Purified L-glutaminase effects against multidrug-resistant Pseudomonas aeruginosa in experimental vaginosis model: An immunological and histopathological observation.

The antimicrobial activity of crude and purified L-glutaminase (EC 3.5.1.2), obtained from Lactobacillus gasseri, was evaluated against multidrug-resistant Pseudomonas aeruginosa in the in vivo vaginosis condition. The L-glutaminase possessed significant antimicrobial and anti-biofilm formation activity against multi-drug resistance P. aeruginosa, which were confirmed in the BALBc rat vaginosis model, together with its effects on the immunological and histopathological aspects. The untreated animals showed heavy vaginitis, characterized by sub-epithelial edema and infiltration of mononuclear leukocytes, perivascular heavy inflammatory cells infiltration in the vaginal tissue, and moderate stromal edema. However, the L-glutaminase treatment exhibited no changes in vaginal tissue structure with normal appearance of the epithelium and lamina propria with marked repair of the vaginal section when compared with normal, uninfected, control group A. The immunomodulatory actions of the L-glutaminase were confirmed by observance of higher concentrations of tumor necrosis factor-γ (TNF-γ), and interleukin -12 (IL-12) in treated animals, while the interleukin-10 (IL-10) was higher in the infected, untreated animals' sera samples. Therefore, the L-glutaminase showed corrective and healing actions, which were observed through histopathological observations of the vaginal tissue. The investigations led to imply that L-glutaminase may have the potential to be an effective antimicrobial agent for preventing and inhibiting bacterial growth, as well as inhibiting the biofilm formation in the P. aeruginosa-originated vaginosis. The observations may be of promising value for future clinical use.

Molecular mechanisms and environmental adaptations of flagellar loss and biofilm growth of Rhodanobacter under environmental stress.

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 suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.5 to 5) and nitrates (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptations for survival and growth at low pH. Biofilms were intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation, findings warranting further investigation. Through random barcode transposon-site sequencing (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 the absence of flagella and chemotaxis genes and the presence of a putative type VI secretion system in the highly biofilm-forming strain FW021-MT20. In this study we identified genetic determinants associated with biofilm growth under metal stress in a predominant environmental genus, Rhodanobacter, and identified traits aiding survival and adaptation to contaminated subsurface environments.

Biofilm growth characteristic and footprint identification in gravity-driven ceramic membrane bioreactor with electro-coagulation under extreme conditions for roofing rainwater purification.

The identification of biofilm growth footprints influencing on the biofilm detachment and breakup can advance research into how biofilms form. Thus, a gravity-driven ceramic membrane bioreactor (GDCMBR) was used to investigate the growth, detachment and breakup of biofilm using rainwater pretreated by electrocoagulation under 70-days continuous operation. The in-situ ultrasonic time-domain reflectometry (UTDR) technique was applied to non-invasively determine the biofilm thickness. Initially, the biofilm was slowly thickening, but it would collapse and became thinner after accumulating to a certain level, and then it thickened again in a later period, following a cyclic pattern of 'thickening - collapsing - thickening'. This is because the biofilm growth is related with the accumulation of flocs, however, excessive floc formation results in the biofilm being overweight till reaching the thickness limit and thus collapsing. Subsequently, the biofilm gradually thickens again due to the floc production and continuous deposition. Although the biofilm was dynamically changing, the water quality of treatment of the biofilm always remained stable. Ammonia nitrogen and total phosphorus have been almost completely removed, while CODMn removal efficiency was around 25%. And total bacteria amount in the membrane concentrate was obviously higher than that in the influent with the greater microbial activity, demonstrating the remarkable enrichment effect on bacteria. The understanding of biofilm growth characteristic and footprint identification enables us to develop rational approaches to control biofilm structure for efficient GDCMBR performance and operation lifespan.

A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

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.

Optimization of culture medium to improve bio-cementation effect based on response surface method.

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.

The mitigation potential of synergistic quorum quenching and antibacterial properties for biofilm proliferation and membrane biofouling.

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.

Optimization of moving bed biofilm reactors for the treatment of municipal wastewater.

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.

Exopolysaccharides metabolism and cariogenesis of Streptococcus mutans biofilm regulated by antisense vicK RNA.

Background: 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). Objective: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans. Methods: 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. Results: 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. Conclusions: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo.

The microbial growth potential of antiscalants used in seawater desalination.

20 years since the first report on the biofouling potential of chemicals used for scale control, still, antiscalants with high bacterial growth potential are used in practice. Evaluating the bacterial growth potential of commercially available antiscalants is therefore essential for a rational selection of these chemicals. Previous antiscalant growth potential tests were conducted in drinking water or seawater inoculated with model bacterial species which do not represent natural bacterial communities. To reflect better on the conditions of desalination systems, we investigated the bacterial growth potential of eight different antiscalants in natural seawater and an autochthonous bacterial population as inoculum. The antiscalants differed strongly in their bacterial growth potential varying from ≤ 1 to 6 µg easily biodegradable C equivalents/mg antiscalant. The six phosphonate-based antiscalants investigated showed a broad range of growth potential, which depended on their chemical composition, whilst the biopolymer and the synthetic carboxylated polymers-based antiscalants showed limited or no significant bacterial growth. Moreover, nuclear magnetic resonance (NMR) scans enabled antiscalant fingerprinting, identifying components and contaminants, providing a rapid and sensitive characterization, and opening opportunities for rational selection of antiscalants for biofouling control.

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air-liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device.

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 alleviation for constructed wetland based on the interaction among biofilm growth and hydrodynamics.

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.

Cytotoxicity and anti-biofilm properties of novel hybrid-glass-based caries infiltrant.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. SIGNIFICANCE: 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.

Effects of the Sex Steroid Hormone Estradiol on Biofilm Growth of Cystic Fibrosis Pseudomonas aeruginosa Isolates.

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.

The effect of biofilm growth on the sulfur oxidation pathway and the synergy of microorganisms in desulfurization reactors under different pH conditions.

Biofilm growth affects the oxygen transfer in biofilm and thus the oxidation pathway of sulfur and the synergy of microorganisms. In this study, the effect of biofilm growth on the oxidation pathway of H2S and the synergy of microorganisms in desulfurization reactors under different pH conditions was first discussed to enhance the understanding of desulfurization process. A biotrickling filter (BTF) was operated for 168 days under acidic condition (pH<4.7) and 32 days under alkaline condition (7.0 89.0%). In alkaline period, the AGM raised to 0.97 g/L-BTF/d, and 77.0% of S-H2S was transferred to elemental sulfur (S0) and polysulfanes (R-Sx-R) accumulated in biofilm. The increase of biofilm and sulfur-oxidizing bacteria activity limited the oxygen transfer in alkaline biofilm, leading to the accumulation of S0 and the emergence of an obligate anaerobe- Acetoanaerobium (8.1%). The formation of R-Sx-R may be due to the reaction of S0 with thiols produced by a thiol-producing bacterium- Pseudomonas (6.7%). The uneven distribution of oxygen in biofilm caused by biofilm growth complicated the transfer pathway of sulfur and the synergy of microorganisms in desulfurization system.

Low maintenance gravity-driven membrane filtration using hollow fibers: Effect of reducing space for biofilm growth and control strategies on permeate flux.

The implementation of centralized drinking water treatment systems necessitates lower operational costs and improved biopolymer removal during ultrafiltration (UF), which can be afforded by gravity-driven membrane (GDM) filtration. However, prior to implementing GDM filtration in centralized systems, biofilm growth in compacted membrane configurations, such as inside-out hollow fiber (HF), and its impact on permeate flux need to be investigated. To this end, we operated modules with distinct limits on available space for biofilm growth: (1) outside-in 1.5 mm 7-capillary HF (non-limited), (2) inside-out 1.5 mm 7-capillary HF (limited), and (3) inside-out 0.9 mm 7-capillary HF (very limited). Here, we observed that the lower the space available for biofilm growth, the lower the permeate flux. To improve GDM performance with inside-out HF, we applied daily shear stress to the biofilm surface with forward flush (FF) or combined relaxation and forward flush (R+FF). We showed that applying shear stress to the biofilm surface was insufficient for controlling flux loss due to low available space for biofilm growth. At the experimental endpoint, we backwashed with a stepwise transmembrane pressure (TMP) increase or a single TMP on all inside-out HF modules, which removed the biofilm from its base. Afterwards, higher fluxes were yielded. We also showed that all modules exhibited a gradual increase in biopolymer removal followed by stabilization between 70 and 90%. Additionally, control of biofilm growth with surface shear stress did not affect biopolymer removal. In summary, the implementation of inside-out HF with GDM filtration is challenged by low available space for biofilm growth, but may be remedied with a regular backwash to remove biofilm from its base. We showed that a wider range of GDM applications are available; making GDM potentially compatible with implementation in centralized systems, if space limitation is taken into consideration for operation optimization.

Method for rapid biofilm cultivation on microplastics and investigation of its effect on the agglomeration and removal of microplastics using organosilanes.

Since microplastics were recognized as a global environmental problem in the early 2000s, research began on possible solutions such as the removal of microplastics from waters. A novel and promising approach for this purpose is microplastics agglomeration-fixation using organosilanes. In this study, it is investigated how biofilm coverage of microplastics affects this process. The biofilm was grown on the microplastics by cultivating it for one week in a packed bed column operated with biologically treated municipal wastewater enriched with glucose. The biofilm was characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and Fourier-Transform infrared spectroscopy (FT-IR). The results show a partial coverage of the microplastics with attached bacteria and extracellular polymeric substances (EPS) after 7 days of incubation. Comparing five polymer types (polyethylene, polypropylene, polyamide, polyester, and polyvinyl chloride) and three organosilanes, the biofilm coverage caused a reduced removal efficiency for all combinations tested as it changes the surface chemistry of the microplastics and therefore the interaction with the organosilanes tested in this study. Treatment of biofilm covered microplastic with ultrasound partly recovers the removal. However, the results underline the importance of simulated environmental exposure when performing experiments for microplastic removal.

Low-Field Nuclear Magnetic Resonance Characteristics of Biofilm Development Process.

To in situ and noninvasively monitor the biofilm development process by low-field nuclear magnetic resonance (NMR), experiments should be made to determine the mechanisms responsible for the T2 signals of biofilm growth. In this paper, biofilms were cultivated in both fluid media and saturated porous media. T2 relaxation for each sample was measured to investigate the contribution of the related processes to T2 relaxation signals. In addition, OD values of bacterial cell suspensions were measured to provide the relative number of bacterial cells. We also obtained SEM photos of the biofilms after vacuum freeze-drying the pure sand and the sand with biofilm formation to confirm the space within the biofilm matrix and identify the existence of biofilm formation. The T2 relaxation distribution is strongly dependent on the density of the bacterial cells suspended in the fluid and the stage of biofilm development. The peak time and the peak percentage can be used as indicators of the biofilm growth states.

Inhibition of AdeB, AceI, and AmvA Efflux Pumps Restores Chlorhexidine and Benzalkonium Susceptibility in Acinetobacter baumannii ATCC 19606.

The management of infections caused by Acinetobacter baumannii is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different A. baumannii efflux pumps (EPs) in tolerance to chlorhexidine (CHX) and benzalkonium (BZK) and identify non-toxic compounds, which can restore susceptibility to CHX and BZK in A. baumannii. A. baumannii ATCC 19606 strain was tolerant to both CHX and BZK with MIC and MBC value of 32 mg/L. CHX subMIC concentrations increased the expression of adeB and adeJ (RND superfamily), aceI (PACE family) and amvA (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in ΔadeB and twofold in ΔamvA or ΔaceI mutants, respectively, while not affected in ΔadeJ mutant; EPs double and triple deletion mutants showed an additive effect on CHX MIC. CHX susceptibility was restored in double and triple deletion mutants with inactivation of adeB gene. BZK MIC was decreased by fourfold in ΔadeB mutant, and twofold in ΔamvA and ΔaceI mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in ΔadeB ΔaceI ΔadeJ and ΔamvA ΔadeB ΔadeJ triple mutants. The structural comparison of AdeB and AdeJ protomers showed a more negatively charged entrance binding site and F-loop in AdeB, which may favor the transport of CHX. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) EP inhibitor reduced dose-dependently CHX MIC in A. baumannii ATCC 19606 and in ΔadeJ, ΔaceI, or ΔamvA mutants, but not in ΔadeB mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in A. baumannii ATCC 19606 parental strain and EPs gene deletion mutants, and CHX-induced EP gene expression. Also, RV inhibited BZK MIC and EP genes expression in A. baumannii ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in A. baumannii is mediated by the activation of AdeB, AceI and AmvA EPs, AdeB playing a major role. Importantly, inhibition of EP genes expression by RV restores CHX and BZK susceptibility in A. baumannii.

Bedform characteristics and biofilm community development interact to modify hyporheic exchange.

The physical and biological attributes of riverine ecosystems interact in a complex manner which can affect the hydrodynamic behaviour of the system. This can alter the mixing characteristics of a river at the sediment-water interface. Research on hyporheic exchange has increased in recent years driven by a greater appreciation for the importance of this dynamic ecotone in connecting and regulating river systems. An understanding of process-based interactions driving hyporheic exchange is still limited, specifically the feedbacks between the physical and biological controlling factors. The interplay between bed morphology and sediment size on biofilm community development and the impact on hyporheic exchange mechanisms, was experimentally considered. Purpose built recirculating flume systems were constructed and three profiles of bedform investigated: i) flat, ii) undulating λ = 1 m, ii) undulating λ = 0.2 m, across two different sized sediments (0.5 mm and 5 mm). The influence of biofilm growth and bedform interaction on hyporheic exchange was explored, over time, using discrete repeat injections of fluorescent dye into the flumes. Hyporheic exchange rates were greatest in systems with larger sediment sizes (5 mm) and with more bedforms (undulating λ = 0.2). Sediment size was a dominant control in governing biofilm growth and hyporheic exchange in systems with limited bedform. In systems where bedform was prevalent, sediment size and biofilm appeared to no longer be a control on exchange due to the physical influence of advective pumping. Here, exchange rates within these environments were more consistent overtime, despite greater microbial growth. As such, bedform has the potential to overcome the rate limiting effects of biotic factors on hyporheic exchange and sediment size on microbial penetration. This has implications for pollutant and nutrient penetration; bedforms increase hydrological connectivity, generating the opportunity to support microbial communities at depth and as such, improve the self-purification ability of river systems.

Label-Free Four-Dimensional Visualization of Anaerobically Growing Electroactive Biofilms.

Light sheet fluorescence microscopy (LSFM) allows nondestructive, label-free and in vivo imaging of large specimen, even at nontransparent surfaces. We show that LSFM can be applied for label-free analyses of prokaryotes on the example of electroactive biofilms. Biofilm growth is linked to the production of current serving as measure of metabolic activity in vivo by monitoring with high spatial and temporal resolution. After 35 h of exponential growth, a homogeneous biofilm with a thickness of 9 µm was formed. This was followed by a stratification of the biofilm including the formation of 3D structures over the next 100 h. Light reflection was sufficient to visualize the biofilm structure and development over time and the terminal morphology was confirmed using fluorescence staining. This proof of concept on using LSFM for investigation of biofilms opens the door for its application in the entire field of microbial ecology. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.