Mixed-species Biofilms Research Articles

Associational Resistance to Predation by Protists in a Mixed Species Biofilm.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

Multiomics reveals the mechanism of B. longum in promoting the formation of mixed-species biofilms.

It has been found previously that Bifidobacterium longum, Bacteroides ovatus, Enterococcus faecalis, and Lactobacillus gasseri can form a biofilm better when co-cultured in vitro and B. longum is the core biofilm-formation-promoting strain in this community. B. longum is part of the core microbiota in the gut and is widely recognized as a probiotic. Therefore, it is necessary to explore its role in mixed-species biofilms through transcriptomics and metabolomics. Metabolomics showed that the increase in amino acid and purine content could promote biofilm formation. In transcriptomic analysis, many genes related to carbohydrate metabolism, amino acid metabolism, and environmental tolerance of B. longum were up-regulated. Combined with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis, the differentially expressed genes (DEGs) of B. longum in mixed-species biofilms were mainly correlated to "quorum sensing (QS)", "ABC transporters", "biosynthesis of amino acids", "microbial metabolism in different environments", "carbohydrate metabolism" and "two-component system". In addition, the rpl and rps gene families, which function in the metabolism of organic substances and the biosynthesis of amino acids, were the core DEGs according to the analysis of the protein-protein interaction (PPI) network. Finally, by combining metabolomics and quorum sensing mechanisms, it was found that the metabolism of autoinducer peptides (proliylglycine and glycylleucine), N-acyl homoserine lactone (N-(3-oxo hydroxy) homoserine lactone), and AI-2 can promote the formation of biofilms, both mono- and mixed-species biofilms composed of B. longum. Our research enabled us to understand the critical role of B. longum in mixed-species biofilms and the interactions between biofilm metabolism and gut health. In addition, the generated knowledge will be of great significance for us to develop biofilm products with beneficial functions in future.

Activity of Lipid Loaded Lectin against co-infection of Candida albicans and Staphylococcus aureus using the Zebrafish model.

Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70%), hyphae formation, staphyloxanthin biosynthesis (> 41%), and antioxidant enzyme and hemolysin activity (> 70%). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA.

Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms.

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.

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.

Protoporphyrin IX derived from dual-species anaerobic biofilms of Fusobacterium necrophorum and Porphyromonas levii attenuates bovine neutrophil function

Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of Fusobacterium necrophorum and Porphyromonas levii was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. Porphyromonas species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria.

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.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Extracellular Vesicles Contribute to Mixed-Fungal Species Competition during Biofilm Initiation.

Extracellular vesicles commonly modulate interactions among cellular communities. Recent studies demonstrate that biofilm maturation features, including matrix production, drug resistance, and dispersion, require the delivery of a core protein and carbohydrate vesicle cargo in Candida species. The function of the vesicle cargo for these advanced-phase biofilm characteristics appears to be conserved across Candida species. Mixed-species interactions in mature biofilms indicate that vesicle cargo serves a cooperative role in preserving the community. Here, we define the function of biofilm-associated vesicles for biofilm initiation both within and among five species across the Candida genus. We found similar vesicle cargo functions for several conserved proteins across species, based on the behavior of mutants. Repletion of the adhesion environment with wild-type vesicles returned the community phenotype toward reference levels in intraspecies experiments. However, cross-species vesicle complementation did not restore the wild-type biology and in fact drove the phenotype in the opposite direction for most cross-species interactions. Further study of mixed-species biofilm adhesion and exogenous wild-type vesicle administration similarly demonstrated competitive interactions. Our studies indicate that similar vesicle cargoes contribute to biofilm initiation. However, vesicles from disparate species serve an interference competitive role in mixed-Candida species scenarios. IMPORTANCE Candida species commonly form mixed-species biofilms with other Candida species and bacteria. In the established biofilm state, vesicle cargo delivers public goods to support the mature community. At biofilm initiation, however, vesicles play a negative role in cross-species interactions, presumably to allow species to gain a survival advantage. These observations and recent reports reveal that vesicle cargo has both cooperative and competitive roles among Candida species, depending on the needs of the community biofilm formation.

Cutaneous dysbiosis may amplify barrier dysfunction in patients with atopic dermatitis.

Atopic dermatitis (AD) is associated with cutaneous dysbiosis, barrier defects, and immune dysregulation, but the interplay between these factors needs further study. Early-onset barrier dysfunction may facilitate an innate immune response to commensal organisms and, consequently, the development of allergic sensitization. We aimed to compare the cutaneous microbiome in patients with active dermatitis with and without a history of childhood flexural dermatitis (atopic dermatitis). Next-gen Ion-Torrent deep-sequencing identified AD-associated changes in the skin bacterial microbiome ("bacteriome") and fungal microbiome ("mycobiome") of affected skin in swabs from areas of skin affected by dermatitis. Data were analyzed for diversity, abundance, and inter-kingdom correlations. Microbial interactions were assessed in biofilms using metabolic activity (XTT) assay and scanning electron microscopy (SEM), while host-pathogen interactions were determined in cultured primary keratinocytes exposed to biofilms. Increased richness and abundance of Staphylococcus, Lactococcus, and Alternaria were found in atopics. Staphylococcus and Alternaria formed robust mixed-species biofilms (based on XTT and SEM) that were resistant to antifungals/antimicrobials. Furthermore, their biofilm supernatant was capable of influencing keratinocytes biology (pro-inflammatory cytokines and structural proteins), suggesting an additive effect on AD-associated host response. In conclusion, microbial inter-kingdom and host-microbiome interactions may play a critical role in the modulation of atopic dermatitis to a greater extent than in non-atopic adults with allergic contact dermatitis.

Similarities and Differences among Species Closely Related to Candida albicans: C. tropicalis, C. dubliniensis, and C. auris

Although Candida species are widespread commensals of the microflora of healthy individuals, they are also among the most important human fungal pathogens that under certain conditions can cause diseases (candidiases) of varying severity ranging from mild superficial infections of the mucous membranes to life-threatening systemic infections. So far, the vast majority of research aimed at understanding the molecular basis of pathogenesis has been focused on the most common species—Candida albicans. Meanwhile, other closely related species belonging to the CTG clade, namely, Candida tropicalis and Candida dubliniensis, are becoming more important in clinical practice, as well as a relatively newly identified species, Candida auris. Despite the close relationship of these microorganisms, it seems that in the course of evolution, they have developed distinct biochemical, metabolic, and physiological adaptations, which they use to fit to commensal niches and achieve full virulence. Therefore, in this review, we describe the current knowledge on C. tropicalis, C. dubliniensis, and C. auris virulence factors, the formation of a mixed species biofilm and mutual communication, the environmental stress response and related changes in fungal cell metabolism, and the effect of pathogens on host defense response and susceptibility to antifungal agents used, highlighting differences with respect to C. albicans. Special attention is paid to common diagnostic problems resulting from similarities between these species and the emergence of drug resistance mechanisms. Understanding the different strategies to achieve virulence, used by important opportunistic pathogens of the genus Candida, is essential for proper diagnosis and treatment.

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.

Antimicrobial-free graphene nanocoating decreases fungal yeast-to-hyphal switching and maturation of cross-kingdom biofilms containing clinical and antibiotic-resistant bacteria

Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA) synergize in cross-kingdom biofilms to increase the risk of mortality and morbidity due to high resistance to immune and antimicrobial defenses. Biomedical devices and implants made with titanium are vulnerable to infections that may demand their surgical removal from the infected sites. Graphene nanocoating (GN) has promising anti-adhesive properties against C. albicans. Thus, we hypothesized that GN could prevent fungal yeast-to-hyphal switching and the development of cross-kingdom biofilms. Herein, titanium (Control) was coated with high-quality GN (coverage > 99%). Thereafter, mixed-species biofilms (C. albicans combined with S. aureus or MRSA) were allowed to develop on GN and Control. There were significant reductions in the number of viable cells, metabolic activity, and biofilm biomass on GN compared with the Control (CFU counting, XTT reduction, and crystal violet assays). Also, biofilms on GN were sparse and fragmented, whereas the Control presented several bacterial cells co-aggregating with intertwined hyphal elements (confocal and scanning electronic microscopy). Finally, GN did not induce hemolysis, an essential characteristic for blood-contacting biomaterials and devices. Thus, GN significantly inhibited the formation and maturation of deadly cross-kingdom biofilms, which can be advantageous to avoid infection and surgical removal of infected devices.

Adding a new dimension: Multi-level structure and organization of mixed-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a 4-D wound microenvironment

Biofilms in wounds typically consist of aggregates of bacteria, most often Pseudomonas aeruginosa and Staphylococcus aureus, in close association with each other and the host microenvironment. Given this, the interplay across host and microbial elements, including the biochemical and nutrient profile of the microenvironment, likely influences the structure and organization of wound biofilms. While clinical studies, in vivo and ex vivo model systems have provided insights into the distribution of P. aeruginosa and S. aureus in wounds, they are limited in their ability to provide a detailed characterization of biofilm structure and organization across the host-microbial interface. On the other hand, biomimetic in vitro systems, such as host cell surfaces and simulant media conditions, albeit reductionist, have been shown to support the co-existence of P. aeruginosa and S. aureus biofilms, with species-dependent localization patterns and interspecies interactions. Therefore, composite in vitro models that bring together key features of the wound microenvironment could provide unprecedented insights into the structure and organization of mixed-species biofilms. We have built a four-dimensional (4-D) wound microenvironment consisting of a 3-D host cell scaffold of co-cultured human epidermal keratinocytes and dermal fibroblasts, and an in vitro wound milieu (IVWM); the IVWM provides the fourth dimension that represents the biochemical and nutrient profile of the wound infection state. We leveraged this 4-D wound microenvironment, in comparison with biofilms in IVWM alone and standard laboratory media, to probe the structure of mixed-species P. aeruginosa and S. aureus biofilms across multiple levels of organization such as aggregate dimensions and biomass thickness, species co-localization and spatial organization within the biomass, overall biomass composition and interspecies interactions. In doing so, the 4-D wound microenvironment platform provides multi-level insights into the structure of mixed-species biofilms, which we incorporate into the current understanding of P. aeruginosa and S. aureus organization in the wound bed.

Bacteriophages: A weapon against mixed-species biofilms in the food processing environment.

Mixed-species biofilms represent the most frequent actual lifestyles of microorganisms in food processing environments, and they are usually more resistant to control methods than single-species biofilms. The persistence of biofilms formed by foodborne pathogens is believed to cause serious human diseases. These challenges have encouraged researchers to search for novel, natural methods that are more effective towards mixed-species biofilms. Recently, the use of bacteriophages to control mixed-species biofilms have grown significantly in the food industry as an alternative to conventional methods. This review highlights a comprehensive introduction of mixed-species biofilms formed by foodborne pathogens and their enhanced resistance to anti-biofilm removal strategies. Additionally, several methods for controlling mixed-species biofilms briefly focused on applying bacteriophages in the food industry have also been discussed. This article concludes by suggesting that using bacteriophage, combined with other 'green' methods, could effectively control mixed-species biofilms in the food industry.

P139 Candida : An important partner in polymicrobial colonization on indwelling medical devices

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMIndwelling medical devices are of routine use in hospitals for patient care but they lead to the colonization of microbes on their surface making the patient vulnerable to device-related infections (DRI). Biofilms formed due to the growth of microbes are the most common cause of device-related infections.ObjectivesTo isolate, identify and study the microbial colonization of indwelling medical devices (urinary catheters and central venous catheters).To quantify the biofilm formed by the isolates.MethodIn the present study, microflora was isolated from urinary catheters (n = 28) and central venous catheters (n = 25) that were catheterized in ICU patients. Quantification of biofilm was done by Crystal violet assay.ResultsAmong 28 urinary catheters, 14 catheters had polymicrobial colonisation (n = 10 mixed bacterial and fungal colonization, n = 3 polybacterial, and n = 1 polyfungal), 10 catheters were observed to have mono-species colonization (n = 6 fungal, n = 4 bacterial), while 4 catheters did not show any colonization. Whereas among 25 central venous catheters, only 3 catheters showed mono-bacterial colonization and 2 catheters showed mono-fungal growth and 20 catheters did not show any colonization. A total of 46 isolates were obtained from all urinary and central venous catheters, out of which Candida spp. was found to be highest in number with n = 13 and 30% of total isolates obtained (the Fig. 1 shows the distribution of microflora obtained). The interesting observation in the study is that Candida spp. is found to have multi-species colonization with bacterial isolates (Pseudomonas aeuroginosa, Klebsiella pneumoniae, Staphylococcus haemolyticus, Enterococcus faecium, and Stenotrophomonas maltophilia) as well as fungal isolates (Trichosporon asahii). Quantification of biofilm formed by these isolates was carried out by crystal violet assay and it was observed that 35.6% isolates formed strong biofilm of which Candida spp. was a major contributor. It was also observed that Candida spp. was always the stronger biofilm forming partner in mixed species biofilms.ConclusionIt was concluded that majority of colonization on indwelling medical devices are polymicrobial and Candida spp. appears as an important partner in polymicrobial colonization. Among all obtained isolates maximum were strong biofilm forming of which Candida spp. were the major contributors.

Effects of stabilized hypochlorous acid on oral biofilm bacteria

BackgroundCaries and periodontitis are amongst the most prevalent diseases worldwide, leading to pain and loss of oral function for those affected. Prevention relies heavily on mechanical removal of dental plaque biofilms but for populations where this is not achievable, alternative plaque control methods are required. With concerns over undesirable side-effects and potential bacterial resistance due to the use of chlorhexidine gluconate (CHX), new antimicrobial substances for oral use are greatly needed. Here we have investigated the antimicrobial effect of hypochlorous acid (HOCl), stabilized with acetic acid (HAc), on oral biofilms and compared it to that of CHX. Possible adverse effects of stabilized HOCl on hydroxyapatite surfaces were also examined.MethodsSingle- and mixed-species biofilms of six common oral bacteria (Streptococcus mutans, Streptococcus gordonii, Actinomyces odontolyticus, Veillonella parvula, Parvimonas micra and Porphyromonas gingivalis) within a flow-cell model were exposed to HOCl stabilized with 0.14% or 2% HAc, pH 4.6, as well as HOCl or HAc alone. Biofilm viability was assessed in situ using confocal laser scanning microscopy following LIVE/DEAD® BacLight™ staining. In-situ quartz crystal microbalance with dissipation (QCM-D) was used to study erosion of hydroxyapatite (HA) surfaces by stabilized HOCl.ResultsLow concentrations of HOCl (5 ppm), stabilized with 0.14% or 2% HAc, significantly reduced viability in multi-species biofilms representing supra- and sub-gingival oral communities, after 5 min, without causing erosion of HA surfaces. No equivalent antimicrobial effect was seen for CHX. Gram-positive and Gram-negative bacteria showed no significant differential suceptibility to stabilized HOCl.ConclusionsAt low concentrations and with exposure times which could be achieved through oral rinsing, HOCl stabilized with HAc had a robust antimicrobial activity on oral biofilms, without causing erosion of HA surfaces or affecting viability of oral keratinocytes. This substance thus appears to offer potential for prevention and/or treatment of oral biofilm-mediated diseases.

Potential of Bouea macrophylla kernel extract as an intracanal medicament against mixed-species bacterial-fungal biofilm. An in vitro and ex vivo study

ObjectiveTo investigate the antimicrobial activity of B. macrophylla kernel extract against mixed-species biofilms of E. faecalis, S. gordonii and C. albicans in vitro. To evaluate the efficacy of the extract as an intracanal medicament compared with Ca(OH)2 and chlorhexidine in ex vivo tooth model. MethodsThe antibiofilm effect of B. macrophylla kernel extract was determined by AlamarBlue™ assay and the effect on biofilms was visualized by LIVE/DEAD® BacLight™ viability test. Mixed-species biofilms were incubated into the tooth model (N = 42) for 21 days. The teeth were randomly divided into 4 medicament groups for 7 days: (i) normal saline, (ii) calcium hydroxide (Ca(OH)2), (iii) chlorhexidine gel, (iv) B. macrophylla kernel extract. Dentine samples were collected, qPCR with PMA was used to quantify the viability and species composition of each sample. SEM was used to visualize the effect of medicament on biofilm structure. ResultsThe MBIC was 6.25 mg/mL and the MBEC was 50 mg/mL. The integrity of microbial cells was progressively compromised as concentration increased, resulting in greater cell death. Ex vivo tooth model revealed that biofilm treated with 50 mg/mL of the B. macrophylla extract demonstrated a significantly higher proportions of dead cells than in Ca(OH)2, chlorhexidine and normal saline groups (p < 0.01). Disruption of biofilm structure and enlargement of dentinal tubules was observed in B. macrophylla group on SEM. ConclusionThe extract of B. macrophylla kernel exhibited significant antibiofilm effect against the mixed-species biofilms of E. faecalis, S. gordonii and C. albicans.

Antimicrobial action of four herbal plants over mixed-species biofilms of Candida albicans with four different microorganisms.

This study aimed to evaluate the antimicrobial effect of four herbal plants glycolic extracts over mixed-species biofilm composed of Candida albicans (C.albicans) and another pathogenic bacterium as alternative therapy to be investigated. Four plants extract of Pfaffia paniculata roots; Hamamelis virginiana leaf, Stryphnodendron barbatiman tree bark and Gymnema sylvestre stem and leaves were tested over multi-species biofilm of C.albicans (ATCC 18804) and Streptococcus mutans (ATCC 35688), Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 4083) or Pseudomonas aeruginosa (ATCC 15442) for 5min and 24h and colony forming units per millilitre was calculated. The data were analysed using Kruskal-Wallis with Dunn's test (p≤ 0.05). All tested extracts showed antimicrobial action over the mixed-species biofilms after 24h. Some extracts eliminated totally the biofilms. The glycolic extract of P.paniculata, H.virginiana, S.barbatiman and G.sylvestre are effective over mixed-species biofilms and may be indicated as endodontic irrigant or intracanal medication.

Co-culture with Acinetobacter johnsonii enhances benzalkonium chloride resistance in Salmonella enterica via triggering lipid A modifications

Salmonella enterica is one of the leading causes of foodborne gastroenteritis worldwide. In the food production environment, many bacterial species co-exist on surfaces in biofilm structures, which can act as reservoirs of microbial contamination of food products. Polymicrobial biofilms have been shown to have greater tolerance to antimicrobials, such as disinfectants, however the mechanistic basis of this is poorly understood. In this study, S. enterica subsp. enterica serovar Liverpool was co-cultured in mixed-species biofilms with bacteria isolated from the food production environment and challenged with the cationic biocide disinfectant, benzalkonium chloride (BC). Co-culture with the common environmental bacterium Acinetobacter johnsonii resulted in >200-fold higher resistance of S. Liverpool to BC, compared to mono-culture biofilms. The transcriptional response of S. enterica to biofilm co-culture was determined using a dual RNA-seq strategy. Genes controlled by the PhoPQ and PmrAB two-component systems, involved in lipid A modification and associated with cationic antimicrobial peptide resistance (CAMP) of S. Liverpool, were significantly upregulated. Deletion of either the phoP or pmrA genes resulted in an increase in susceptibility to BC, suggesting that activation of their regulons during co-culture enhances BC resistance. S. Liverpool lipid A profiles changed significantly upon co-culturing, with greater incorporation of both phosphoethanolamine and palmitate, which was dependent upon activation of PhoPQ and PmrAB. We conclude that when grown in the presence of A. johnsonii, S. Liverpool increases its tolerance to cationic BC disinfection by remodelling its cell envelope including reducing the net negative charge of lipid A and increasing lipid A acyl density.