Multispecies Biofilm Research Articles

Exploring Nitrogen-Functionalized Graphene Composites for Urinary Catheter Applications.

Graphene has been broadly studied, particularly for the fabrication of biomedical devices, owing to its physicochemical and antimicrobial properties. In this study, the antibiofilm efficacy of graphene nanoplatelet (GNP)-based composites as coatings for urinary catheters (UCs) was investigated. GNPs were functionalized with nitrogen (N-GNP) and incorporated into a polydimethylsiloxane (PDMS) matrix. The resulting materials were characterized, and the N-GNP/PDMS composite was evaluated against single- and multi-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Both biofilm cell composition and structure were analyzed. Furthermore, the antibacterial mechanisms of action of N-GNP were explored. The N-GNP/PDMS composite showed increased hydrophobicity and roughness compared to PDMS. In single-species biofilms, this composite significantly reduced the number of S. aureus, P. aeruginosa, and K. pneumoniae cells (by 64, 41, and 29%, respectively), and decreased S. aureus biofilm culturability (by 50%). In tri-species biofilms, a 41% reduction in total cells was observed. These results are aligned with the outcomes of the biofilm structure analysis. Moreover, N-GNP caused changes in membrane permeability and triggered reactive oxygen species (ROS) synthesis in S. aureus, whereas in Gram-negative bacteria, it only induced changes in cell metabolism. Overall, the N-GNP/PDMS composite inhibited biofilm development, showing the potential of these carbon materials as coatings for UCs.

A mini-review on indigenous microbial biofilm from various wastewater for heavy-metal removal - new trends.

Biofilm, as a form of the microbial community in nature, represents an evolutionary adaptation to the influence of various environmental conditions. In nature, the largest number of microorganisms occur in the form of multispecies biofilms. The ability of microorganisms to form a biofilm is one of the reasons for antibiotic resistance. The creation of biofilms resistant to various contaminants, on the other hand, improves the biological treatment process in wastewater treatment plants. Heavy metals cannot be degraded, but they can be transformed into non-reactive and less toxic forms. In this process, microorganisms are irreplaceable as they interact with the metals in a variety of ways. The environment polluted by heavy metals, such as wastewater, is also a source of undiscovered microbial diversity and specific microbial strains. Numerous studies show that biofilm is an irreplaceable strategy for heavy metal removal. In this review, we systematize recent findings regarding the bioremediation potential of biofilm-forming microbial species isolated from diverse wastewaters for heavy metal removal. In addition, we include some mechanisms of action, application possibilities, practical issues, and future prospects.

Killing of a Multispecies Biofilm Using Gram-Negative and Gram-Positive Targeted Antibiotic Released from High Purity Calcium Sulfate Beads.

Multispecies biofilm orthopedic infections are more challenging to treat than mono-species infections. In this in-vitro study, we aimed to determine if a multispecies biofilm, consisting of Gram positive and negative species with different antibiotic susceptibilities could be treated more effectively using high purity antibiotic-loaded calcium sulfate beads (HP-ALCSB) containing vancomycin (VAN) and tobramycin (TOB) in combination than alone. Three sets of species pairs from bioluminescent strains of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) and clinical isolates, Enterococcus faecalis (EF) and Enterobacter cloacae were screened for compatibility. PA + EF developed intermixed biofilms with similar cell concentrations and so were grown on 316L stainless steel coupons for 72 h or as 24 h agar lawn biofilms and then treated with HP-ALCSBs with single or combination antibiotics and assessed by viable count or bioluminescence and light imaging to distinguish each species. Replica plating was used to assess viability. The VAN + TOB bead significantly reduced the PA + EF biofilm CFU and reduced the concentration of surviving antibiotic tolerant variants by 50% compared to single antibiotics. The combination of Gram-negative and positive targeted antibiotics released from HP-ALCSBs may be more effective in treating multispecies biofilms than monotherapy alone.

Effect of ginger essential oil and 6-gingerol on a multispecies biofilm of Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas aeruginosa.

The objective of this study was to evaluate the potential antimicrobial and antibiofilm effect of ginger essential oil (GEO) and 6-gingerol on a multispecies biofilm formed by Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas aeruginosa on a polypropylene surface. The minimum inhibitory concentration concentrations obtained for GEO were 100 and 50mg/mL and for 6-gingerol 1.25mg/mL. Sessile cell counts ranged within 5.35-7.35 log CFU/cm2 in the control biofilm, with the highest sessile growth at 72h. GEO treatments acted on the total population regardless of concentration at 1 and 48h. L. monocytogenes behaved similarly to the total population, showing GEO action at 1h and keeping the same pattern at 48, 72, and 96h. Better action on S. Typhimurium was obtained at times of 1, 72, and 96h. P. aeruginosa showed logarithmic reduction only when treated with GEO 50mg at 24h. As for 6-gingerol, in general, there was no significant action (p > 0.05) on the evaluated sessile cells. GEO showed antimicrobial activity againstL. monocytogenes, S. Typhimurium, and P. aeruginosa, acting as an inhibitor of biofilm formation. As for 6-gingerol, it was considered a possible antimicrobial agent but without efficacy during biofilm formation.

NaCl-induced modulation of species distribution in a mixed P. aeruginosa / S. aureus /B.cepacia biofilm

Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections.In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations.Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix.Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Pseudomonas aeruginosa increases the susceptibility of Candida albicans to amphotericin B in dual-species biofilms.

Biofilms are the leading cause of nosocomial infections and are hard to eradicate due to their inherent antimicrobial resistance. Candida albicans is the leading cause of nosocomial fungal infections and is frequently co-isolated with the bacterium Pseudomonas aeruginosa from biofilms in the cystic fibrosis lung and severe burn wounds. The presence of C. albicans in multispecies biofilms is associated with enhanced antibacterial resistance, which is largely mediated through fungal extracellular carbohydrates sequestering the antibiotics. However, significantly less is known regarding the impact of polymicrobial biofilms on antifungal resistance. Here we show that, in dual-species biofilms, P. aeruginosa enhances the susceptibility of C. albicans to amphotericin B, an effect that was biofilm specific. Transcriptional analysis combined with gene ontology enrichment analysis identified several C. albicans processes associated with oxidative stress to be differentially regulated in dual-species biofilms, suggesting that P. aeruginosa exerts oxidative stress on C. albicans, likely through the secretion of phenazines. However, the mitochondrial superoxide dismutase SOD2 was significantly down-regulated in the presence of P. aeruginosa. Monospecies biofilms of the sod2Δ mutant were more susceptible to amphotericin B, and the susceptibility of these biofilms was further enhanced by exogenous phenazines. We propose that in dual-species biofilms, P. aeruginosa simultaneously induces mitochondrial oxidative stress, while down-regulating key detoxification enzymes, which prevents C. albicans mounting an appropriate oxidative stress response to amphotericin B, leading to fungal cell death. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility.

Lactobacillus acidophilus impairs the establishment of pathogens in a subgingival multispecies biofilm

The present study evaluated the antibiofilm effects of Lactobacillus acidophilus within a subgingival multispecies biofilm. Lactobacillus acidophilus (La5) at 1 × 102, 1 × 104, and 1 × 106 were included at the beginning of biofilm formation, which lasted 7 days. The biofilms comprised 33 periodontitis-related bacterial species and the Calgary Biofilm device was used. At the end, DNA–DNA hybridization (checkerboard) was performed. A Kruskal–Wallis test followed by a Dunn post hoc test were performed (p ≤ 0.05). La5 at 1 × 104 and 1 × 106 reduced the total counts of biofilm and the proportions of red and green complexes when compared to the control biofilm without La5 (p ≤ 0.05). La5 at 1 × 104 increased the proportions of Actinomyces complex compared to the controls (p ≤ 0.05). Both La5 at 1 × 104 and 1 × 106 decreased levels of 20 and 14 distinct species, respectively, including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum polymorphum, and Parvimonas micra compared to the control (p ≤ 0.05). Only La5 at 1 × 104 reduced the levels of Tannerella forsythia, Fusobacterium periodonticum, and Aggregatibacter actinomycetencomytans compared to the control (p ≤ 0.05). L. acidophilus inhibited establishing periodontic pathogens from red complex such as P. gingivalis and T. forsythia in a subgingival multispecies biofilm.

Anticariogenic Activity of Celastrol and Its Enhancement of Streptococcal Antagonism in Multispecies Biofilm.

Dental caries is a chronic disease resulting from dysbiosis in the oral microbiome. Antagonism of commensal Streptococcus sanguinis and Streptococcus gordonii against cariogenic Streptococcus mutans is pivotal to keep the microecological balance. However, concerns are growing on antimicrobial agents in anticaries therapy, for broad spectrum antimicrobials may have a profound impact on the oral microbial community, especially on commensals. Here, we report celastrol, extracted from Traditional Chinese Medicine's Tripterygium wilfordii (TW) plant, as a promising anticaries candidate. Our results revealed that celastrol showed antibacterial and antibiofilm activity against cariogenic bacteria S. mutans while exhibiting low cytotoxicity. By using a multispecies biofilm formed by S. mutans UA159, S. sanguinis SK36, and S. gordonii DL1, we observed that even at relatively low concentrations, celastrol reduced S. mutans proportion and thereby inhibited lactic acid production as well as water-insoluble glucan formation. We found that celastrol thwarted S. mutans outgrowth through the activation of pyruvate oxidase (SpxB) and H2O2-dependent antagonism between commensal oral streptococci and S. mutans. Our data reveal new anticaries properties of celastrol that enhance oral streptococcal antagonism, which thwarts S. mutans outgrowth, indicating its potential to maintain oral microbial balance for prospective anticaries therapy.

Chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion enhances photodynamic therapy against multi-species biofilms related to periodontitis

In our previous studies, Chlorin-e6 (Ce6) demonstrated a significant reduction of microorganisms’ viability against multi-species biofilm related to periodontitis while irradiated with blue light. However, the conjugation of Ce6 and antimicrobial peptides, and the incorporation of this photosensitizer in a nanocarrier, is still poorly explored. We hypothesized that chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion could inhibit a multi-species biofilm related to periodontitis during photodynamic therapy (PDT), the pre-treatment with hydrogen peroxide was also tested. The nanoemulsion (NE) incorporated with Ce6 was characterized regarding the physiochemical parameters. Images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Later, the Ce6 and LL-37 incorporated in NE was submitted to UV–Vis analysis and Reactive Oxygen Species (ROS) assay. Finally, the combined formulation (Ce6+LL-37 in nanoemulsion) was tested against multi-species biofilm related to periodontitis. The formed nanoformulation was kinetically stable, optically transparent with a relatively small droplet diameter (134.2 unloaded and 146.9 loaded), and weak light scattering. The NE system did not impact the standard UV–VIS spectra of Ce6, and the ROS production was improved while Ce6 was incorporated in the NE. The combination of Ce6 and LL-37 in NE was effective to reduce the viability of all bacteria tested. The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability. The current aPDT regimen was the best already tested against periodontal biofilm by our research team. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections such as periodontal disease.- Nanoemulsion demonstrated to be an excellent nanocarrier for photodynamic application.- Chlorin-e6 incorporated in nanoemulsion showed great physicochemical and biophotonic parameters.- The combination of chlorin-e6 and LL-37 peptide in nanoemulsion is effective to eliminate periodontal pathogenic bacteria.- The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability.

Decontamination of biofilm-contaminated implant surfaces: An in vitro evaluation.

The aim of the present study was to evaluate the cleaning efficacy of two mechanical and two chemical protocols in the decontamination of implant surfaces. In total, 123 commercially available implants were mounted in plastic models mimicking peri-implant circumferential intra-bony defects. A multispecies biofilm was grown on implant surfaces. Mechanical (air-polishing (AP), rotating titanium brush (TiB)) and chemical decontamination (alkaline electrolyzed water, N-acetyl-L-cysteine) protocols were used. Cleaning efficacy in terms of residual biofilm area, chemical surface properties, and bacterial counts were analyzed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and quantitative polymerase chain reaction. Surface decontamination protocols including use of an AP device or a rotating TiB were superior in terms of biofilm removal and in reducing atomic% of Carbon on implant surfaces when compared to methods restricted to wiping with gauze. The use of chemical agents as adjuncts to the mechanical cleaning protocols provided no relevant overall benefit over saline. No treatment modality, however, resulted in complete biofilm removal. Air-polishing and rotating TiB were more effective implant surface decontamination protocols than wiping with gauzes. Use of chemical agents did not improve cleaning efficacy.

Antimicrobial Solutions for Endotracheal Tubes in Prevention of Ventilator-Associated Pneumonia.

Ventilator-associated pneumonia is one of the most frequently encountered hospital infections and is an essential issue in the healthcare field. It is usually linked to a high mortality rate and prolonged hospitalization time. There is a lack of treatment, so alternative solutions must be continuously sought. The endotracheal tube is an indwelling device that is a significant culprit for ventilator-associated pneumonia because its surface can be colonized by different types of pathogens, which generate a multispecies biofilm. In the paper, we discuss the definition of ventilator-associated pneumonia, the economic burdens, and its outcomes. Then, we present the latest technological solutions for endotracheal tube surfaces, such as active antimicrobial coatings, passive coatings, and combinatorial methods, with examples from the literature. We end our analysis by identifying the gaps existing in the present research and investigating future possibilities that can decrease ventilator-associated pneumonia cases and improve patient comfort during treatment.

Staphylococcus aureus in Polymicrobial Skinand Soft Tissue Infections: Impact of Inter-Species Interactionsin Disease Outcome.

Polymicrobial biofilms provide a complex environment where co-infecting microorganisms can behave antagonistically, additively, or synergistically to alter the disease outcome compared to monomicrobial infections. Staphylococcus aureus skin and soft tissue infections (Sa-SSTIs) are frequently reported in healthcare and community settings, and they can also involve other bacterial and fungal microorganisms. This polymicrobial aetiology is usually found in chronic wounds, such as diabetic foot ulcers, pressure ulcers, and burn wounds, where the establishment of multi-species biofilms in chronic wounds has been extensively described. This review article explores the recent updates on the microorganisms commonly found together with S. aureus in SSTIs, such as Pseudomonas aeruginosa, Escherichia coli, Enterococcus spp., Acinetobacter baumannii, and Candida albicans, among others. The molecular mechanisms behind these polymicrobial interactions in the context of infected wounds and their impact on pathogenesis and antimicrobial susceptibility are also revised.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms

ObjectiveTo investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. MethodsThe inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. ResultsLAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. ConclusionsLAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

Oxygen Availability on the Application of Antimicrobial Photodynamic Therapy against Multi-Species Biofilms.

Methylene blue (MB)- or Curcumin (Cur)-based photodynamic therapy (PDT) has been used as an adjunctive treatment for periodontitis. Its actual clinical efficacy is still in question because the lack of oxygen in a deep periodontal pocket might reduce the PDT efficacy. We aim to investigate the effect of oxygen on PDT efficacy and to examine if the addition of hydrogen peroxide (HP) could improve PDT performance anaerobically. To this end, we cultured 48 h saliva-derived multi-species biofilms and treated the biofilms with 25 µM MB or 40 µM Cur, HP (0.001%, 0.01% and 0.1%), light (L-450 nm or L-660 nm), or combinations thereof under ambient air or strictly anaerobic conditions. MB- and Cur-PDTs significantly reduced biofilm viability in air but not under anaerobic conditions. HP at 0.1% significantly enhanced the killing efficacies of both MB- and Cur-PDTs anaerobically. The killing efficacy of Cur-PDT combined with 0.1% HP was higher anaerobically than in air. However, this was not the case for MB-PDT combined with 0.1% HP. In conclusion, this study demonstrated that the biofilm killing efficacies of MB- and Cur-PDTs diminished when there was no oxygen. HP at 0.1% can enhance the efficacy of PDT performed anaerobically, but the level of enhancement is photosensitizer-dependent.

Influence Of Surface Treatment On The Physical Properties And Biofilm Formation Of Zirconia-Reinforced Lithium Silicate Ceramics: In Vitro Trial.

To evaluate the performance of fully (Celtra Duo, Dentsply Sirona) and partially (VITA Suprinity, VITA) crystallized zirconia-reinforced lithium silicate and partially sintered lithium disilicate (IPS e.max CAD, Ivoclar Vivadent) glass-ceramics submitted to polishing, glazing, or no surface treatment after aging. Specimens of each glass-ceramic were subjected to polishing with rubber cups (POL), glazing (GL), or control (C; no treatment) and afterwards aged with 18,000 thermal cycles (5°C to 55°C). The average roughness (Ra), 2D and 3D morphology, contact angle, multispecies biofilm formation (Streptococcus mutans and Candida albicans), and mechanical strength were evaluated by atomic force microscopy (AFM, n = 5), sessile-drop goniometry (n = 5), spectrophotometry (n = 5), and flexural strength test (n = 10), respectively. Data were analyzed using two-way analysis of variance and Tukey test (α = 5%). POL promoted lower Ra than glazing, and Celtra Duo presented higher Ra than IPS e.max CAD (P < .05). Surfaces without polishing promoted higher Ra than the POL group (P < .001), greater contact angle (P < .001), and significant morphologic changes, regardless of the glass-ceramic. Irrespective of the treatment, the contact angle was higher in Celtra Duo, and regardless of the material, there was higher biofilm formation and lower flexural strength of unpolished compared to POL or GL ceramics. Polishing with rubber cups promoted lower roughness and minor morphologic surface alterations, but biofilm formation and flexural strength were similar to the glazed surface. In general, Celtra Duo and VITA Suprinity showed similar behavior to IPS e.max CAD, which makes zirconia-reinforced lithium silicate glass-ceramics a good option for indirect restorations.

Multichannel Microfluidic Platform for Temporal-Spatial Investigation of Niche Roles of Pseudomonas aeruginosa and Escherichia coli within a Dual-Species Biofilm.

In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. In this study, a microfluidic device designed with multiple channels and a gradient generator was used for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. We compared the structural parameters of monospecies and dual-species biofilms containing Pseudomonas aeruginosa (expressing mCherry) and Escherichia coli (expressing green fluorescent protein [GFP]) to understand the interactions in the biofilm. The rate of biovolume increase of each species in monospecies biofilm (2.7 × 105 μm3) was higher than those in a dual-species biofilm (9.68 × 104 μm3); however, synergism was still observed in the dual-species biofilm due to overall increases in biovolume for both species. Synergism was also observed in a dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. The microfluidic chip was useful for monitoring the dual-species biofilm in the microenvironment, indicating that different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. Finally, we demonstrated that the nucleic acids can be extracted from the dual-species biofilm in situ after biofilm imaging analysis. In addition, gene expression supported that the activation and suppression of different quorum sensing genes resulted in the different phenotype seen in the biofilm. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously. IMPORTANCE In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. Here, we demonstrate that a microfluidic device with multiple channels and a gradient generator can be useful for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. Our study revealed synergism in the dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. Furthermore, different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously.

Mild magnetic hyperthermia is synergistic with an antibiotic treatment against dual species biofilms consisting of S. aureus and P. aeruginosa by enhancing metabolic activity

Objective The wound biofilm infections that develop tolerance to standard-of-care antimicrobial treatment has been increasing. The objective of this study was to demonstrate a proof-of-concept of mild magnetic nanoparticle (MNP)/alternating magnetic field (AMF) hyperthermia as an anti-biofilm therapy against multispecies biofilm infections. Methods Using both an in vitro cell culture and in vivo murine model of wound infection, we investigated whether MNP/AMF hyperthermia applied at a mild thermal dosage would be synergistically effective against dual species biofilm infection consisting of S. aureus and P. aeruginosa when combined with a broad-spectrum antibiotic, ciprofloxacin (CIP). Results The combined treatment of MNP/AMF hyperthermia and CIP to the wounds of diabetic mice (db/db mice) significantly reduced the CFU number of S. aureus and P. aeruginosa by 2-log and 3-log, respectively, compared to the untreated control group, whereas either mild MNP/AMF hyperthermia or CIP treatment alone had little effect on the eradication of both bacteria. Our gene microarray data obtained from the culture of S. aureus biofilm suggest that mild MNP/AMF could shift the expression of genes for cellular respiration from anaerobic fermentation to an aerobic glycolytic/tricarboxylic acid cycle (TCA) pathway, implicating that the beneficial effect of mild MNP/AMF hyperthermia on the increased susceptibility of biofilm bacteria to an antibiotic treatment is associated with an increased metabolic activity. Conclusion Our results support the translational potential of mild MNP/AMF as an adjunctive therapy that can be combined with a broad-spectrum antibiotic treatment for the management of wound biofilm infections associated with multispecies bacteria.

Bacterial Adhesion of TESPSA and Citric Acid on Different Titanium Surfaces Substrate Roughness: An In Vitro Study with a Multispecies Oral Biofilm Model.

This in vitro study analyzed the influence of substrate roughness on biofilm adhesion and cellular viability over triethoxysilylpropyl succinic anhydride silane (TESPSA)- and citric acid (CA)-coated surfaces at 12 and 24 h, respectively. A multispecies biofilm composed of S. oralis, A. naslundii, V. parvula, F. nucleatum, P. intermedia, P. gingivalis, P. endodontalis and F. alocis was developed over titanium discs grouped depending on their roughness (low, medium, high) and antibacterial coating (low-TESPSA, medium-TESPSA, high-TESPSA, and CA). The biofilm was quantified by means of quantitative polymerase chain reaction (PCR) and viability PCR and assessed through confocal laser scanning microscope (CLSM). Quantitative PCR revealed no significant differences in bacterial adhesion and biofilm mortality. CA was the surface with the lowest bacterial counts and highest mortality at 12 and 24 h, respectively, while high harbored the highest amount of biofilm at 24 h. By CLSM, CA presented significant amounts of dead cells compared to medium-TESPSA and high-TESPSA. A significantly greater volume of dead cells was found at 12 h in low-TESPSA compared to medium-TESPSA, while CA also presented significant amounts of dead cells compared to medium-TESPSA and high-TESPSA. With regard to the live/dead ratio, low-TESPSA presented a significantly higher ratio at 12 h compared to medium-TESPSA and high-TESPSA. Similarly, CA exhibited a significantly higher live/dead ratio compared to medium-TESPSA and high-TESPSA at 12 h. This multispecies in vitro biofilm did not evidence clear antiadhesive and bactericidal differences between surfaces, although a tendency to reduce adhesion and increase antibacterial effect was observed in the low-TESPSA and CA.

Process optimization of a moving bed bioreactor undergoing simultaneous nitrification and denitrification for wastewater in the absence of organic carbon

ABSTRACT Moving bed bioreactors are efficient for the removal of nitrogen effluents by simultaneous nitrification and denitrification (SND), which is particularly challenging for effluents with low C/N ratio due to insufficient carbon available for denitrification. The addition of external organic carbon in such cases increases treatment costs. Endogenous degradation of biomass offers a cost-effective and persistent source of carbon. Internal carbon leads to low denitrification rates and optimizing the biomass content leads to a sustainable solution for treating such effluents. The present study aims to optimize the process of SND using endogenous carbon by investigating the effect of operational parameters like dissolved oxygen, influent ammonium (NH4–N) loading, hydraulic retention time (HRT) and carrier filling ratio (CFR). The synthetic wastewater used in the study does not inherently contain any organic carbon, thus indicating the responsible carbon for denitrification to be of internal origin. Using a novel carrier for multispecies biofilm development, initial NH4–N concentration variation showed an insignificant effect on SND efficiency. At higher loading, nitrite accumulation due to incomplete denitrification was observed. The effect of CFR was investigated in a range of 20–35%, beyond which non-homogeneous mixing was caused in the reactor. Under controlled DO and temperature of about 5 mg/L and 28 ± 2°C, respectively, an optimum filling ratio of 35% was finalised, resulting in an SND efficiency of 97.9% at 22 h HRT for high influent NH4–N concentration of 500 mg/L. Bacteriological species identification revealed the coexistence of nitrogen-fixing, aerobic nitrifying and anaerobic denitrifying bacteria in the biofilm growing on the biocarrier. Mechanism of SND occurring in aerobic moving bed bioreactors: Laboratory-scale aerobic Moving Bed Bioreactor setup for a continuous study evaluating the influence of operational parameters on SND Occurrence of aerobic and anoxic zones in biofilm grown on the surface of novel carriers due to diffusional gradient of dissolved oxygen

Development of a Prevotella bivia PNA probe and a multiplex approach to detect three relevant species in bacterial vaginosis-associated biofilms

Bacterial vaginosis (BV) is the most common vaginal infection worldwide. We developed a peptide nucleic acid (PNA) probe targeting Prevotella bivia, a common BV-associated bacteria, and optimized a multiplex approach for detection of Gardnerella spp., P. bivia and Fannyhessea vaginae. Our P. bivia PNA probe specifically detected the target species, and the optimized multiplex approach was able to detect the presence of the three species in multi-species BV biofilms.