planktonic bacteria after 6 and 24-h exposure respectively, compared to control biofilms. This suggests that ISMN induces the transition of sessile biofilm cells to free-swimming planktonic cells. In addition, ISMN exhibits synergistic effects against S. aureus biofilms with ciprofloxacin when tested above its minimum inhibitory concentration (MIC). Specifically, exposure to ISMN significantly enhanced the efficacy of ciprofloxacin by reducing the number of CFU (~3log 10 or ~2log 10 ) of biofilm-associated and planktonic bacteria respectively, compared to drug alone. Combined exposure to both ISMN and certain antimicrobial agents may therefore offer an innovative approach to control persistent biofilm and biofilm-associated infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is unknown whether bacterioplankton and biofilm communities are structured by the same ecological processes, and whether they influence each other through continuous dispersal (known as mass effects). Using a hierarchical sampling approach we compared the relative importance of ecological processes structuring the dominant fraction (relative abundance ≥0.1%) of bacterioplankton and biofilm communities from three microhabitats (open water, Nuphar and Phragmites sites) at within- and among-pond scale in a set of 14 interconnected shallow ponds. Our results demonstrate that while bacterioplankton and biofilm communities are highly distinct, a similar hierarchy of ecological processes is acting on them. For both community types, most variation in community composition was determined by pond identity and environmental variables, with no effect of space. The highest β-diversity within each community type was observed among ponds, while microhabitat type (Nuphar, Phragmites, open water) significantly influenced biofilm communities but not bacterioplankton. Mass effects among bacterioplankton and biofilm communities were not detected, as suggested by the absence of within-site covariation of biofilm and bacterioplankton communities. Both biofilm and plankton communities were thus highly structured by environmental factors (i.e., species sorting), with among-lake variation being more important than within-lake variation, whereas dispersal limitation and mass effects were not observed., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cellular functions and responses to external stimuli, but this analysis remains challenging because of various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition of E. faecalis grown as planktonic bacteria and biofilm by developing "stable isotope labeling by amino acids in bacterial culture" (SILAB), optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was performed by labeling E. faecalis in biofilm with heavy Lys (l-[ 13 C 6 , 2 D 9 , 15 N 2 ]Lys) and planktonic bacteria with natural abundance l-Lys, then mixing equal amounts of bacteria from each condition, and performing cell wall isolation and mutanolysin digestion necessary for liquid chromatography and mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy Lys isotopic enrichment (98.33 ± 0.05%) and incorporation (83.23 ± 1.16%). Forty-seven pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for SILAB analysis. We found that the PG in biofilm showed an increased level of PG cross-linking, an increased level of N-deacetylation of GlcNAc, a decreased level of O-acetylation of MurNAc, and an increased number of stem modifications by d,d- and l,d-carboxypeptidases.

A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa , biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors., (Copyright © 2018 American Society for Microbiology.)

Here, we investigated novel interactions of three global regulators of the network that controls biofilm formation in the model bacterium Escherichia coli using computational network analysis, an in vivo reporter assay and physiological validation experiments. We were able to map critical nodes that govern planktonic to biofilm transition and identify 8 new regulatory interactions for CRP, IHF or Fis responsible for the control of the promoters of rpoS, rpoE, flhD, fliA, csgD and yeaJ. Additionally, an in vivo promoter reporter assay and motility analysis revealed a key role for IHF as a repressor of cell motility through the control of FliA sigma factor expression. This investigation of first stage and mature biofilm formation indicates that biofilm structure is strongly affected by IHF and Fis, while CRP seems to provide a fine-tuning mechanism. Taken together, the analysis presented here shows the utility of combining computational and experimental approaches to generate a deeper understanding of the biofilm formation process in bacteria.

Electron transfer is a rate-limiting step in microbiologically influenced corrosion (MIC) caused by microbes that utilize extracellular electrons. Cross-cell wall electron transfer is necessary to transport the electrons released from extracellular iron oxidation into the cytoplasm of cells. Electron transfer mediators were found to accelerate the MIC caused by sulfate reducing bacteria. However, there is no publication in the literature showing the effect of electron transfer mediators on MIC caused by nitrate reducing bacteria (NRB). This work demonstrated that the corrosion of anaerobic Pseudomonas aeruginosa (PAO1) grown as a nitrate reducing bacterium biofilm on C1018 carbon steel was enhanced by two electron transfer mediators, riboflavin and flavin adenine dinucleotide (FAD) separately during a 7-day incubation period. The addition of either 10ppm (w/w) (26.6μM) riboflavin or 10ppm (12.7μM) FAD did not increase planktonic cell counts, but they increased the maximum pit depth on carbon steel coupons considerably from 17.5μm to 24.4μm and 25.0μm, respectively. Riboflavin and FAD also increased the specific weight loss of carbon steel from 2.06mg/cm 2 to 2.34mg/cm 2 and 2.61mg/cm 2 , respectively. Linear polarization resistance, electrochemical impedance spectroscopy and potentiodynamic polarization curves all corroborated the pitting and weight loss data., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: To identify, isolate, and characterize a lytic bacteriophage against the multiple-drug resistant clinical strain of Pseudomonas aeruginosa-2995 and to determine the phage efficacy against the bacterial planktonic cells and the biofilm., Main Methods: Wastewater was used to isolate a bacteriophage. The phage was characterized with Transmission electron microscopy (TEM). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to identify the expressed proteins. Bacteria were cultured in both suspension and biofilm to check and compare their susceptibility to phage lytic action. The activity of the phage (determined as AZ1) was determined against P. aeruginosa-2995 in both planktonic cells and the biofilm., Key Findings: A bacteriophage, designated as AZ1, was isolated from waste water showing a narrow host range. AZ1 was characterized by TEM and could be identified as an isolate in the family Siphoviridae [order Caudovirals]. Seventeen structural proteins ranging from about 12 to 110kDa were found through SDS-PAGE analysis. Its genome was confirmed as dsDNA with a length of approx. 50kb. The log-phase growth of P. aeruginosa-2995 was significantly reduced after treatment with AZ1 (4.50×10 8 to 2.1×10 3 CFU/ml) as compared to control. Furthermore, phage AZ1 significantly reduced 48h old biofilm biomass about 3-fold as compared to control., Significance: Pseudomonas aeruginosa is a ubiquitous free-living opportunistic human pathogen characterized by high antibiotic tolerance and tendency for biofilm formation. The phage, identified in this study, AZ1, showed promising activity in the destruction of both planktonic cells and biofilm of P. aeruginosa-2995. However, complete eradication may require a combination of phages., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen and forms biofilm easily. Bacteria inside biofilms display an increased resistance to antibiotics and disinfectants. The objective of the current study was to assess the antimicrobial activities of emodin, 1,2,8-trihydroxy-6-methylanthraquinone, an anthraquinone derivative isolated from Polygonum cuspidatum and Rheum palmatum, against S. aureus CMCC26003 grown in planktonic and biofilm cultures in vitro. In addition, a possible synergistic effect between emodin and berberine chloride was evaluated. As quantified by crystal violet method, emodin significantly decreased S. aureus biofilm growth in a dose-dependent manner. The above findings were further supported by scanning electron microscopy. Moreover, the present study demonstrated that sub-MICs emodin obviously intervened the release of extracellular DNA and inhibited expression of the biofilm-related genes (cidA, icaA, dltB, agrA, sortaseA and sarA) by real-time RT-PCR. These results revealed a promising application for emodin as a therapeutic agent and an effective strategy to prevent S. aureus biofilm-related infections.

Candida spp. adhere to medical devices, such as catheters, forming drug-tolerant biofilms that resist killing by the immune system. Little is known about how C. glabrata, an emerging pathogen, resists attack by phagocytes. Here we show that upon encounter with planktonic (non-biofilm) C. glabrata, human neutrophils initially phagocytose the yeast and subsequently release neutrophil extracellular traps (NETs), complexes of DNA, histones, and proteins capable of inhibiting fungal growth and dissemination. When exposed to C. glabrata biofilms, neutrophils also release NETs, but significantly fewer than in response to planktonic cells. Impaired killing of biofilm parallels the decrease in NET production. Compared to biofilm, neutrophils generate higher levels of reactive oxygen species (ROS) when presented with planktonic organisms, and pharmacologic inhibition of NADPH-oxidase partially impairs NET production. In contrast, inhibition of phagocytosis nearly completely blocks NET release to both biofilm and planktonic organisms. Imaging of the host response to C. glabrata in a rat vascular model of infection supports a role for NET release in vivo. Taken together, these findings show that C. glabrata triggers NET release. The diminished NET response to C. glabrata biofilms likely contributes to the resilience of these structured communities to host defenses.

Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3'-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with β-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following β-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (β-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks β-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of β-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the β-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP) and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST) combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and tachyplesin I linear analogue (TP-I-L) was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxaemias in animal species. Recently, C. perfringens was shown to form biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. However, very little is known on the subject and no information is available on gene expression in C. perfringens biofilms. To gain insights into the differences between free-living C. perfringens cells and those in biofilms, we used RNA sequencing. In total, 25.7% of genes showed differential expression in the two growth modes; about 12.8% of genes were up-regulated and about 12.9% were down-regulated in biofilms. We show that 772 genes were significantly differentially expressed between biofilms and planktonic cells from the supernatant of biofilms. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in virulence, energy production, amino acid, nucleotide and carbohydrate metabolism, and in translation and ribosomal structure. Genes up-regulated in biofilm cells were mainly involved in amino acid and carbohydrate metabolism, transcription, inorganic ion metabolism and in defence mechanisms. This study provides new insights into the transcriptomic response of C. perfringens during biofilm formation.

Social interactions in which bacteria respond to one another by modifying their phenotype are central determinants of microbial communities. It is known that interspecific interactions influence the biofilm phenotype of bacteria; a phenotype that is central to the fitness of bacteria. However, the underlying role of fundamental ecological factors, specifically coexistence and phylogenetic history, in biofilm formation remains unclear. This study examines how social interactions affect biofilm formation in multi-species co-cultures from five diverse environments. We found prevalence of increased biofilm formation among co-cultured bacteria that have coexisted in their original environment. Conversely, when randomly co-culturing bacteria across these five consortia, we found less biofilm induction and a prevalence of biofilm reduction. Reduction in biofilm formation was even more predominant when co-culturing bacteria from environments where long-term coexistence was unlikely to have occurred. Phylogenetic diversity was not found to be a strong underlying factor but a relation between biofilm induction and phylogenetic history was found. The data indicates that biofilm reduction is typically correlated with an increase in planktonic cell numbers, thus implying a behavioral response rather than mere growth competition. Our findings suggest that an increase in biofilm formation is a common adaptive response to long-term coexistence., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Aim: Study the effect of water temperature from 40 to 70 degrees C on viability of plankton forms and model Legionella pneumophila under experimental conditions., Materials and Methods: Monospecies legionella biofilms, obtained in plates for enzyme immunoassay during 96 hours at 28 degrees C, and water suspension of BCYE agar cultivated cells of L. pneumophila at a concentration of 10(3) - 10(5) CFU per liter were used in the study for evaluation of bactericidal effect of temperature on various legionella forms., Results: Analysis of effects of various temperature regimens on plankton forms and model legionella biofilms has shown that at a temperature range from 50 to 60 degrees C a significant reduction of quantity of viable legionella cells occurs. Model legionella biofilms have partially conserved viability at a temperature of 60 degrees C and only exposition to a temperature of 70 degrees C resulted in death of legionella biofilms and plankton forms of bacteria. A dependence of viability conservation of legionella from the initial concentration of the causative agent in water and duration of exposition at varying temperature was shown., Conclusion: Short-term heating at a temperature of at least 70 degrees C has the most pronounced bactericidal effect on plankton forms and model L. pneumophila biofilms under experimental conditions. Such temperature regimen could be used as one of the prophylaxis approaches during maintenance of especially dangerous water system and, fist of all, systems of hot water supply.

Carbon steels are widely used in the oil and gas industry from downhole tubing to transport trunk lines. Microbes form biofilms, some of which cause the so-called microbiologically influenced corrosion (MIC) of carbon steels. MIC by sulfate reducing bacteria (SRB) is often a leading cause in MIC failures. Electrogenic SRB sessile cells harvest extracellular electrons from elemental iron oxidation for energy production in their metabolism. A previous study suggested that electron mediators riboflavin and flavin adenine dinucleotide (FAD) both accelerated the MIC of 304 stainless steel by the Desulfovibrio vulgaris biofilm that is a corrosive SRB biofilm. Compared with stainless steels, carbon steels are usually far more prone to SRB attacks because SRB biofilms form much denser biofilms on carbon steel surfaces with a sessile cell density that is two orders of magnitude higher. In this work, C1018 carbon steel coupons were used in tests of MIC by D. vulgaris with and without an electron mediator. Experimental weight loss and pit depth data conclusively confirmed that both riboflavin and FAD were able to accelerate D. vulgaris attack against the carbon steel considerably. It has important implications in MIC failure analysis and MIC mitigation in the oil and gas industry.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest., (© 2014 SETAC.)

Bacterial respiration is an essential driving force in biogeochemical cycling and bioremediation processes. Electron acceptors respired by bacteria often have solid and soluble forms that typically coexist in the environment. It is important to understand how sessile bacteria attached to solid electron acceptors respond to ambient soluble alternative electron acceptors. Microbial fuel cells (MFCs) provide a useful tool to investigate this interaction. In MFCs with Shewanella decolorationis, azo dye was used as an alternative electron acceptor in the anode chamber. Different respiration patterns were observed for biofilm and planktonic cells, with planktonic cells preferred to respire with azo dye while biofilm cells respired with both the anode and azo dye. The additional azo respiration dissipated the proton accumulation within the anode biofilm. There was a large redox potential gap between the biofilms and anode surface. Changing cathodic conditions caused immediate effects on the anode potential but not on the biofilm potential. Biofilm viability showed an inverse and respiration-dependent profile when respiring with only the anode or azo dye and was enhanced when respiring with both simultaneously. These results provide new insights into the bacterial respiration strategies in environments containing multiple electron acceptors and support an electron-hopping mechanism within Shewanella electrode-respiring biofilms.

Although free-swimming planktonic bacteria historically have been the typical focus of microbiological studies, the natural state of many or most bacteria is one where they instead are associated with surfaces and/or each other. For many pathogenic as well as nuisance bacteria, including biofouling bacteria, it consequently is within the context of this biofilm state that antibacterial strategies must be implemented. For reasons that are not fully understood, however, biofilm-associated bacteria tend to be less susceptible to treatments with standard chemical antibacterial agents than are planktonic bacteria, and this appears to be especially an issue with the use of less-harsh agents such as antibiotics. Within a variety of contexts the development of less- or selectively toxic antibacterial agents capable of clearing biofilms therefore would be welcome. In this review we consider the use of three categories of such agents as anti-biofilm antibacterials. These are lytic viruses of bacteria, that is, bacteriophages, effecting phage-mediated biocontrol of bacteria (a.k.a., phage therapy); purified phage-encoded enzymes that digest bacterial cell-wall material (endolysins or simply lysins); and a second category of phage-encoded enzymes that digest the extracellular polymeric substance (EPS) that are particularly notable components of bacterial biofilms (EPS depolymerases). These agents have been shown to reduce the bacterial density of a diversity of biofilms and, in many cases, tend to be lacking in inherent toxicity against the tissues of animals. Here we consider these phage-based anti-biofilm strategies with emphasis on ecological aspects of their action and with particular consideration of EPS depolymerases.

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG1 and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.

This study evaluated the effectiveness of selected disinfectants against bacterial cells within a biofilm using flow cytometry, the conventional total viable count test and scanning electron microscopy (SEM). A flow cytometric procedure based on measurement of the cellular redox potential (CRP) was demonstrated to have potential for the rapid evaluation of activity against biofilm and planktonic forms of microbes. Quaternary ammonium compound-based disinfectant (QACB) demonstrated a higher level of anti-microbial activity than a performic acid preparation (PAP), with mean CRP values against P. aeruginosa cells of 2 and 1.33 relative fluorescence units (RFU) vs 63.33 and 61.33 RFU for 8 and 24 h cultures respectively. Flow cytometric evaluation of the anti-biofilm activity demonstrated a higher efficacy of QACB compared to PAP for P. aeruginosa cells of 1 and 0.66 RFU vs 18.33 and 22.66 RFU for 8 and 24 h cultures respectively. SEM images of treated P. aeruginosa cells demonstrated disinfectant-specific effects on cell morphology.

Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.

Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.

Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.