Comparative analysis of biofilm characterization of probiotic Escherichia coli.

Biofilms are thought to play a vital role in the beneficial effects of probiotic bacteria. However, the structure and function of probiotic biofilms are poorly understood. In this work, biofilms of Escherichia coli (E. coli) Nissle 1917 were investigated and compared with those of pathogenic and opportunistic strains (E. coli MG1655, O157:H7) using crystal violet assay, confocal laser scanning microscopy, scanning electron microscopy and FTIR microspectroscopy. The study revealed significant differences in the morphological structure, chemical composition, and spatial heterogeneity of the biofilm formed by the probiotic E. coli strain. In particular, the probiotic biofilm can secrete unique phospholipid components into the extracellular matrix. These findings provide new information on the morphology, architecture and chemical heterogeneity of probiotic biofilms. This information may help us to understand the beneficial effects of probiotics for various applications.

A meta-analysis on the role of sonication in the diagnosis of cardiac implantable electronic device-related infections.

Introduction: One of the biggest obstacles in diagnosing Implant-Associated Infections is the lack of infection criteria and standardized diagnostic methods. These infections present a wide range of symptoms, and their diagnosis can be hampered by the formation of microbial biofilms on the surface of implants. This study aimed to provide insight into the performance of sonication in the diagnosis of infections associated with Cardiac Implantable Electronic Devices, to help define a consensus on the algorithm for the microbial diagnosis of these infections. Methods: We carried out a systematic review with meta-analysis. The PRISMA methodology guidelines were followed, and an advanced search was carried out in PubMed and Web of Science, which enabled 8 articles to be included in the review, in which a meta-analysis was also carried out. QUADAS-2 was used to assess the risk of bias and effect measures were calculated to assess publication bias. Results: The overall sensitivity of the method was 0.823 (95% CI: 0.682-0.910) and the specificity was 0.632 (95% CI: 0.506-0.743). Discussion: These results suggest that sonication may offer advantages in diagnosing these infections. However, it is essential to approach these findings carefully and take into account the recommendations provided in the EHRA 2019 guidelines. This study highlights the importance of more effective diagnostic approaches for implantable medical device-associated infections to improve the quality of treatment and minimize the risks associated with these challenging medical conditions.

Cassava pomace-based biodegradable packaging materials: a review.

Starch-based biodegradable packaging materials are gaining popularity as an alternative to the adverse environmental effects caused by conventional packaging materials. Despite the fact that cassava can withstand harsh environmental conditions and convert a greater quantity of solar energy into carbohydrates, its postharvest shelf life is extremely short. The preparation of cassava starch is an important method for extending the storage life of cassava. When one ton of cassava is processed, approximately 900 kg of cassava pomace, also known as cassava bagasse and cassava pulp, are produced. Due to the high residual starch and fibre content, reinforced packaging materials made from cassava pomace predominate. In the present manuscript, many possible uses of cassava pomace in packaging materials are discussed.  Furthermore, the performance attributes of packing materials assume a crucial role in the evaluation of the quality of the respective materials. The manuscript discusses various performance characteristics of packaging materials derived from cassava pomace. The features discussed include water vapour permeability, moisture content, solubility, thickness, colour, light barrier properties, mechanical properties, FT-IR analysis, thermal stability, biodegradation, contact angle, and the presence of plasticizers. Though cassava starch film has become a favourable substitute for conventional packaging materials, commercialization is limited due to having drawbacks, and the current solutions are also catalogued in this review.

Evaluation of visible light and natural photosensitizers against Staphylococcus epidermidis and Staphylococcus saprophyticus planktonic cells and biofilm.

Antimicrobial photoinactivation (API) has shown some promise in potentially treating different nosocomial bacterial infections, however, its application on staphylococci, especially other than Staphylococcus aureus or methicillin-resistant S. aureus (MRSA) species is still limited. Although S. aureus is a well-known and important nosocomial pathogen, several other species of the genus, particularly coagulase-negative Staphylococcus (CNS) species such as Staphylococcus epidermidis and Staphylococcus saprophyticus, can also cause healthcare-associated infections and foodborne intoxications. CNS are often involved in resilient biofilm formation on medical devices and can cause infections in patients with compromised immune systems or those undergoing invasive procedures. In this study, the effects of chlorophyllin and riboflavin-mediated API on S. epidermidis and S. saprophyticus planktonic cells and biofilm are demonstrated for the first time. Based on the residual growth determination and metabolic reduction ability changes, higher inactivating efficiency of chlorophyllin-mediated API was determined against the planktonic cells of both tested species of bacteria and against S. saprophyticus biofilm. Some insights on whether aqueous solutions of riboflavin and chlorophyllin, when illuminated with optimal exciting wavelength (440 nm and 402 nm, respectively) generate O2-•, are also provided in this work.

Metal-based nanoparticles: an alternative treatment for biofilm infection in hard-to-heal wounds.

Metal-based nanoparticles (MNPs) are promoted as effective compounds in the treatment of bacterial infections and as possible alternatives to antibiotics. These MNPs are known to affect a broad spectrum of microorganisms using a multitude of strategies, including the induction of reactive oxygen species and interaction with the inner structures of the bacterial cells. The aim of this review was to summarise the latest studies about the effect of metal-based nanoparticles on pathogenic bacterial biofilm formed in wounds, using the examples of Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Pseudomonas aeruginosa, as well as provide an overview of possible clinical applications.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries.

Compact and water flushing resistant mesh biofilms forming at short SRT disappeared naturally under extended SRT in dynamic membrane bioreactor.

Extending the solids retention time (SRT) has been demonstrated to mitigate membrane biofouling. Nevertheless, it remains an intriguing question whether the compact and water flushing resistant mesh biofilms developed at short SRT can undergo biodegradation and be removed with extended SRT. In present study, the bio-fouled mesh filter in the 10d-SRT dynamic membrane bioreactor (DMBR), with mesh surfaces and pores covered by compact and water flushing resistant biofilms exhibiting low water permeability, was reused in the 40d-SRT DMBR without any cleanings. After being reused at 40d-SRT, its flux driven by gravity occurred from the 10th day and recovered to a regular level of 36.7 L m-2·h-1 on the 27th day. Both scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) analyses indicated that the compact mesh biofilms formed at10d-SRT biodegraded and were removed at 40d-SRT, with the residual biofilms becoming removable by water flushing. As a result, the hydraulic resistance of the bio-fouled mesh filter decreased from 4.36 × 108 to 6.97 × 107 m-1, and its flux fully recovered. The protein and polysaccharides densities in mesh-biofilms decreased from 24.4 to 9.7 mg/cm2 and from 10.7 to 0.10 mg/cm2, respectively, which probably have contributed to the disappearance of compact biofilms and the decrease in adhesion. Furthermore, the sludge and mesh-biofilms in the 40d-SRT reactor contained a higher relative abundance of dominant quorum quenching bacteria, such as Rhizobium (3.52% and 1.35%), compared to those in the 10d-SRT sludge (0.096%) and mesh biofilms (0.79%), which might have been linked to a decline in extracellular polymeric substances and, consequently, the biodegradation and disappearance of compact biofilms.

Substrate geometry affects population dynamics in a bacterial biofilm.

Biofilms inhabit a range of environments, such as dental plaques or soil micropores, often characterized by noneven surfaces. However, the impact of surface irregularities on the population dynamics of biofilms remains elusive, as most experiments are conducted on flat surfaces. Here, we show that the shape of the surface on which a biofilm grows influences genetic drift and selection within the biofilm. We culture Escherichia coli biofilms in microwells with a corrugated bottom surface and observe the emergence of clonal sectors whose size corresponds to that of the corrugations, despite no physical barrier separating different areas of the biofilm. The sectors are remarkably stable and do not invade each other; we attribute this stability to the characteristics of the velocity field within the biofilm, which hinders mixing and clonal expansion. A microscopically detailed computer model fully reproduces these findings and highlights the role of mechanical interactions such as adhesion and friction in microbial evolution. The model also predicts clonal expansion to be limited even for clones with a significant growth advantage-a finding which we confirm experimentally using a mixture of antibiotic-sensitive and antibiotic-resistant mutants in the presence of sublethal concentrations of the antibiotic rifampicin. The strong suppression of selection contrasts sharply with the behavior seen in range expansion experiments in bacterial colonies grown on agar. Our results show that biofilm population dynamics can be affected by patterning the surface and demonstrate how a better understanding of the physics of bacterial growth can be used to control microbial evolution.

Oral bacterial insights from a comparative study between healthy and comorbid diseased human individuals.

The human oral cavity is colonized by a diverse microbial community, which includes both native and transient colonizers. The microbial composition is crucial for maintaining oral homeostasis, but due to overgrowth or imbalances of these microbial communities, dysbiosis can occur. There is a lack of understanding of the research of native and transient colonizers in the oral cavity of the Indian subpopulation Therefore, in our present study, we explored the role and prevalence of transient and native colonizers between healthy and comorbid oral diseased human individuals. Culture-dependent techniques and culture-independent 16S r DNA metagenomic analyses were employed to isolate and study the interactions of native and transient colonizers from human oral samples. Among the 66 human individuals of both healthy and comorbid individuals, the most abundant isolate was found to be Bacillus amyloliquefaciens MCC 4424. In addition, the more prevalent culturable isolate from the healthy samples was Streptococcus salivarius MTCC 13009, whereas in comorbid samples Staphylococcus pasteuri MTCC 13076, Rothia dentocariosa MTCC 13010 and Pseudomonas aeruginosa MTCC 13077 were prevalent to a greater extent. 16S rDNA metagenomic analyses revealed the prevalence and abundance of genera such as Bacteroidetes and Proteobacteria in healthy individuals; consequently, Fusobacteria and Firmicutes were observed mostly in comorbid individuals. The significant differences in bacterial population density were observed in terms of the Shannon index (p = 0.5145) and Simpson index (p = 0.9061) between the healthy and comorbid groups. B. amyloliquefaciens MCC 4424 exhibits antagonistic behavior when grown as a dual-species with native and transient colonizers. This result is very consistent with the findings of antibiofilm studies using confocal laser scanning microscopy, which revealed a significant reduction in biofilm biovolume (73 %) and maximum thickness (80 %) and an increase in the rough coefficient of biofilms (30 %). Our data suggested that B. amyloliquefaciens MCC 4424 can be a native colonizer of Indian sub-populations. It may act as a novel candidate for oral healthcare applications and greatly aids in the regulation of transient species in the oral cavity.

Cell density and extracellular matrix composition mitigate bacterial biofilm sensitivity to UV-C LED irradiation.

Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm2) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 105 and 109 CFU/cm2, and biofilms at 108 CFU/cm2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm2) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties.

Optimization of periodic treatment strategies for bacterial biofilms using an agent-based in silico approach.

Biofilms are responsible for most chronic infections and are highly resistant to antibiotic treatments. Previous studies have demonstrated that periodic dosing of antibiotics can help sensitize persistent subpopulations and reduce the overall dosage required for treatment. Because the dynamics and mechanisms of biofilm growth and the formation of persister cells are diverse and are affected by environmental conditions, it remains a challenge to design optimal periodic dosing regimens. Here, we develop a computational agent-based model to streamline this process and determine key parameters for effective treatment. We used our model to test a broad range of persistence switching dynamics and found that if periodic antibiotic dosing was tuned to biofilm dynamics, the dose required for effective treatment could be reduced by nearly 77%. The biofilm architecture and its response to antibiotics were found to depend on the dynamics of persister cells. Despite some differences in the response of biofilm governed by different persister switching rates, we found that a general optimized periodic treatment was still effective in significantly reducing the required antibiotic dose. As persistence becomes better quantified and understood, our model has the potential to act as a foundation for more effective strategies to target bacterial infections.

Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization.

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

DeepSeeded: Volumetric Segmentation of Dense Cell Populations with a Cascade of Deep Neural Networks in Bacterial Biofilm Applications.

Accurate and automatic segmentation of individual cell instances in microscopy images is a vital step for quantifying the cellular attributes, which can subsequently lead to new discoveries in biomedical research. In recent years, data-driven deep learning techniques have shown promising results in this task. Despite the success of these techniques, many fail to accurately segment cells in microscopy images with high cell density and low signal-to-noise ratio. In this paper, we propose a novel 3D cell segmentation approach DeepSeeded, a cascaded deep learning architecture that estimates seeds for a classical seeded watershed segmentation. The cascaded architecture enhances the cell interior and border information using Euclidean distance transforms and detects the cell seeds by performing voxel-wise classification. The data-driven seed estimation process proposed here allows segmenting touching cell instances from a dense, intensity-inhomogeneous microscopy image volume. We demonstrate the performance of the proposed method in segmenting 3D microscopy images of a particularly dense cell population called bacterial biofilms. Experimental results on synthetic and two real biofilm datasets suggest that the proposed method leads to superior segmentation results when compared to state-of-the-art deep learning methods and a classical method.

Impact of lactic acid bacteria inoculation on fungal diversity during Spanish-style green table olive fermentations.

This study delved into the evolution of fungal population during the fermentation of Spanish-style green table olives (Manzanilla cultivar), determining the influence of different factors such as fermentation matrix (brine or fruit) or the use of a lactic acid bacteria inoculum, on its distribution. The samples (n = 24) were directly obtained from industrial fermentation vessels with approximately 10.000 kg of fruits and 6.000 L of brines. Our findings showcased a synchronized uptick in lactic acid bacteria counts alongside fungi proliferation. Metataxonomic analysis of the Internal Transcribed Spacer (ITS) region unearthed noteworthy disparities across different fermentation time points (0, 24, and 83 days). Statistical analysis pinpointed two Amplicon Sequence Variants (ASV), Candida and Aureobasidium, as accountable for the observed variances among the different fermentation time samples. Notably, Candida exhibited a marked increase during 83 days of fermentation, opposite to Aureobasidium, which demonstrated a decline. Fungal biodiversity was slightly higher in brines than in fruits, whilst no effect of inoculation was noticed. At the onset of fermentation, prominently detected genera were also Mycosphaerella (19.82 %) and Apohysomyces (16.31 %), hitherto unreported in the context of table olive processing. However, their prevalence dwindled to nearly negligible levels from 24th day fermentation onwards (<2 %). On the contrary, they were replaced by the fermentative yeasts Saccharomyces and Isstachenkia. Results obtained in this work will be useful for designing new strategies for better control of table olive fermentations.

Nitrate increases the capacity of an aerobic moving-bed biofilm reactor (MBBR) for winery wastewater treatment.

We used bench-scale tests and mathematical modeling to explore chemical oxygen demand (COD) removal rates in a moving-bed biofilm reactor (MBBR) for winery wastewater treatment, using either urea or nitrate as a nitrogen source. With urea addition, the COD removal fluxes ranged from 34 to 45 gCOD/m2-d. However, when nitrate was added, fluxes increased up to 65 gCOD/m2-d, twice the amount reported for aerobic biofilms for winery wastewater treatment. A one-dimensional biofilm model, calibrated with data from respirometric tests, accurately captured the experimental results. Both experimental and modelling results suggest that nitrate significantly increased MBBR capacity by stimulating COD oxidation in the deeper, oxygen-limited regions of the biofilm. Our research suggests that the addition of nitrate, or other energetic and broadly used electron acceptors, may provide a cost-effective means of covering peak COD loads in biofilm processes for winery or another industrial wastewater treatment.

Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

OBJECTIVE: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization. METHODS: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05). RESULTS: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium. CONCLUSIONS: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed. CLINICAL SIGNIFICANCE: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss.

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater.

Organic contaminants enter aquatic ecosystems from various sources, including wastewater treatment plant effluent. Freshwater biofilms play a major role in the removal of organic contaminants from receiving water bodies, but knowledge of the molecular mechanisms driving contaminant biotransformations in complex stream biofilm (periphyton) communities remains limited. Previously, we demonstrated that biofilms in experimental flume systems grown at higher ratios of treated wastewater (WW) to stream water displayed an increased biotransformation potential for a number of organic contaminants. We identified a positive correlation between WW percentage and biofilm biotransformation rates for the widely-used insect repellent, N,N-diethyl-meta-toluamide (DEET) and a number of other wastewater-borne contaminants with hydrolyzable moieties. Here, we conducted deep shotgun sequencing of flume biofilms and identified a positive correlation between WW percentage and metagenomic read abundances of DEET hydrolase (DH) homologs. To test the causality of this association, we constructed a targeted metagenomic library of DH homologs from flume biofilms. We screened our complete metagenomic library for activity with four different substrates, including DEET, and a subset thereof with 183 WW-related organic compounds. The majority of active hydrolases in the metagenomic library preferred aliphatic and aromatic ester substrates while, remarkably, only a single reference enzyme was capable of DEET hydrolysis. Of the 626 total enzyme-substrate combinations tested, approximately 5% were active enzyme-substrate pairs. Metagenomic DH family homologs revealed a broad substrate promiscuity spanning 22 different compounds when summed across all enzymes tested. We biochemically characterized the most promiscuous and active enzymes identified based on metagenomic analysis from uncultivated Rhodospirillaceae and Planctomycetaceae. In addition to characterizing new DH family enzymes, we exemplified a framework for linking metagenome-guided hypothesis generation with experimental validation. Overall, this study expands the scope of known enzymatic contaminant biotransformations for metagenomic hydrolases from WW-receiving stream biofilm communities.

Interspecies interactions in dairy biofilms drive community structure and response against cleaning and disinfection.

Interspecies interactions within a biofilm community influence population dynamics and community structure, which in turn may affect the bacterial stress response to antimicrobials. This study was conducted to assess the impact of interactions between Kocuria salsicia and a three-species biofilm community (comprising Stenotrophomonas rhizophila, Bacillus licheniformis, and Microbacterium lacticum) on biofilm mass, the abundance of individual species, and their survival under a laboratory-scale cleaning and disinfection (C&D) regime. The presence of K. salsicia enhanced the cell numbers of all three species in pairwise interactions. The outcomes derived from summing up pairwise interactions did not accurately predict the bacterial population dynamics within communities of more than two species. In four-species biofilms, we observed the dominance of S. rhizophila and B. licheniformis, alongside a concurrent reduction in the cell counts of K. salsicia and M. lacticum. This pattern suggests that the underlying interactions are not purely non-transitive; instead, a more complex interplay results in the dominance of specific species. We observed that bacterial spatial organization and matrix production in different mixed-species combinations affected survival in response to C&D. Confocal microscopy analysis of spatial organization showed that S. rhizophila localized on the biofilm formed by B. licheniformis and M. lacticum, and S. rhizophila was more susceptible to C&D. Matrix production in B. licheniformis, evidenced by alterations in biofilm mass and by scanning electron microscopy, demonstrated its protective role against C&D, not only for this species itself, but also for neighbouring species. Our findings emphasise that various social interactions within a biofilm community not only affect bacterial population dynamics but also influence the biofilm community's response to C&D stress.

Bactericidal and biofilm eradication efficacy of a fluorinated benzimidazole derivative, TFBZ, against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major inducement of nosocomial infections and its biofilm formation render the high tolerance to conventional antibiotics, which highlights the requirement to develop new antimicrobial agents urgently. In this study, we identified a fluorinated benzimidazole derivative, TFBZ, with potent antibacterial efficacy toward planktonic MRSA (MIC = 4 µg/mL, MBC = 8 µg/mL) and its persistent biofilms (≥99%, MBEC = 8 µg/mL). TFBZ manifested significant irreversible time-dependent killing against MRSA as characterized by diminished cell viability, bacterial morphological change and protein leakage. Furthermore, the results from CBD devices, crystal violet assay in conjunction with live/dead staining and scanning electron microscopy confirmed that TFBZ was capable of eradicating preformed MRSA biofilms with high efficiency. Simultaneously, TFBZ reduced the bacterial invasiveness and exerted negligible hemolysis and cytotoxicity toward mammalian cells, which ensuring the robust therapeutic effect on mouse skin abscess model. The transcriptome profiling and quantitative RT-PCR revealed that a set of encoding genes associated with cell adhesion, biofilm formation, translation process, cell wall biosynthesis was consistently downregulated in MRSA biofilms upon exposure to TFBZ. In conclusion, TFBZ holds promise as a valuable candidate for therapeutic applications against MRSA chronic infections.