Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in sub-optimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, nitrate or metal concentrations is underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimes of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse pH (3.5 to 5) and nitrate levels (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptation to survive and grow at low pH. Biofilms intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation warranting further investigation. Through RB-TnSeq, proteomics, use of specific mutants and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed absence of flagella and chemotaxis genes, and presence of putative Type VI secretion system in the high biofilm-forming strain FW021-MT20. This study identifies genetic determinants associated with biofilm growth in a predominant environmental genus, Rhodanobacter, under metal stress and identifies traits aiding survival and adaptation to contaminated subsurface environments.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

The main challenge in the large-scale application of MICP lies in its low efficiency and promoting biofilm growth can effectively address this problem. In the present study, a prediction model was proposed using the response surface method. With the prediction model, optimum concentrations of nutrients in the medium can be obtained. Moreover, the optimized medium was compared with other media via bio-cementation tests. The results show that this prediction model was accurate and effective, and the predicted results were close to the measured results. By using the prediction model, the optimized culture media was determined (20.0 g/l yeast extract, 10.0 g/l polypeptone, 5.0 g/l ammonium sulfate, and 10.0 g/l NaCl). Furthermore, the optimized medium significantly promoted the growth of biofilm compared to other media. In the medium, the effect of polypeptone on biofilm growth was smaller than the effect of yeast extract and increasing the concentration of polypeptone was not beneficial in promoting biofilm growth. In addition, the sand column solidified with the optimized medium had the highest strength and the largest calcium carbonate contents. The prediction model represents a platform technology that leverages culture medium to impart novel sensing, adjustive, and responsive multifunctionality to structural materials in the civil engineering and material engineering fields.

Biofouling has been a persistent problem hindering the application of membranes in water treatment, and quorum quenching has been identified as an effective method for mitigating biofouling, but surface accumulation of live bacteria still induces biofilm secretion, which poses a significant challenge for sustained prevention of membrane biofouling. In this study, we utilized quercetin, a typical flavonoid with the dual functions of quorum quenching and bacterial inactivation, to evaluate its role in preventing biofilm proliferation and against biofouling. Quercetin exhibited excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and the decreased bioactivity was positively correlated with the quercetin concentration, with inhibition rates of 53.1 % and 57.4 %, respectively, at the experimental concentrations. The RT-qPCR results demonstrated that quercetin inhibited AI-2 of E. coli and AGR of S. aureus mediated quorum sensing system, and reduced the expression of genes such as adhesion, virulence, biofilm secretion, and key regulatory proteases. As a result, the bacterial growth cycle was retarded and the biomass and biofilm maturation cycles were alleviated with the synergistic effect of quorum quenching and antibacterial activity. In addition, membrane biofouling was significantly declined in the dynamic operation experiments, dead cells in the biofilm overwhelmingly dominated, and the final normalized water fluxes were increased by more than 49.9 % and 34.5 % for E. coli and S. aureus, respectively. This work demonstrates the potential for mitigating biofouling using protocols that quorum quenching and inactivate bacteria, also provides a unique and long-lasting strategy to alleviate membrane fouling.

The properties of biocarriers significantly influence the performance of a moving bed-biofilm reactor (MBBR). This study aimed to assess the impact of media type, filling ratio, and hydraulic retention time (HRT) on biofilm formation and MBBR performance in both batch and continuous setups using real municipal wastewater. Two different media, high-density polyethylene (HDPE) and polypropylene (PPE), with varying surface area and properties were used. Biofilm growth and MBBR performance were monitored and optimized using response surface methodology. The effect of different media was investigated for three filling ratios of 20%, 40% and 60% and HRT of 4, 6 and 8 h. Results depicted a better biofilm growth on HDPE media in comparison to PPE carriers due to difference in media structure and surface properties. At all the conditions tested, HDPE media showed comparatively better performance for the removal of organic matter and nutrients than PPE media. The maximum organic matter removal efficiency was found as 77% and 75% at an HRT of 6 h and filling ratio of 40% for HDPE and PPE media, respectively. The ammonia removal was also found better for HDPE media due to its geometry and structure favoring the anoxic conditions with maximum removal of 89% achieved at 6-h HRT and 40% filling ratio. Overall, the system with HDPE media indicated more stability in terms of reactor performance than PPE carriers with variations in the operating conditions.

UNASSIGNED: Streptococcus mutans (S. mutans) is a pivotal cariogenic pathogen contributing to its multiple virulence factors, one of which is synthesizing exopolysaccharides (EPS). VicK, a sensor histidine kinase, plays a major role in regulating genes associated with EPS synthesis and adhesion. Here we first identified an antisense vicK RNA (ASvicK) bound with vicK into double-stranded RNA (dsRNA).
UNASSIGNED: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans.
UNASSIGNED: The phenotypes of biofilm were detected by scanning electron microscopy (SEM), gas chromatography-mass spectrometery (GC-MS) , gel permeation chromatography (GPC) , transcriptome analysis and Western blot. Co-immunoprecipitation (Co-ip) assay and enzyme activity experiment were adopted to investigate the mechanism of ASvicK regulation. Caries animal models were developed to study the relationship between ASvicK and cariogenicity of S. mutans.
UNASSIGNED: Overexpression of ASvicK can inhibit the growth of biofilm, reduce the production of EPS and alter genes and protein related to EPS metabolism. ASvicK can adsorb RNase III to regulate vicK and affect the cariogenicity of S. mutans.
UNASSIGNED: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo.

To assess the cytotoxicity of an experimental hybrid-glass-based infiltrant and its effect on biofilm attachment, growth and metabolic activity, and to compare it to the resin-based infiltrant Icon.
Cytotoxicity of hybrid-glass-based material (EXP) and resin-based infiltrant Icon (Icon) was tested in direct contact tests on freshly cured (direct_mat) and on materials kept for 24 h in cell culture medium (direct_exmat), and extract test with materials 24-h extracts (extract). Cell viability of L929 mouse fibroblast cell line was measured with MTT assay, according to ISO10993-5:2009. Biofilm attachment (5 h), growth (24 h and 48 h) and lactic-acid production (24 h and 48 h) on glass-disk specimens coated with EXP or Icon, or uncoated (control), were assessed using a microcosm biofilm model and Amsterdam Active Attachment system. At indicated time points, biofilms were harvested, plated, and CFU counts were determined, while lactic-acid production was measured colorimetrically.
Cell viability reduction by EXP was below 30%-threshold in direct contact tests, while in extract test an increased cell viability was observed. Icon reduced cell viability substantially in all three tests. Significantly less bacteria attached to the surface of EXP after 5 h compared to Icon and control. Biofilm growth was significantly lower on EXP than on Icon and control after 24 h, but this difference was smaller and statistically insignificant after 48 h. There was no difference in lactic-acid production among groups.
Novel hybrid-glass-based infiltrant seems to have a better biocompatibility and accumulates on its surface less bacteria than resin-based infiltrant, which makes it an attractive resin-free alternative.

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid-liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air-liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.

Bioclogging is the most crucial operation problem of the constructed wetlands, which reduce its removal efficiency and life span. A strategy through properly increasing hydraulic loading is proposed in this study to alleviate the bioclogging for CWs. The two-dimensional porous media flow cell (2D PMFC) test indicated that a quadratic correlation was found between local biofilms growth rate and the near-wall Reynolds number (r > 0.765, p < 0.05). The biofilm growth rate declined with the flowrate when Re exceeded about 6.0. It was also found that the higher flowrate (6 mL/min) lead to the homogeneous biofilm and velocity distribution in the PMFC. The column test indicated that the highest hydraulic loading (9.2 cm/h) produced the smallest decrease in hydraulic conductivity, which was 80 times more than that of low hydraulic load (3.0 cm/h) at the end (40 days) of experiment. Moreover, the relatively homogenized distribution of biofilm was found along the column with the highest hydraulic loading, which confirmed that the proper increase in hydraulic loading can alleviate bioclogging.

Women with cystic fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization with biofilm formed by Pseudomonas aeruginosa. There is growing evidence that blood serum concentrations of the steroid sex hormone estradiol (E2) correlate with the occurrence of pulmonary exacerbations in CF but also play a role in the mucoid switch of P. aeruginosa. This study aims to shed light on possible microbiological reasons for sexual dimorphism in CF by investigating the influence of E2 on biofilm formation of P. aeruginosa CF isolates. For this purpose, 10 CF isolates of the respiratory tract derived from different CF patients have been treated with E2 in a microtiter plate biofilm model. Biofilms have been examined by crystal violet assays, field emission scanning electron microscopy (FE-SEM), 3D laser scanning microscopy (LSM), and quorum sensing (QS) reporter assays of the supernatants taken from biofilms. This allowed us to simultaneously investigate the effects of E2 on attached biofilm mass, biofilm ultrastructure, and QS activity. Upon E2 treatment, six out of 10 investigated CF isolates showed an increase of attached biofilm mass, whereas biofilms from two tested non-CF laboratory strains (PAO1 and ATCC19660) did not. Moreover, FE-SEM and 3D LSM analyses of the E2 responsive CF biofilms revealed ultrastructural remodeling of biofilm structure at different scales with increased formation of prominent biofilm spots, enhanced coverage with extracellular polymeric substance (EPS), and extended average surface roughness. QS activity measurements performed in biofilm supernatants via luminescence acyl homoserine lactone (AHL) reporter assays further showed that E2 treatment may also modulate QS signaling, as shown in an E2 sensitive CF isolate. Together, our results suggest the biofilm modulating effects of E2 on various clinical CF isolates that are documented by both biomass and ultrastructural changes of biofilms. The gained new insight into the influence of steroid hormones on P. aeruginosa biofilm phenotypes might pave the way for novel future approaches in personalized medicine based on the patients\' sex and hormonal status.