Dual-species Biofilms Research Articles

A desiccated dual-species subaerial biofilm reprograms its metabolism and affects water dynamics in limestone

Understanding the impact of sessile communities on underlying materials is of paramount importance in stone conservation. Up until now, the critical role of subaerial biofilms (SABs) whether they are protective or deteriorative remains unclear, especially under desiccation. The interest in desiccated SABs is raised by the prediction of an increase in drought events in the next decades that will affect the Mediterranean regions' rich stone heritage as never before. Thus, the main goal of this research is to study the effects of desiccation on both the biofilms' eco-physiology and its impacts on the lithic substrate. To this end, we used a dual-species model system composed of a phototroph and a chemotroph to simulate biofilm behavior on stone heritage. We found that drought altered the phototroph-chemotroph balance and enriched the biofilm matrix with proteins and DNA. Desiccated SABs underwent a shift in metabolism to fermentation and a decrease in oxidative stress. Additionally, desiccated SABs changed the water-related dynamics (adsorption, evaporation, and wetting properties) in limestone. Water absorption experiments showed that desiccated SABs protected the stone from rapid water uptake, while a thermographic survey indicated a delay in water evaporation. Spilling-drop tests revealed a change in the wettability of the stone-SAB interface, which affected the water transport properties of the stone. Finally, desiccated SABs reduced stone swelling in the presence of water vapor. The biodeteriorative and bioprotective implications of desiccated SABs on the stone were ultimately assessed.

VT-1161-A Tetrazole for Management of Mono- and Dual-Species Biofilms.

VT-1161 is a novel tetrazole antifungal agent with high specificity for fungal CYP51 (compared to human CYP enzymes) which has been proven to have fewer adverse effects and drug-drug interaction profiles due to fewer off-target inhibitors. In this study, we evaluated the anti-biofilm potential of VT-1161 against mono- and dual-species biofilms of Candida albicans, Klebsiella pneumoniae and Staphylococcus aureus. VT-1161 inhibited planktonic growth of all three strains, with an MIC value of 2 µg mL-1 for C. albicans and 0.5 µg mL-1 for K. pneumoniae and S. aureus, and killed 99.9% of the microbial populations, indicating a cytocidal action. Additionally, VT-1161 showed an excellent anti-biofilm action, since it inhibited mono-microbial biofilms by 80% at 0.5 µg mL-1, and dual-species biofilms of C. albicans/K. pneumoniae and C. albicans/S. aureus by 90% at the same concentration. Additionally, the eradication of mature biofilms after 24 h of VT-1161 exposure was excellent, reaching 90% at 2 μg mL-1 for both mono- and dual-species biofilms. In such mixed biofilms, the use of VT-1161 was revealed to be an alternative treatment because it was able to reduce the number of cells of each species during both inhibition and eradication. Since long-term therapy is necessary for most fungal biofilm infections due to their recurrence and obstinacy, VT-1161 showed low cytotoxicity against normal human cell lines and also against the invertebrate model Caenorhabditis elegans. Considering the excellent anti-biofilm potential and its GRAS (generally recognized as safe) status, VT-1161 may find use in the prevention or therapeutic treatment of mono- or poly-microbial biofilms.

Potential Use of Brazilian Green Propolis Extracts as New Photosensitizers for Antimicrobial Photodynamic Therapy against Cariogenic Microorganisms

The synergic effect of Streptococcus mutans and Candida albicans increases dental caries severity. Antimicrobial photodynamic therapy (aPDT) is a non-invasive treatment for antimicrobial aspects. However, the current photosensitizers (PS) have many downsides for dental applications. This study aimed to evaluate the efficiency of two different Brazilian green propolis (BGP-AF and BGP-AG) as PS for aPDT against these microorganisms. A single-species biofilm was irradiated with crude extracts and their fractions and controls. Such extracts showed the best results and were evaluated in dual-species biofilms. Photodegradation, reactive oxygen species (ROS), cytotoxicity, and color stability assays were also investigated. Reductions higher than 3 log10 CFU/mL (p < 0.0001) occurred for crude BGP in single- and dual-species biofilms. Singlet oxygen was produced in BGP (p < 0.0001). BGP-mediated aPDT delayed S. mutans and C. albicans regrowth after 24 h of treatment (p < 0.0001). Both BGP did not change the color of dental materials (p > 0.05). BGP-AF-mediated aPDT showed 72.41% of oral keratinocyte viability (p < 0.0001). BGP extracts may be used in aPDT against S. mutans and C. albicans. Specifically, BGP-AF may represent a promising PS for dental applications.

Formation of a biofilm matrix network shapes polymicrobial interactions.

Staphylococcus aureus colonizes the same ecological niche as many commensals. However, little is known about how such commensals modulate staphylococcal fitness and persistence. Here we report a new mechanism that mediates dynamic interactions between a commensal streptococcus and S. aureus. Commensal Streptococcus parasanguinis significantly increased the staphylococcal biofilm formation in vitro and enhanced its colonization in vivo. A streptococcal biofilm-associated protein BapA1, not fimbriae-associated protein Fap1, is essential for dual-species biofilm formation. On the other side, three staphylococcal virulence determinants responsible for the BapA1-dependent dual-species biofilm formation were identified by screening a staphylococcal transposon mutant library. The corresponding staphylococcal mutants lacked binding to recombinant BapA1 (rBapA1) due to lower amounts of eDNA in their culture supernatants and were defective in biofilm formation with streptococcus. The rBapA1 selectively colocalized with eDNA within the dual-species biofilm and bound to eDNA in vitro, highlighting the contributions of the biofilm matrix formed between streptococcal BapA1 and staphylococcal eDNA to dual-species biofilm formation. These findings have revealed an additional new mechanism through which an interspecies biofilm matrix network mediates polymicrobial interactions.

Activity of Sodium Trimetaphosphate Nanoparticles on Cariogenic-Related Biofilms In Vitro.

In light of the promising effect of sodium trimetaphosphate nanoparticles (TMPn) on dental enamel, in addition to the scarce evidence of the effects of these nanoparticles on biofilms, this study evaluated the activity of TMPn with/without fluoride (F) on the pH, inorganic composition and extracellular matrix (ECM) components of dual-species biofilms of Streptococcus mutans and Candida albicans. The biofilms were cultivated in artificial saliva in microtiter plates and treated with solutions containing 1% or 3% conventional/microparticulate TMP (TMPm) or TMPn, with or without F. After the last treatment, the protein and carbohydrate content of the ECM was analyzed, and the pH and F, calcium (Ca), phosphorus (P), and TMP concentrations of the biofilms were determined. In another set of experiments, after the last treatment, the biofilms were exposed to a 20% sucrose solution, and their matrix composition, pH, and inorganic component contents were evaluated. 3% TMPn/F significantly reduced ECM carbohydrate and increased biofilm pH (after sucrose exposure) than other treatments. Also, it significantly increased P and F levels before sucrose exposure in comparison to 3% TMPm/F. In conclusion, 3% TMPn/F affected the biofilm ECM and pH, besides influencing inorganic biofilm composition by increasing P and F levels in the biofilm fluid.

The Bio-Aging of Biofilms on Behalf of Various Oral Status on Different Titanium Implant Materials.

The properties of titanium implants are affected by bio-aging due to long-term exposure to the oral microenvironment. This study aimed to investigate probable changes in titanium plates after different biofilm bio-aging processes, representing various oral status. Titanium plates with different surface treatments were used, including polish, sandblasted with large grit and acid etched (SLA), microarc oxidation (MAO), and hydroxyapatite coating (HA). We established dual-species biofilms of Staphylococcus aureus (S. aureus)-Candida albicans (C. albicans) and saliva biofilms from the healthy and patients with stage III-IV periodontitis, respectively. After bio-aging with these biofilms for 30 days, the surface morphology, chemical composition, and water contact angles were measured. The adhesion of human gingival epithelial cells, human gingival fibroblasts, and three-species biofilms (Streptococcus sanguis, Porphyromonas gingivalis, and Fusobacterium nucleatum) were evaluated. The polished specimens showed no significant changes after bio-aging with these biofilms. The MAO- and SLA-treated samples showed mild corrosion after bio-aging with the salivary biofilms. The HA-coated specimens were the most vulnerable. Salivary biofilms, especially saliva from patients with periodontitis, exhibited a more distinct erosion on the HA-coating than the S. aureus-C. albicans dual-biofilms. The coating became thinner and even fell from the substrate. The surface became more hydrophilic and more prone to the adhesion of bacteria. The S. aureus-C. albicans dual-biofilms had a comparatively mild corrosion effect on these samples. The HA-coated samples showed more severe erosion after bio-aging with the salivary biofilms from patients with periodontitis compared to those of the healthy, which emphasized the importance of oral hygiene and periodontal health to implants in the long run.

Carbon/nitrogen ratios determine biofilm formation and characteristics in model microbial cultures

The influence of growth medium water chemistry, specifically carbon/nitrogen (C/N) molar ratios, on the characteristics and development of biofilms of the model microorganism Pseudomonas aeruginosa was investigated. C/N = 9 had a unique effect on biofilm composition as well as quorum sensing (QS) pathways, with higher concentrations of carbohydrates and proteins in the biofilm and a significant upregulation of the QS gene lasI in planktonic cells. The effect of C/N ratio on total attached biomass was negligible. Principal component analysis revealed a different behavior of most outputs such as carbohydrates and QS chemicals at C/N = 9, and pointed to correlations between parameters of biofilm formation and steady state distribution of cells and extracellular components. C/N ratio was also shown to influence organic compound utilization by both planktonic and sessile organisms, with a maximum chemical oxygen demand (COD) removal of 83% achieved by biofilms at C/N = 21. Planktonic cells achieved higher COD removal rates, but greater overall rates after six days occurred in biofilms. The development of a dual-species biofilm of P. aeruginosa and Nitrobacter winogradskyi was also influenced by C/N, with increase in the relative abundance of the slower-growing N. winogradskyi above C/N = 9. These results indicate that altering operational parameters related to C/N would be relevant for mitigating or promoting biofilm formation and function depending on the desired industrial application or treatment configuration.

A model for microbial interactions and metabolomic alterations in Candida glabrata-Staphylococcus epidermidis dual-species biofilms.

The fungus Candida glabrata and the bacterium Staphylococcus epidermidis are important biofilm-forming microorganisms responsible of nosocomial infections in patients. In addition to causing single-species disease, these microorganisms are also involved in polymicrobial infections leading to an increased antimicrobial resistance. To expand knowledge about polymicrobial biofilms, in this study we investigate the formation of single- and dual-species biofilms of these two opportunistic pathogens employing several complementary approaches. First, biofilm biomass, biofilm metabolic activity and the microbial composition in single- and dual-species biofilms were assessed and compared. Then, the expression of three genes of C. glabrata and three genes of S. epidermidis positively related to the process of biofilm formation was evaluated. Although S. epidermidis is a stronger biofilm producer than C. glabrata, both biological and genetic data indicate that S. epidermidis growth is inhibited by C. glabrata which dominates the dual-species biofilms. To better understand the mechanisms of the interactions between the two microorganisms, a broad GC-MS metabolomic dataset of extracellular metabolites for planktonic, single- and dual-species biofilm cultures of C. glabrata and S. epidermidis was collected. As demonstrated by Partial Least Squares Discriminant Analysis (PLS-DA) of GC-MS metabolomic data, planktonic cultures, single- and dual-species biofilms can be sharply differentiated from each other by the nature and levels of an assortment of primary and secondary metabolites secreted in the culture medium. However, according to our data, 2-phenylethanol (secreted by C. glabrata) and the synergistically combined antifungal activity of 3-phenyllactic acid and of the cyclic dipeptide cyclo-(l-Pro-l-Trp) (secreted by S. epidermidis) play a major role in the race of the two microorganisms for predominance and survival.

Antibacterial and Antibiofilm Activity of Carvacrol against Oral Pathogenic Bacteria.

Faced with the current situation of high rates of microbial resistance, together with the scarcity of new antibiotics, it is necessary to search for and identify new antimicrobials, preferably natural, to alleviate this situation. The aim of this work was to evaluate the antibacterial activity of carvacrol (CAR), a phenolic compound of essential oils, against pathogenic microorganisms causing oral infections, such as Streptococcus mutans and S. sanguinis, never evaluated before. The minimum inhibitory and the minimum bactericidal concentration were 93.4 μg/mL and 373.6 μg/mL, respectively, for the two strains. The growth kinetics under different concentrations of CAR, as well as the bactericidal power were determined. The subinhibitory concentrations delayed and decreased bacterial growth. Its efficacy on mature biofilms was also tested. Finally, the possible hemolytic effect of CAR, not observable at the bactericidal concentrations under study, was evaluated. Findings obtained point to CAR as an excellent alternative agent to safely prevent periodontal diseases. In addition, it is important to highlight the use of an experimental methodology that includes dual-species biofilm and subinhibitory concentration models to determine optimal CAR treatment concentrations. Thus, CAR could be used preventively in mouthwashes or biomaterials, or in treatments to avoid existing antibiotic resistance.

Phytolectin conjugated positively charged fatty acid amide impairs virulence factors and inhibits cross-kingdom biofilm formation of Candida albicans and uropathogenic Escherichia coli.

Polymicrobial biofilm encasing cross-kingdom micro-organisms are apparent in medicine, which imposes serious resistance to conventional antimicrobial treatment. The objective of the study was to explore Butea monosperma seed lectin (BMSL) conjugated antimicrobial lipid, 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) to inhibit mixed-species biofilm of uropathogenic Escherichia coli-Candida albicans. Antimicrobial activity and antibiofilm of cN16E and cN16E-BMSL conjugate (BcN16E) were analysed against single- and mixed microbial cultures. The minimum inhibitory concentration (MIC) indicates that the MIC of cN16E-BMSL conjugate (BcN16E) against cohabiting UPEC-C. albicans was eightfold lower than the cN16E. BcN16E affects membrane integrity to elicit antimicrobial activity. BcN16E inhibits the dual-species biofilm even with 16 times lower MIC of cN16E. BcN16E impairs the biofilm-associated virulence factors which include extracellular polysaccharides, cell surface hydrophobicity, swimming, swarming motilities, hyphal filamentous morphology, curli formation and haemolysin activity. As a proof of concept, we demonstrated BcN16E ability to inhibit dual-species biofilm formation on a urinary catheter. The study revealed that the BcN16E is better than cN16E in impairing biofilm-associated virulence factors and exerting antimicrobial activity. The findings emphasize that phytolectin has the potential to enhance the anti-virulence strategies of antimicrobials against cross-kingdom biofilm-related infections.

Characterization of Mixed-Species Biofilms Formed by Four Gut Microbiota.

In natural settings, approximately 40-80% of bacteria exist as biofilms, most of which are mixed-species biofilms. Previous studies have typically focused on single- or dual-species biofilms. To expand the field of study on gut biofilms, we found a group of gut microbiota that can form biofilms well in vitro: Bifidobacterium longum subsp. infantis, Enterococcus faecalis, Bacteroides ovatus, and Lactobacillus gasseri. The increase in biomass and bio-volume of the mixed-species biofilm was confirmed via crystal violet staining, field emission scanning electron microscopy, and confocal laser scanning microscopy, revealing a strong synergistic relationship in these communities, with B. longum being the key biofilm-contributing species. This interaction may be related to changes in the cell number, biofilm-related genes, and metabolic activities. After quantifying the cell number using quantitative polymerase chain reaction, B. longum and L. gasseri were found to be the dominant flora in the mixed-species biofilm. In addition, this study analyzed biological properties of mixed-species biofilms, such as antibiotic resistance, cell metabolic activity, and concentration of water-insoluble polysaccharides. Compared with single-species biofilms, mixed-species biofilms had higher metabolic activity, more extracellular matrix, and greater antibiotic resistance. From these results, we can see that the formation of biofilms is a self-protection mechanism of gut microbiota, and the formation of mixed-species biofilms can greatly improve the survival rate of different strains. Finally, this study is a preliminary exploration of the biological characteristics of gut biofilms, and the molecular mechanisms underlying the formation of biofilms warrant further research.

Impact of N-Acetylcysteine and Antibiotics Against Single and Dual Species Biofilms of Pseudomonas aeruginosa and Achromobacter xylosoxidans.

Lungs of cystic fibrosis patients are often colonized or infected with organisms, such as Pseudomonas aeruginosa and other emerging pathogenic bacteria such as Achromobacter xylosoxidans. Further, it is well established that infections of the cystic fibrosis lung airways are caused by polymicrobial infections, although its composition and diversity may change throughout the patient's life. In the present study, we investigated the effects of N-acetylcysteine (NAC) and amikacin, aztreonam, ciprofloxacin, and tobramycin alone and in combination against single- and dual-species biofilms of P. aeruginosa and A. xylosoxidans, in vitro and in the Caenorhabditis elegans infection model. Results showed that tobramycin and ciprofloxacin were the most effective antibiotics, while aztreonam was the least effective antibiotic against both single- and dual-species biofilms of P. aeruginosa and A. xylosoxidans. However, NAC showed little effect on both single- and dual-species, even with a combination of antibiotics. Increased survival was observed in C. elegans when treated with NAC in combination with tobramycin or ciprofloxacin, compared to no treatment or NAC alone. Tobramycin and ciprofloxacin were found effective in biofilms, but more research is needed to better understand the effects of NAC and antibiotics against single- and dual-species biofilms.

Evaluation of efficacy of new chalcone-based endodontic irrigant against dual biofilm Enterococcus faecalis and Candida albicans: a study in vitro.

The aim of this research was to develop a chalcone-based endodontic irrigant for cleaning and disinfecting the root canal. Minimal inhibitory concentration (MIC) experiments in C. albicans and E. faecalis strains with different aminochalcones (AM) were carried out, and the compound that presented the best activity against both pathogens was chosen. The formulation of an endodontic irrigant was elaborated, tested in mono and dual specie biofilms. Disks were sterilized and then incubated with E. faecalis, C. albicans and E. faecalis and C. albicans mixed for 72h for biofilm maturation. After contamination, samples were divided in 4 experimental groups and 2 positive control group as follows: Group1: Irrigant; Group2: Irrigant + AM-38; Group3: Chlorhexidine 2% (positive control) and, Group 4: 1.0% sodium hypochlorite (positive control). The samples were analyzed by CFU/ml count. The sample was taken to sonicador to remove the cells and then plated. The toxicity was determined in vitro with human gingival fibroblast cells (HGF) and in vivo using the Galleria mellonella model. Formulation showed antimicrobial activity, with MIC on C. albicans 15.6 and E. faecalis 7.8µg/ml. Treatment with formulation in concentration 156µg/ml significantly reduced mono or dual species biofilm formation and viability (p < 0.05). The results were significant against C. albicans and E. faecalis and did not show toxicity in cells and G. mellonella. In general, the formulation showed effective antibiofilm activity, significantly reducing microorganisms, opening paths in search of new endodontic irrigants.

Surface Dependent Inhibition of Mycobacterium abscessus by Diverse Pseudomonas aeruginosa Strains.

Both Pseudomonas aeruginosa and Mycobacterium abscessus are bacteria that cause pulmonary infection in people with inflammatory lung disease, including individuals with cystic fibrosis (CF). These bacterial species inhabit the same environmental reservoirs (soil and water) and can be coisolated in the lungs of people with CF. We investigated the interaction of these bacteria and found an antagonistic interaction favoring P. aeruginosa that was observed in biofilms but not in planktonic cultures. This antagonism extended to multiple P. aeruginosa strains and against Mycobacterium smegmatis. We tested known P. aeruginosa mutants for genes that can play roles in interbacterial contact-dependent (type III and type VI secretion systems) and contact-independent (quorum sensing, type II secretion) antagonism pathways to interrogate the mechanism of action. Our results indicate that well-known mechanisms of interbacterial competition are not responsible for the antagonism of P. aeruginosa toward M. abscessus, suggesting a novel antibacterial strategy. IMPORTANCE The biofilm lifestyle is favored by many organisms, and understanding interbacterial interactions that occur between coisolated bacterial species can provide new information regarding bacterial defense mechanisms and antibacterial targets. This may also provide insights into possible interbacterial interactions impacting host immunity during coinfection. Here, we investigate an antagonistic interaction favoring P. aeruginosa over M. abscessus exclusively in dual-species biofilms and not in liquid coculture.

Enterococcus faecalis thrives in dual-species biofilm models under iron-rich conditions.

Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) are pathogenic strains that often coexist in intestinal flora of humans and are prone to cause biofilm-associated infections, such as gastrointestinal tract and urinary tract infections. Earlier studies have demonstrated that E. faecalis biofilm can metabolize ferrous ions in iron-rich environments and promote biofilm growth under in-vivo conditions. However, the influence of iron transporters on dual-species biofilm growth and the nature of molecular-level interactions between iron transporter proteins and Fe2+ remains unknown. Therefore, in this work, co-culture studies were performed and the study indicates that Fe2+ at concentrations of 50-150µM promotes the colonization of E. coli, and Fe2+ concentrations of 50-200µM promote the growth of E. faecalis and dual-species colonies. Atomic absorption spectroscopy results reveal that Fe2+ ion augmentation in bacterial cells was increased to 4 folds in the single-species model and 11 folds in the dual-species model under iron-supplemented conditions. Furthermore, Fe2+ augmentation increased the antibiotic resistance of E. faecalis in both single- and dual-species bacterial cultures. In addition, in-silico docking were performed to determine a three-dimensional (3D) structure of ferrous iron-transporter proteins FeoB of E. faecalis and its affinity to extracellular Fe2+. Our model suggests that the FeoB facilitates the Fe2+ uptake in E. faecalis cells in the absence of iron chelator, 2,2-bipyridyl.

The Potential of Phenothiazines against Endodontic Pathogens: A Focus on Enterococcus-Candida Dual-Species Biofilm.

Persistent apical periodontitis occurs when the endodontic treatment fails to eradicate the intraradicular infection, and is mainly caused by Gram-positive bacteria and yeasts, such as Enterococcus faecalis and Candida albicans, respectively. Phenothiazines have been described as potential antimicrobials against bacteria and fungi. This study aimed to investigate the antimicrobial potential of promethazine (PMZ) and chlorpromazine (CPZ) against E. faecalis and C. albicans dual-species biofilms. The susceptibility of planktonic cells to phenothiazines, chlorhexidine (CHX) and sodium hypochlorite (NaOCl) was initially analyzed by broth microdilution. Interaction between phenothiazines and CHX was examined by chequerboard assay. The effect of NaOCl, PMZ, CPZ, CHX, PMZ + CHX, and CPZ + CHX on biofilms was investigated by susceptibility assays, biochemical and morphological analyses. Results were evaluated through one-way ANOVA and Tukey's multiple comparison post-test. PMZ, alone or in combination with irrigants, was the most efficient phenothiazine, capable of reducing cell counts, biomass, biovolume, carbohydrate and protein contents of dual-species biofilms. Neither PMZ nor CPZ increased the antimicrobial activity of CHX. Further investigations of the properties of phenothiazines should be performed to encourage their use in endodontic clinical practice.

Candida albicans Promotes the Antimicrobial Tolerance of Escherichia coli in a Cross-Kingdom Dual-Species Biofilm

Cross-kingdom multi-species biofilms consisting of fungi and bacteria are often resistant to antimicrobial treatment, leading to persistent infections. We evaluated whether the presence of Candida albicans affects the antibacterial tolerance of Escherichia coli in dual-species biofilms and explored the underlying mechanism. We found that the survival of E. coli in the presence of antibacterial drugs was higher in dual-species biofilms compared to single-species biofilms. This tolerance-inducing effect was observed in E. coli biofilms that were treated with a C. albicans culture supernatant. To explore the antibacterial tolerance-inducing factor contained in the culture supernatant and identify the tolerance mechanism, a heated supernatant, a supernatant treated with lyticase, DNase, and proteinase K, or a supernatant added to a drug efflux pump inhibitor were used. However, the tolerance-inducing activity was not lost, indicating the existence of some other mechanisms. Ultrafiltration revealed that the material responsible for tolerance-inducing activity was <10 kDa in size. This factor has not yet been identified and needs further studies to understand the mechanisms of action of this small molecule precisely. Nevertheless, we provide experimental evidence that Candida culture supernatant induces E. coli antibacterial tolerance in biofilms. These findings will guide the development of new treatments for dual-species biofilm infections.

Carbon nanotube-based surfaces: Effect on the inhibition of single- and dual-species biofilms of Escherichia coli and Enterococcus faecalis

This study aimed to investigate the influence of multi-walled carbon nanotube (MWCNT) loading on the antibiofilm activity of MWCNT/poly(dimethylsiloxane) (PDMS) composites against single- and dual-species biofilms of Escherichia coli and Enterococcus faecalis. MWCNT/PDMS composites containing different MWCNT loadings (1, 2, and 3 wt%) were synthesized and tested for their ability to reduce biofilm formation. Surfaces were evaluated using a parallel plate flow chamber for 24 h under the hydrodynamic conditions prevailing in urinary catheters. Biofilm formation by E. coli and E. faecalis was assessed by determining the number of total, viable and culturable cells, and the biofilm architecture was analyzed by confocal laser scanning microscopy (CLSM). Results revealed that the composite with the highest MWCNT loading (3%) was the most effective against E. coli and E. faecalis biofilms. When compared with bare PDMS, it significantly reduced the number of total (63%), viable (74%), and culturable (77%) cells of E. faecalis biofilms, and the number of viable (50%) and culturable (64%) cells of E. coli biofilms. The effect of these MWCNT-based composites on the inhibition of biofilm formation was corroborated by CLSM, where reductions of up to 57% and 64% in biofilm biovolume and thickness, respectively, were observed. Even though dual-species biofilms presented lower susceptibility to the antimicrobial activity of MWCNT/PDMS composites, a reduction of 32% in the number of culturable cells was obtained with 3% MWCNT/PDMS surfaces. Taken together, these results suggest that MWCNT/PDMS composites are promising materials for developing novel antimicrobial and antifouling coatings for urinary tract devices.

Dual-Species Biofilms Formed by Escherichia coli and Salmonella Enhance Chlorine Tolerance.

In this research, mono- and dual-species biofilms of Escherichia coli (O45:H2 and O121:H19) and Salmonella enterica serovar Typhimurium formed on stainless-steel coupons were treated with 100 mg/L NaClO for 1 min. Confocal laser scanning microscopy (CLSM) was applied to investigate the spatial structural dynamics of mono- and dual-species biofilms, and nuclear magnetic resonance (NMR) spectroscopy was employed to further investigate their metabolic responses toward chlorine. CLSM results indicated that mixed-species biofilms (total biovolume, 148,000 to 167,000 μm3) stimulated the growth of biomass 2 to 6 times that of single-species biofilms. Upon chlorine treatment, E. coli O45 and S. Typhimurium achieved less reduction (P < 0.05) when coexisting in mixed biofilms (0.70 and 1.17 log CFU/coupon reductions, respectively) compared with their corresponding single-species biofilms (1.97 and 2.01 log CFU/coupon reductions, respectively), while for E. coli O121, more reduction (P < 0.05) was achieved in a mixed biofilm (1.37 log CFU/coupon reductions) compared with its single-species biofilm (0.59 log CFU/coupon reductions). Moreover, NMR results suggested that the increase of putrescine (antioxidation regulator) and the decrease of glucose (enhanced glycolysis for energy replenishment) might contribute to the improved chlorine tolerance in mixed biofilms. Overall, dual-species biofilms promoted biofilm growth and their chlorine tolerance. This study improved our knowledge of the metabolic difference of single- and mixed-species biofilms of E. coli and Salmonella to chlorine sanitization and raised an urgency to investigate the effectiveness of common disinfectants against multispecies consortia. IMPORTANCE Outbreaks of Escherichia coli and Salmonella in food might be associated with the cross-contamination of biofilms on food-contact surfaces. The knowledge of the sanitization of mono-species biofilm on the food-contact surface is well established, while mixed-species biofilm occurs more naturally, which could profoundly affect the efficacy of sanitizer. Therefore, this research aims to evaluate the efficacy of using chlorine against single- and dual-species biofilms of E. coli and Salmonella along with the underlying bacterial metabolic responses. The responses of a mixed biofilm of E. coli and Salmonella to chlorine sanitization were clarified, providing insights to develop a targeted and green sanitization strategy against specific pathogens by perturbing their most susceptible metabolism pathway without sanitizer residue.

Pseudomonas fluorescens group bacterial strains interact differently with pathogens during dual-species biofilm formation on stainless steel surfaces in milk.

In order to develop strategies for preventing biofilm formation in the dairy industry, a deeper understanding of the interaction between different species during biofilm formation is necessary. Bacterial strains of the P. fluorescens group are known as the most important biofilm-formers on the surface of dairy processing equipment that may attract and/or shelter other spoilage or pathogenic bacteria. The present study used different strains of the P. fluorescens group as background microbiota of milk, and evaluated their interaction with Staphylococcus aureus, Bacillus cereus, Escherichia coli O157:H7, and Salmonella Typhimurium during dual-species biofilm formation on stainless steel surfaces. Two separate scenarios for dual-species biofilms were considered: concurrent inoculation of Pseudomonas and pathogen (CI), and delayed inoculation of pathogen to the pre-formed Pseudomonas biofilm (DI). The gram-positive pathogens used in this study did not form dual-species biofilms with P. fluorescens strains unless they were simultaneously inoculated with Pseudomonas strains. E. coli O157:H7 was able to form dual-species biofilms with all seven P. fluorescens group strains, both in concurrent (CI) and delayed (DI) inoculation. However, the percentage of contribution varied depending on the P. fluorescens strains and the inoculation scenario. S. Typhimurium contributed to biofilm formation with all seven P. fluorescens group strains under the CI scenario, with varying degrees of contribution. However, under the DI scenario, S. Typhimurium did not contribute to the biofilm formed by three of the seven P. fluorescens group strains. Overall, these are the first results to illustrate that the strains within the P. fluorescens group have significant differences in the formation of mono-or dual-species biofilms with pathogenic bacteria. Furthermore, the possibility of forming dual-species biofilms with pathogens depends on whether the pathogens form the biofilm simultaneously with the P. fluorescens group strains or whether these strains have already formed a biofilm.