Mixed Biofilms Research Articles

In Vitro Biofilm-Mediated Biodegradation of Pesticides and Dye-Contaminated Effluents Using Bacterial Biofilms.

Overuse of pesticides in agricultural soil and dye-polluted effluents severely contaminates the environment and is toxic to animals and humans making their removal from the environment essential. The present study aimed to assess the biodegradation of pesticides (cypermethrin (CYP) and imidacloprid (IMI)), and dyes (malachite green (MG) and Congo red (CR)) using biofilms of bacteria isolated from pesticide-contaminated soil and dye effluents. Biofilms of indigenous bacteria, i.e., Bacillus thuringiensis 2A (OP554568), Enterobacter hormaechei 4A (OP723332), Bacillus sp. 5A (OP586601), and Bacillus cereus 6B (OP586602) individually and in mixed culture were tested against CYP and IMI. Biofilms of indigenous bacteria i.e., Lysinibacillus sphaericus AF1 (OP589134), Bacillus sp. CF3 (OP589135) and Bacillus sp. DF4 (OP589136) individually and in mixed culture were tested for their ability to degrade dyes. The biofilm of a mixed culture of B. thuringiensis + Bacillus sp. (P7) showed 46.2% degradation of CYP compared to the biofilm of a mixed culture of B. thuringiensis + E. hormaechei + Bacillus sp. + B. cereus (P11), which showed significantly high degradation (70.0%) of IMI. Regarding dye biodegradation, a mixed culture biofilm of Bacillus sp. + Bacillus sp. (D6) showed 86.76% degradation of MG, which was significantly high compared to a mixed culture biofilm of L. sphaericus + Bacillus sp. (D4) that degraded only 30.78% of CR. UV-VIS spectroscopy revealed major peaks at 224 nm, 263 nm, 581 nm and 436 nm for CYP, IMI, MG and CR, respectively, which completely disappeared after treatment with bacterial biofilms. Fourier transform infrared (FTIR) analysis showed the appearance of new peaks in degraded metabolites and disappearance of a peak in the control spectrum after biofilm treatment. Thin layer chromatography (TLC) analysis also confirmed the degradation of CYP, IMI, MG and CR into several metabolites compared to the control. The present study demonstrates the biodegradation potential of biofilm-forming bacteria isolated from pesticide-polluted soil and dye effluents against pesticides and dyes. This is the first report demonstrating biofilm-mediated bio-degradation of CYP, IMI, MG and CR utilizing soil and effluent bacterial flora from Multan and Sheikhupura, Punjab, Pakistan.

A Re-Purposing Strategy: Sub-Lethal Concentrations of an Eicosanoid Derived from the Omega-3-Polyunsaturated Fatty Acid Resolvin D1 Affect Dual Species Biofilms.

The fungal species Candida parapsilosis and the bacterial species Staphylococcus aureus may be responsible for hospital-acquired infections in patients undergoing invasive medical interventions or surgical procedures and often coinfect critically ill patients in complicating polymicrobial biofilms. The efficacy of the re-purposing therapy has recently been reported as an alternative to be used. PUFAs (polyunsaturated fatty acids) may be used alone or in combination with currently available traditional antimicrobials to prevent and manage various infections overcoming antimicrobial resistance. The objectives of the study were to evaluate the effects of Resolvin D1 (RvD1) as an antimicrobial on S. aureus and C. parapsilosis, as well as the activity against the mixed biofilm of the same two species. Microdilution assays and time-kill growth curves revealed bacterial and fungal inhibition at minimum concentration values between 5 and 10 μg mL-1. In single-species structures, an inhibition of 55% and 42% was reported for S. aureus and C. parapsilosis, respectively. Moreover, RvD1 demonstrated an eradication capacity of 60% and 80% for single- and mixed-species biofilms, respectively. In association with the inhibition activity, a downregulation of genes involved in biofilm formation as well as ROS accumulation was observed. Eradication capability was confirmed also on mature mixed biofilm grown on silicone platelets as shown by scanning electron microscopy (SEM). In conclusion, RvD1 was efficient against mono and polymicrobial biofilms in vitro, being a promising alternative for the treatment of mixed bacterial/fungal infections.

Toward a low-energy bioelectrochemical fixation of N2 via mixed cultures electroactive biofilms

Toward a low-energy bioelectrochemical fixation of N2 via mixed cultures electroactive biofilms

Does quorum quenching matter to microbial community dynamics in long-term membrane bioreactor operation?

Quorum quenching (QQ) has effectively prevented biofouling in membrane bioreactors (MBRs) employing isolated QQ bacterial strains. However, the influence of QQ on the microbial population still needs to be fully understood. This research aims to analyze the microbial population in MBRs over an extended period (>250 days) under different conditions, such as varying aeration intensities and doses of QQ bacteria, QQ media, and types of feed. Results show that no significant changes occurred in the structure and diversity of the microbial community in the mixed liquor and biofilm due to QQ treatment. Canonical correspondence analysis did reveal that the microbial communities were strongly influenced by feed types and phases. The microbial community composition varied between bacterial habitats (i.e., mixed liquor and biofilm), showing the two dominant phyla Proteobacteria and Bacteroidota in the former and Proteobacteria and Chloroflexi in the latter. The co-occurrence network analysis indicated that the biofilm (with 163 edges) in the MBR fed with real wastewater exhibited a more intricate network than the biofilm (with 53 edges) in the MBR fed with synthetic wastewater. With QQ, the biofilm exhibited more positive edges than negative ones. The phylogenetic investigation of communities showed that QQ barely affects functional gene-related quorum sensing (e.g., bacterial chemotaxis, motility proteins, and secretion) in mixed liquor but in biofilms at relatively large QQ doses (> 75 mg/L BH4). This research sheds light on the bacterial QQ's role in reducing MBR biofouling and provides crucial insights into its underlying mechanisms.

Process optimization of malachite green degradation by mixed biofilm positive bacteria: Application of Box–Behnken designs, RSM and whole genome sequences analysis

Bacterial biofilm positive strains recovered from engine oil polluted-soil was investigated for its capacity to remove malachite green (MG) in mineral salt solution. Box–Behnken designs combining four factors (glucose, yeast extract, temperature and agitation) with three levels (−1, 0, +1) was used to found the best biotic and abiotic conditions for MG removal. The biodegradation potential of the selected microbial consortium was also confirmed using analytical techniques. Then, whole genome sequencing (WGS) analysis were performed to detect genes related to biofilm formation and chemicals biodegradation. Our results showed that the selected strains (G5, G13 and G15) were able to form biofilm on congo red agar plates developing almost black, very black and almost black colonies respectively. Experimental results using Box-Behnken designs showed that decolorization rate of MG reach 89.35% in these optimized conditions: glucose (2%), yeast extract (1%), temperature (40 °C), and agitation (150 rpm). Molecular investigation revealed that the isolated strains were identified as Cronobacter sakazakii G5, Enterobacter hormaechei G13, and Klebsiella pneumoniae G15 based on the whole genome sequences . Whole genome sequences analysis demonstrated the presence of genes involved in biofilm formation as well as dyes and chemical detoxification. The findings presented here provide useful information for development an efficient microbial consortium, formed by adherent bacteria, able to remove dye from coloured wastewater.

Исследование антибактериальной, антиадгезивной и антибиопленкообразующей активности растительных комплексов в отношении пародонтопатогенных бактерий in vitro

Periodontitis is a problem urgent in Russia and throughout the world in general. Because of the dynamically changing flora causing this diseases, the treatment methods designed against it should be adapted on a regular basis. The classic approach to arresting development of the acute process relies on 0.2–0.12% chlorhexidine, a chemical antiseptic, but after 3 weeks of use, its efficacy drops drastically because pathogenic flora adjusts thereto. In the recent years, plantbased complexes with antiseptic properties have shown their capacity to challenge the classic approach. Obviously, efficacy of active ingredients depends on the form of the final product. The marker of periodontitis in the oral cavity is Staphylococcus aureus. Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii have virulence markers that are copathogens for periodontitis. This study aimed to find plant-based preparations capable of eliminating the said microbes and Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius, Enterococcus faecalis. We compared antibacterial, adhesion and biofilm formation preventing properties of Phytodent plant-based products in various forms: water solution, water-alcohol solution, oil solution, gel. Long exposure form — gel — proved to be the most effective in terms of the properties tested. Products with synthetic and plant-based antiseptics, as well as those with plant-based antiseptics in maximum concentration (elixir), had comparable efficacy. Water and oil solutions are less effective because of the lower active ingredient concentration and relatively brief exposure. Our results support the results of clinical studies dedicated to the use of Phytodent products as oral care products in the context of periodontitis prevention and treatment. We recommend conducting further studies comparing compositions, cross- and comparative studies investigating the effect of frequency of application and time of exposure, such studies registering titers of active ingredient concentrations, and with subjects thereof including mixed biofilms.

Inhibitory effects of vorinostat (SAHA) against food-borne pathogen Salmonella enterica serotype Kentucky mixed culture biofilm with virulence and quorum-sensing relative expression

Salmonella is a food-borne microorganism that is also a zoonotic bacterial hazard in the food sector. This study determined how well a mixed culture of Salmonella Kentucky formed biofilms on plastic (PLA), silicon rubber (SR), rubber gloves (RG), chicken skin and eggshell surfaces. In vitro interactions between the histone deacetylase inhibitor-vorinostat (SAHA)-and S. enterica serotype Kentucky were examined utilizing biofilms. The minimum inhibitory concentration (MIC) of SAHA was 120 µg mL−1. The addition of sub-MIC (60 µg mL−1) of SAHA decreased biofilm formation for 24 h on PLA, SR, RG, Chicken skin, and eggshell by 3.98, 3.84, 4.11, 2.86 and 3.01 log (p < 0.05), respectively. In addition, the initial rate of bacterial biofilm formation was higher on chicken skin than on other surfaces, but the inhibitory effect was reduced. Consistent with this conclusion, virulence genes expression (avrA, rpoS and hilA) and quorum-sensing (QS) gene (luxS) was considerably downregulated at sub-MIC of SAHA. SAHA has potential as an anti-biofilm agent against S. enterica serotype Kentucky biofilm, mostly by inhibiting virulence and quorum-sensing gene expression, proving the histone deacetylase inhibitor could be used to control food-borne biofilms in the food industry.

In Vitro and Ex Vivo Biofilm-Forming Ability of Rhinocladiella similis and Trichophyton rubrum Isolated from a Mixed Onychomycosis Case.

Infections caused by biofilm-forming agents have important implications for world health. Mixed infections, caused by more than one etiological agent, are also an emerging problem, especially regarding the standardization of effective diagnosis and treatment methods. Cases of mixed onychomycosis (OM) have been reported; however, studies on the microbial interactions between the different fungi in biofilms formed on nails are still scarce. We describe a case of mixed OM caused by the dermatophyte Trichophyton rubrum and the black yeast-like fungus Rhinocladiella similis. Identical growths of both fungi were observed in more than 50 cultures from different nail samples. Additionally, both species were able to form organized single and mixed biofilms, reinforcing the participation of both fungi in the etiology of this OM case. R. similis seemed to grow faster during the process, suggesting that T. rubrum benefits from biofilm development when in combination. Moreover, the biofilm of the Rhinocladiella isolate exhibited exacerbated production of the extracellular matrix, which was not observed with that of a Rhinocladiella reference strain, suggesting that the isolate had natural abilities that were possibly perfected during development in the nail of the patient.

Spatially-ordered layer-by-layer biofilms of a two-species microbial consortium promote hydrogen production

The low hydrogen production efficiency caused by volatile fatty acids stress is the main technical barrier in the field of biohydrogen production. To enhance the resistance to acid stress and improve the synergistic efficiency of hydrogen-producing bacteria, spatially-ordered layer-by-layer biofilms of a two-species microbial consortium, consisting of Bacillus cereus A1 and Brevumdimonas naejangsanensis B1, were developed. The results showed that the spatially-ordered biofilms (SBs) significantly improved the cooperation efficiency of the two strains, and the hydrogen production increased by 103.27% and 66.50% compared with suspended fermentation and mixed biofilms (MBs) fermentation, respectively. Furthermore, the mixed ratios of the two strains were further optimized, and the highest hydrogen accumulation of 1755 mL L−1 was obtained at the inoculation volume ratio of A1:B1 = 1:1. Finally, the biofilms were used for 5 repeat batches of fermentation, and the hydrogen production performance was stable, with an average hydrogen accumulation of 1736 mL L−1. This study proposed a layer-by-layer biofilm hydrogen production system based on the spatially ordered distribution of functional bacteria, which significantly improved the cooperation efficiency and stability of the two-species microbial consortium.

In vitro evaluation of biofilm formation by Candida parapsilosis and Enterobacter cloacae. Scanning electron microscopy and efficacy of antimicrobial combinations study

Fungal–bacterial infections are being increasingly recognized in clinical settings, and the interaction between these species in polymicrobial biofilms often lead to infections that are highly resistant to treatment. In this in vitro study, we analyzed the formation of mixed biofilms using clinically isolated Candida parapsilosis and Enterobacter cloacae. Additionally, we assessed the potential of conventional antimicrobials, both alone and in combination, for treating polymicrobial biofilms built by these human pathogens. Our results demonstrate that C. parapsilosis and E. cloacae are capable of forming mixed biofilms, as confirmed by scanning electron microscopy. Interestingly, we found that colistin alone or in combination with antifungal drugs was highly effective reducing up to 80% of the total biomass of polymicrobial biofilms.

Interspecies interactions in mixed-species biofilms formed by Enterococcus faecalis and gram-negative bacteria isolated from polymicrobial diabetic foot ulcers

Diabetic foot ulcers (DFU) are exacerbated by bacterial colonisation. Here, a high prevalence of Enterococcus faecalis was observed in DFU patients from an Argentinean hospital. E. faecalis was frequently co-isolated with Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa. The effect of interspecies interactions on bacterial growth was investigated in mixed-species macrocolony biofilms developed in Lubbock-Glc-agar. Similar cell counts were found for E. faecalis and M. morganii growing in mixed and single-species biofilms. An E. faecalis strain showed 1 Log higher cell counts in mixed biofilms with E. coli. Remarkably, E. faecalis strains showed 2 to 4 Log higher cell counts in mixed biofilms with P. aeruginosa. This effect was not observed in planktonic growth or biofilms developed in tryptic soy agar. The present findings reveal bacterial interactions that benefit E. faecalis in mixed-species biofilms, mainly with P. aeruginosa, in a medium that partially mimics the nutrients found in DFU.

Case of Mixed Infection of Toenail Caused by Candida parapsilosis and Exophiala dermatitidis and In Vitro Effectiveness of Propolis Extract on Mixed Biofilm

Onychomycosis is a chronic fungal nail infection caused by several filamentous and yeast-like fungi, such as the genus Candida spp., of great clinical importance. Black yeasts, such as Exophiala dermatitidis, a closely related Candida spp. species, also act as opportunistic pathogens. Fungi infectious diseases are affected by organisms organized in biofilm in onychomycosis, making treatment even more difficult. This study aimed to evaluate the in vitro susceptibility profile to propolis extract and the ability to form a simple and mixed biofilm of two yeasts isolated from the same onychomycosis infection. The yeasts isolated from a patient with onychomycosis were identified as Candida parapsilosis sensu stricto and Exophiala dermatitidis. Both yeasts were able to form simple and mixed (in combination) biofilms. Notably, C. parapsilosis prevailed when presented in combination. The susceptibility profile of propolis extract showed action against E. dermatitidis and C. parapsilosis in planktonic form, but when the yeasts were in mixed biofilm, we only observed action against E. dermatitidis, until total eradication.

Dual Burst and Sustained Release of p-Coumaric Acid from Shape Memory Polymer Foams for Polymicrobial Infection Prevention in Trauma-Related Hemorrhagic Wounds.

Hemorrhage is the primary cause of trauma-related death. Of patients that survive, polymicrobial infection occurs in 39% of traumatic wounds within a week of injury. Moreover, traumatic wounds are susceptible to hospital-acquired and drug-resistant bacterial infections. Thus, hemostatic dressings with antimicrobial properties could reduce morbidity and mortality to enhance traumatic wound healing. To that end, p-coumaric acid (PCA) was incorporated into hemostatic shape memory polymer foams by two mechanisms (chemical and physical) to produce dual PCA (DPCA) foams. DPCA foams demonstrated excellent antimicrobial and antibiofilm properties against native Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis; co-cultures of E. coli and S. aureus; and drug-resistant S. aureus and S. epidermidis at short (1 h) and long (7 days) time points. Resistance against biofilm formation on the sample surfaces was also observed. In ex vivo experiments in a porcine skin wound model, DPCA foams exhibited similarly high antimicrobial properties as those observed in vitro, indicating that PCA was released from the DPCA foam to successfully inhibit bacterial growth. DPCA foams consistently showed improved antimicrobial properties relative to those of clinical control foams containing silver nanoparticles (AgNPs) against single and mixed species bacteria, single and mixed species biofilms, and bacteria in the ex vivo wound model. This system could allow for physically incorporated PCA to first be released into traumatic wounds directly after application for instant wound disinfection. Then, more tightly tethered PCA can be continuously released into the wound for up to 7 days to kill additional bacteria and protect against biofilms.

Streptococcus salivarius as an Important Factor in Dental Biofilm Homeostasis: Influence on Streptococcus mutans and Aggregatibacter actinomycetemcomitans in Mixed Biofilm.

A disturbed balance within the dental biofilm can result in the dominance of cariogenic and periodontopathogenic species and disease development. Due to the failure of pharmacological treatment of biofilm infection, a preventive approach to promoting healthy oral microbiota is necessary. This study analyzed the influence of Streptococcus salivarius K12 on the development of a multispecies biofilm composed of Streptococcus mutans, S. oralis and Aggregatibacter actinomycetemcomitans. Four different materials were used: hydroxyapatite, dentin and two dense polytetrafluoroethylene (d-PTFE) membranes. Total bacteria, individual species and their proportions in the mixed biofilm were quantified. A qualitative analysis of the mixed biofilm was performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results showed that in the presence of S. salivarius K 12 in the initial stage of biofilm development, the proportion of S. mutans was reduced, which resulted in the inhibition of microcolony development and the complex three-dimensional structure of the biofilm. In the mature biofilm, a significantly lower proportion of the periodontopathogenic species A. actinomycetemcomitans was found in the salivarius biofilm. Our results show that S. salivarius K 12 can inhibit the growth of pathogens in the dental biofilm and help maintain the physiological balance in the oral microbiome.

Synergistic interactions in multispecies biofilm combinations of bacterial isolates recovered from diverse food processing industries.

Most biofilms within the food industry are formed by multiple bacterial species which co-exist on surfaces as a result of interspecies interactions. These ecological interactions often make these communities tolerant against antimicrobials. Our previous work led to the identification of a large number (327) of highly diverse bacterial species on food contact surfaces of the dairy, meat, and egg industries after routine cleaning and disinfection (C&D) regimes. In the current study, biofilm-forming ability of 92 bacterial strains belonging to 26 genera and 42 species was assessed and synergistic interactions in biofilm formation were investigated by coculturing species in all possible four-species combinations. Out of the total 455 four-species biofilm combinations, greater biofilm mass production, compared to the sum of biofilm masses of individual species in monoculture, was observed in 34 combinations. Around half of the combinations showed synergy in biofilm mass > 1.5-fold and most of the combinations belonged to dairy strains. The highest synergy (3.13-fold) was shown by a combination of dairy strains comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus. The observed synergy in mixed biofilms turned out to be strain-specific rather than species-dependent. All biofilm combinations showing remarkable synergy appeared to have certain common species in all combinations which shows there are keystone industry-specific bacterial species which stimulate synergy or antagonism and this may have implication for biofilm control in the concerned food industries.

Inhibition of Mixed Biofilms of Candida albicans and Staphylococcus aureus by β-Caryophyllene-Gold Nanoparticles.

Polymicrobial biofilms, consisting of fungal and bacterial pathogens, often contribute to the failure of antimicrobial treatment. The growing resistance of pathogenic polymicrobial biofilms to antibiotics has led to the development of alternative strategies to combat polymicrobial diseases. To this end, nanoparticles synthesized using natural molecules have received significant attention for disease treatment. Here, gold nanoparticles (AuNPs) were synthesized using β-caryophyllene, a bioactive compound isolated from various plant species. The shape, size, and zeta potential of the synthesized β-c-AuNPs were found to be non-spherical, 17.6 ± 1.2 nm, and -31.76 ± 0.73 mV, respectively. A mixed biofilm of Candida albicans and Staphylococcus aureus was used to test the efficacy of the synthesized β-c-AuNPs. The results revealed a concentration-dependent inhibition of the initial stages of formation of single-species as well as mixed biofilms. Furthermore, β-c-AuNPs also eliminated mature biofilms. Therefore, using β-c-AuNPs to inhibit biofilm and eradicate bacterial-fungal mixed biofilms represents a promising therapeutic approach for controlling polymicrobial infections.

Inhibitory effects of Stevioside on Streptococcus mutans and Candida albicans dual-species biofilm.

Streptococcus mutans is the most prevalent biofilm-forming pathogen in dental caries, while Candida albicans is often detected in the presence of S. mutans. We aimed to evaluate the anti-caries effect of stevioside in medium trypticase soy broth (TSB) with or without sucrose supplementation compared with the same sweetness sucrose and xylitol in a dual-species model of S. mutans and C. albicans, based on planktonic growth, crystal violet assay, acid production, biofilm structural imaging, confocal laser scanning microscopy, and RNA sequencing. Our results showed that compared with sucrose, stevioside significantly inhibited planktonic growth and acid production, changed the structure of the mixed biofilm, and reduced the viability of biofilm and the production of extracellular polysaccharides in dual-species biofilm. Through RNA-seq, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact analysis showed that stevioside decreased sucrose metabolism and increased galactose and intracellular polysaccharide metabolism in S. mutans, and decreased genes related to GPI-modified proteins and secreted aspartyl proteinase (SAP) family in C. albicans. In contrast to xylitol, stevioside also inhibited the transformation of fungal morphology of C. albicans, which did not form mycelia and thus had reduced pathogenicity. Stevioside revealed a superior suppression of dual-species biofilm formation compared to sucrose and a similar anti-caries effect with xylitol. However, sucrose supplementation diminished the suppression of stevioside on S. mutans and C. albicans. Our study is the first to confirm that stevioside has anticariogenic effects on S. mutans and C. albicans in a dual-species biofilm. As a substitute for sucrose, it may help reduce the risk of developing dental caries.

Acetate modulates the inhibitory effect of Lactobacillus gasseri against the pathogenic yeasts Candida albicans and Candida glabrata

The exploration of the interference prompted by commensal bacteria over fungal pathogens is an interesting alternative to develop new therapies. In this work we scrutinized how the presence of the poorly studied vaginal species Lactobacillus gasseri affects relevant pathophysiological traits of Candida albicans and Candida glabrata. L. gasseri was found to form mixed biofilms with C. albicans and C. glabrata resulting in pronounced death of the yeast cells, while bacterial viability was not affected. Reduced viability of the two yeasts was also observed upon co-cultivation with L. gasseri under planktonic conditions. Either in planktonic cultures or in biofilms, the anti-Candida effect of L. gasseri was augmented by acetate in a concentration-dependent manner. During planktonic co-cultivation the two Candida species counteracted the acidification prompted by L. gasseri thus impacting the balance between dissociated and undissociated organic acids. This feature couldn't be phenocopied in single-cultures of L. gasseri resulting in a broth enriched in acetic acid, while in the co-culture the non-toxic acetate prevailed. Altogether the results herein described advance the design of new anti-Candida therapies based on probiotics, in particular, those based on vaginal lactobacilli species, helping to reduce the significant burden that infections caused by Candida have today in human health.

Rapid Inactivation of mixed biofilms of Candida albicans and Candida tropicalis using antibacterial photodynamic therapy: Based on PAD™ Plus

BackgroundTo investigate the sterilizing effect of antimicrobial photodynamic therapy (aPDT) based on PAD™ Plus on mixed biofilms of Candida albicans and Candida tropicalis. MethodsA mature mixed biofilm model of C. albicans and C. tropicalis was constructed in vitro. FITC-concanavalin A staining was conducted to observe the formation of the extracellular matrix. MTT assay was performed to determine biofilm viability. The chromogenic medium was used to examine the Candida composition of the mixed biofilms. For aPDT treatment, based on PAD™ Plus, the biofilms were incubated with 1 mg/mL TBO for 1, 5, or 10 min, followed by 500 or 750 mW LED illumination for 1 or 2 min. The live/dead fungi were detected by SYTO9/propidium iodide staining. A multivariate factorial design was conducted to analyze the correlations of parameters with the inactivation effect of the mixed biofilms. ResultsMature mixed biofilms formed at 24 h after seeding. Compared with untreated biofilms, following 1-min TBO incubation, 500 mW LED illumination for 1 min inactivated more than 90% of the fungi. Extending the incubation time did not significantly improve the inactivation effect. Application of 750 mW output power or 2 min LED illumination inactivated more than 99% of the fungi without increasing other parameters. ConclusionsPAD™ Plus combined with 1 mg/mL TBO can rapidly inactivate the mature mixed biofilms of C. albicans and C. tropicalis, serving as a robust platform for the treatment of mixed infections of C. albicans and C. tropicalis.

Biological and synthetic surfactant exposure increases antimicrobial gene occurrence in a freshwater mixed microbial biofilm environment.

Aquatic habitats are particularly susceptible to chemical pollution, such as antimicrobials, from domestic, agricultural, and industrial sources. This has led to the rapid increase of antimicrobial resistance (AMR) gene prevalence. Alternate approaches to counteract pathogenic bacteria are in development including synthetic and biological surfactants such as sodium dodecyl sulfate (SDS) and rhamnolipids. In the aquatic environment, these surfactants may be present as pollutants with the potential to affect biofilm formation and AMR gene occurrence. We tested the effects of rhamnolipid and SDS on aquatic biofilms in a freshwater stream in Northern Ireland. We grew biofilms on contaminant exposure substrates deployed within the stream over 4 weeks. We then extracted DNA and carried out shotgun sequencing using a MinION portable sequencer to determine microbial community composition, with 16S rRNA analyses (64,678 classifiable reads identified), and AMR gene occurrence (81 instances of AMR genes over 9 AMR gene classes) through a metagenomic analysis. There were no significant changes in community composition within all systems; however, biofilm exposed to rhamnolipid had a greater number of unique taxa as compared to SDS treatments and controls. AMR gene prevalence was higher in surfactant-treated biofilms, although not significant, with biofilm exposed to rhamnolipids having the highest presence of AMR genes and classes compared to the control or SDS treatments. Our results suggest that the presence of rhamnolipid encourages an increase in the prevalence of AMR genes in biofilms produced in mixed-use water bodies.