A microfluidic device for high throughput bacterial biofilm studies

Bacteria are almost always found in ecological niches as matrix-encased, surface-associated, multi-species communities known as biofilms. It is well established that soluble chemical signals produced by the bacteria influence the organization and structure of the biofilm; therefore, there is significant interest in understanding how different chemical signals are coordinately utilized for community development. Conventional methods for investigating biofilm formation such as macro-scale flow cells are low-throughput, require large volumes, and do not allow spatial and temporal control of biofilm community formation. Here, we describe the development of a PDMS-based two-layer microfluidic flow cell (μFC) device for investigating bacterial biofilm formation and organization in response to different concentrations of soluble signals. The μFC device contains eight separate microchambers for cultivating biofilms exposed to eight different concentrations of signals through a single diffusive mixing-based concentration gradient generator. The presence of pneumatic valves and a separate cell seeding port that is independent from gradient-mixing channels offers complete isolation of the biofilm microchamber from the gradient mixer, and also performs well under continuous, batch or semi-batch conditions. We demonstrate the utility of the μFC by studying the effect of different concentrations of indole-like biofilm signals (7-hydroxyindole and isatin), either individually or in combination, on biofilm development of pathogenic E. coli. This model can be used for developing a fundamental understanding of events leading to bacterial attachment to surfaces that are important in infections and chemicals that influence the biofilm formation or inhibition.     

硫脲壳聚糖微量银配合物的制备及抑菌活性

由硫脲壳聚糖和微量的AgNO3反应得到硫脲壳聚糖Ag+配合物。 通过红外光谱对其结构进行了表征。 研究了壳聚糖、硫脲壳聚糖、硫脲壳聚糖-Ag+配合物及AgNO3对大肠杆菌和金黄葡萄球菌的抑菌活性,并测定了其最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。 结果表明,硫脲壳聚糖-Ag+配合物的抑菌活性强于壳聚糖和硫脲壳聚糖,且其MIC和MBC均为100 mg/L（游离Ag+含量为0.032 mg/L）,低于AgNO3的MIC和MBC（均为120 mg/L）。     

Disruption of urogenital biofilms by lactobacilli

The process that changes a relatively sparse vaginal microbiota of healthy women into a dense biofilm of pathogenic and potentially pathogenic bacteria is poorly understood. Likewise, the reverse step whereby an aberrant biofilm is displaced and returns to a healthy lactobacilli dominated microbiota is unclear. In order to study these phenomena, in vitro experiments were performed to examine the structure of biofilms associated with aerobic vaginosis, urinary tract infections, and bacterial vaginosis (BV). Uropathogenic Escherichia coli were able to form relatively thin biofilms within five days (6 μm height), while Atopobium vaginae and Gardnerella vaginalis formed thicker biofilms 12 μm in height within two days. Challenge of E. coli biofilms with lactobacilli did not result in pathogen displacement. However, the resulting thicker lactobacilli infused biofilms, caused significant E. coli killing. E. coli biofilms challenged with secreted products of L. rhamnosus GR-1 caused a marked decrease in cell density, and increased cell death. Similarly challenge of BV biofilms with lactobacilli infiltrated BV biofilms and caused bacterial cell death. Metronidazole produced holes in the biofilm but did not eradicate the organisms. The findings provide some evidence of how lactobacilli probiotics might interfere with an aberrant vaginal microbiota, and strengthen the position that combining probiotics with antimicrobials could better eradicate pathogenic biofilms.     

Anti-Biofilm and Anti-Hemolysis Activities of 10-Hydroxy-2-decenoic Acid against Staphylococcus aureus

Persistent infections caused by Staphylococcus aureus biofilms pose a major threat to global public health. 10-Hydroxy-2-decenoic acid (10-HDA), a main fatty acid in royal jelly, has been shown to possess various biological activities. The purpose of this study was to explore the effects of 10-HDA on the biofilms and virulence of S. aureus and its potential molecular mechanism. Quantitative crystal violet staining indicated that 10-HDA significantly reduced the biofilm biomass at sub-minimum inhibitory concentration (MIC) levels (1/32MIC to 1/2MIC). Scanning electron microscope (SEM) observations demonstrated that 10-HDA inhibited the secretion of extracellular polymeric substances, decreased bacterial adhesion and aggregation, and disrupted biofilm architecture. Moreover, 10-HDA could significantly decrease the biofilm viability and effectively eradicated the mature biofilms. It was also found that the hemolytic activity of S. aureus was significantly inhibited by 10-HDA. qRT-PCR analyses revealed that the expressions of global regulators sarA, agrA, and α-hemolysin gene hla were downregulated by 10-HDA. These results indicate that 10-HDA could be used as a potential natural antimicrobial agent to control the biofilm formation and virulence of S. aureus.     

Surface-enhanced Raman scattering (SERS) revealing chemical variation during biofilm formation: from initial attachment to mature biofilm

Surface-enhanced Raman scattering (SERS) has recently been proved to be a promising technique for characterizing the chemical composition of the biofilm matrix. In the present study, to fully understand the chemical variations during biofilm formation, SERS based on silver colloidal nanoparticles was applied to evaluate the chemical components in the matrix of biofilm at different growth phases, including initial attached bacteria, colonies, and mature biofilm. Meanwhile, atomic force microscopy was also applied to study the changes of biofilm morphology. Three model bacteria, including Escherichia coli, Pseudomonas putida, and Bacillus subtilis, were used to cultivate biofilms. The results showed that the content of carbohydrates, proteins, and nucleic acids in the biofilm matrix increased significantly along with the biofilm growth of the three bacteria judging from the intensities and appearance probabilities of related marker peaks in the SERS spectra. The content of lipids, however, only increased in the Gram-negative biofilms (E. coli and P. putida) rather than the Gram-positive biofilm (B. subtilis). Our findings strongly suggest the SERS has significant potential for studying chemical variations during biofilm formation.     

In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device

Antibiotic resistance of biofilms is a growing public health concern due to overuse and improper use of antibiotics. Thus, determining an effective minimal concentration of antibiotics to eradicate bacterial biofilms is crucial. Here we present a simple, novel one-pot assay for the analysis of antibiotic susceptibility of bacterial biofilms using a microfluidics system where continuous concentration gradients of antibiotics are generated. The results of minimal biofilm eradication concentration (MBEC) clearly confirm that the concentration required to eradicate biofilm-grown Pseudomonas aeruginosa is higher than the minimal inhibitory concentration (MIC) that has been widely used to determine the lowest concentration of antibiotics against planktonically grown bacteria.     

Antimicrobial properties of some bis(iminoacenaphthene (BIAN)-supported N-heterocyclic carbene complexes of silver and gold

The AgCl, AgOAc, AuCl, and AuOAc complexes of the new bis(imino)acenaphthene(BIAN)-supported N-heterocyclic carbene ligand and the precursor imidazolium salt have been investigated with respect to their antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Psudomonas aeruginosa. The most active antimicrobial is the precursor imidazolium salt, which has a minimum inhibitory concentration (MIC) value of <40 μg/mL. The MIC values for the silver complexes IPr(BIAN)AgCl and IPr(BIAN)AgOAc against Gram-positive S. aureus are comparable to that for AgNO?, while those against Gram-negative E. coli and P.aeroginosa are significantly larger. Similar behavior was evident for the gold acetate complex IPr(BIAN)AuOAc. However, in the case of the gold chloride analogue, the MIC values are virtually identical for both the Gram-positive and the Gram-negative bacteria.     

Inhibitory Effect of Nepeta deflersiana on Climax Bacterial Community Isolated from the Oral Plaque of Patients with Periodontal Disease

Background: The red-complex bacteria are one of the most significant complexes found simultaneously in subgingival plaque next to the periodontal pocket. The current antibacterial treatment is not adequate, and multidrug resistance to it is developing. Henceforth, the antibacterial effect of the ethanolic extract of Nepeta deflersiana was put to test against red-complex bacteria in patients with chronic periodontitis. Methods: Well diffusion and micro broth dilution procedure by Alamar blue were applied to assess the zone of inhibition (ZOI), the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC). Anti-virulence efficacies of the plant extract that comprise of adherence and formation of biofilms were examined by the process of adherence and biofilm production assay. Results: The crude extract of Nepeta deflersiana exhibited significant inhibitory outcome against periodontopathic bacteria with noteworthy MIC (0.78–3.12 mg/mL), inhibitory zone (12–20 mm), as well as MBC (3.12–12.50 mg/mL). The N. deflersiana extract inhibited bacterial adhesion ranging from 41% to 52%, 53% to 66%, and 60% to 79% at the given MIC × 0.5, MIC × 1, and MIC × 2 in succession. Substantial suppression was also developed in the biofilm production of the investigated periodontopathic strains following exposure to numerous concentrations of N. deflersianan extract for a period of 24 and 48 h. Conclusion: These outcomes divulge a new concept that N. deflersiana extract can be utilized to manufacture valuable antibacterial compounds to treat chronic and acute periodontitis. This identifies N. deflersiana as an essential natural source for future drug development.     

Decrease in skin permeation and antibacterial effect of parabens by a polymeric additive, poly(2-methacryloyloxyethyl phosphorylcholine-co-butylmetacrylate)

The inhibitory effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-butylmetacrylate) (PMB) on the in vitro skin permeation of p-hydroxybenzoic acid and its esters (parabens; methylparaben, ethylparaben, n-propylparaben and n-butylparaben) as model compounds was evaluated. Solubility of the parabens in distilled water was increased by addition of PMB, and the increasing ratio was dependent on the concentration of PMB. The increment of the ratio was more marked in lipophilic parabens than hydrophilic ones. Skin permeability of parabens from their aqueous suspensions was decreased by addition of PMB, and the decreasing ratio was dependent on the PMB concentration. Then, antibacterial effect of parabens of their aqueous solutions was determined with and without PMB by measuring minimum inhibitory concentration (MIC) against E. coli and S. aureus. As results, MIC and antibacterial effect were increased and decreased, respectively, by addition of PMB. The contribution of PMB was more marked in butylparaben than methylparaben. These decreases in skin permeation and antibacterial effect of parabens by PMB are probably due to a higher solubility in water and a lower partition to the skin and bacterial membranes of parabens by addition of PMB.     

壳多糖抑制细菌生长的构效关系

运用化学结构已清楚, 分属4大系列的29种壳多糖, 以4种不同类型的细菌(革兰氏阳性菌Ecoli K1、革兰氏阴性菌Bacillus cereus、Bacillus megaterium和Staphlylococcu aureus)为研究对象, 进行了壳多糖抑菌能力构效关系的研究. 在实验中采用96孔平板, 用计算机\|吸光值读数仪直接测定每个孔的吸光值, 获得了各个细菌在不同壳多糖浓度中的生长曲线和壳多糖抑制细菌生长的最低抑制浓度(MIC, Minimum inhibit concentration). 通过比较同一(各个)系列的壳多糖在这些相同(不同)细菌的MIC变化规律与壳多糖的化学结构的关系, 发现同一壳多糖对不同的细菌的MIC值是不相同的, 因而壳多糖抑制细菌生长的能力首先与细菌本身特点有关, 但与是否为革兰氏阳性菌或阴性菌无直接的相关性; 同一细菌对不同化学结构的壳多糖有一定的相关性, 在壳多糖的聚合程度(DP)相同的条件下, 壳多糖中氨基被乙酰化(DA)的程度越低, 壳多糖抑制细菌生长的MIC值越低, 壳多糖抑制细菌生长的能力就越强; 同样,在DA相同的情况下, 分子越小, 壳多糖抑制细菌生长的MIC值越低, 抑制细菌生长的能力越强. 根据上述实验结果, 初步推测壳多糖抑制细菌生长的机制可能与其在溶液中所带的正电荷多少有关.     

Polycationic chitosan-conjugated photosensitizer for antibacterial photodynamic therapy

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan-conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7-day-old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.     

Salpianthus macrodontus Extracts,a Novel Source of Phenolic Compounds with Antibacterial Activity against Potentially Pathogenic Bacteria Isolated from White Shrimp

This study aimed to evaluate the antibacterial activity in vitro of Salpianthus macrodontus and Azadirachta indica extracts against potentially pathogenic bacteria for Pacific white shrimp. Furthermore, the extracts with higher inhibitory activity were analyzed to identify compounds responsible for bacterial inhibition and evaluate their effect on motility and biofilm formation. S. macrodontus and A. indica extracts were prepared using methanol, acetone, and hexane by ultrasound. The minimum inhibitory concentration (MIC) of the extracts was determined against Vibrio parahaemolyticus, V. harveyi, Photobacterium damselae and P. leiognathi. The polyphenol profile of those extracts showing the highest bacterial inhibition were determined. Besides, the bacterial swimming and swarming motility and biofilm formation were determined. The highest inhibitory activity against the four pathogens was found with the acetonic extract of S. macrodontus leaf (MIC of 50 mg/mL for Vibrio spp. and 25 mg/mL for Photobacterium spp.) and the methanol extract of S. macrodontus flower (MIC of 50 mg/mL for all pathogens tested). Both extracts affected the swarming and swimming motility and the biofilm formation of the tested bacteria. The main phenolic compounds related to Vibrio bacteria inhibition were naringin, vanillic acid, and rosmarinic acid, whilst hesperidin, kaempferol pentosyl-rutinoside, and rhamnetin were related to Photobacterium bacteria inhibition.     

Efflux Pump Inhibitor Potentiates Antimicrobial Photodynamic Inactivation of Enterococcus faecalis Biofilm

Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2-2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm-derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (P < 0.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (P < 0.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure.     

Boronic Acid Functionalized Photosensitizers: A Strategy To Target the Surface of Bacteria and Implement Active Agents in Polymer Coatings

Advanced methods for preventing and controlling hospital‐acquired infections via eradication of free‐floating bacteria and bacterial biofilms are of great interest. In this regard, the attractiveness of unconventional treatment modalities such as antimicrobial photodynamic therapy (aPDT) continues to grow. This study investigated a new and innovative strategy for targeting polysaccharides found on the bacterial cell envelope and the biofilm matrix using the boronic acid functionalized and highly effective photosensitizer (PS) silicon(IV) phthalocyanine. This strategy has been found to be successful in treating planktonic cultures and biofilms of Gram‐negative E. coli. An additional advantage of boronic acid functionality is a possibility to anchor the tailor made PS to poly(vinyl alcohol) and to fabricate a self‐disinfecting coating.     

Synthesis of 3,5-Bis(trifluoromethyl)phenyl-Substituted Pyrazole Derivatives as Potent Growth Inhibitors of Drug-Resistant Bacteria

Enterococci and methicillin-resistant S. aureus (MRSA) are among the menacing bacterial pathogens. Novel antibiotics are urgently needed to tackle these antibiotic-resistant bacterial infections. This article reports the design, synthesis, and antimicrobial studies of 30 novel pyrazole derivatives. Most of the synthesized compounds are potent growth inhibitors of planktonic Gram-positive bacteria with minimum inhibitory concertation (MIC) values as low as 0.25 µg/mL. Further studies led to the discovery of several lead compounds, which are bactericidal and potent against MRSA persisters. Compounds 11, 28, and 29 are potent against S. aureus biofilms with minimum biofilm eradication concentration (MBEC) values as low as 1 µg/mL.     

Evaluation of Total Phenolic and Flavonoid Contents,Antibacterial and Antibiofilm Activities of Hungarian Propolis Ethanolic Extract against Staphylococcus aureus

Propolis is a natural bee product that is widely used in folk medicine. This study aimed to evaluate the antimicrobial and antibiofilm activities of ethanolic extract of propolis (EEP) on methicillin-resistant and sensitive Staphylococcus aureus (MRSA and MSSA). Propolis samples were collected from six regions in Hungary. The minimum inhibitory concentrations (MIC) values and the interaction of EEP-antibiotics were evaluated by the broth microdilution and the chequerboard broth microdilution methods, respectively. The effect of EEP on biofilm formation and eradication was estimated by crystal violet assay. Resazurin/propidium iodide dyes were applied for simultaneous quantification of cellular metabolic activities and dead cells in mature biofilms. The EEP1 sample showed the highest phenolic and flavonoid contents. The EEP1 successfully prevented the growth of planktonic cells of S. aureus (MIC value = 50 µg/mL). Synergistic interactions were shown after the co-exposition to EEP1 and vancomycin at 10⁸ CFU/mL. The EEP1 effectively inhibited the biofilm formation and caused significant degradation of mature biofilms (50–200 µg/mL), as a consequence of the considerable decrement of metabolic activity. The EEP acts effectively as an antimicrobial and antibiofilm agent on S. aureus. Moreover, the simultaneous application of EEP and vancomycin could enhance their effect against MRSA infection.     

Essential oil analysis and antibacterial activity of Rosmarinus tournefortii from Algeria

The volatile compounds obtained by hydrodistillation of the aerial parts of Rosmarinus tournefortii De Noé. growing wild in the occidental region of Algeria were analyzed by GC/MS. Thirty-six compounds were characterized representing 95.6% of the essential oil, with camphor (37.6%), 1,8-cineole (10.0%), p-cymene-7-ol (7.8%), and borneol (5.4%) as the major components. The antimicrobial activity was evaluated against three pathogenic bacteria: Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus). The minimum inhibitory concentration (MIC; mg/mL) was determined by sub-culture on Muller Hinton agar plates. The essential oil exhibited strong antibacterial activity against E. coli and P. aeruginosa, and was also active against Staphylococcus aureus.     

Antimicrobial evaluation of selected naturally occurring oxyprenylated secondary metabolites

This study tested the antimicrobial activity of eight selected naturally occurring oxyprenylated secondary metabolites against Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 35984, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. Results showed a moderate antimicrobial activity. The most active compounds were 3-(4-geranyloxyphenyl)-1-ethanol (4) and 3-(4-isopentenyloxyphenyl)-1-propanol (5) that were tested on mature and in-formation biofilms of all micro-organisms, moreover the cytotoxic activity was evaluated. Except for S. epidermidis, both compounds reduced significantly (p < 0.05) the microbial biofilm formation at 1/2 MIC and 1/4 MIC, in particular, compounds 4 and 5 at each concentration, inhibited E. coli biofilm formation to a greater extent, the biofilm formation was never more than 44% in respect to the control, moreover both compounds showed a low cytotoxic effect. Oxyprenylated derivatives may be of great interest for the development of novel antimicrobial therapeutic strategies and the synthesis of semi-synthetic analogues with anti-biofilm efficacy.     

Biofilms as bio-indicator for polluted waters?

The aim of this work was to investigate the heavy metal accumulation by natural biofilms living in the catchment area of the Tisza river in Hungary, as well as in biofilms cultivated in vitro. Laboratory tests have demonstrated that metals can be adsorbed on biofilms, depending on their concentration and on the availability of free sorptive places. Biofilms were cultivated in vitro in natural freshwater from the Saale river, Germany. After reaching the plateau phase, Cu was added to reach a concentration of 100 µg/L. An increase of its mass fraction in the biofilm was observed, which caused the decrease of the concentration in the water phase. Unfortunately, the reactor wall was also found to act as adsorbent for Cu. More detailed results of our in vitro experiments will be published in a forthcoming paper. Naturally grown biofilm samples from exposed as well as background places at the Hungarian rivers Szamos and Tisza were collected in 2000 and 2002 after the cyanide spill, and analysed using total reflection X-ray fluorescence analysis (TXRF). Metal mass fraction differences as high as two orders of magnitude were found between polluted and unpolluted (background) sampling points. Extremely high concentration values, e.g. 5600 µg/g Zn in biofilm, were found at highly polluted sampling points. This means an enrichment factor of ca. 10,000 compared to the water phase.     

Ag/Fe_3O_4/rGO纳米复合材料的制备及抗菌性能

以四水合氯化亚铁和硝酸银为原料,硼氢化钠为还原剂,氧化石墨烯(GO)为载体,通过原位还原法制备了具有磁分离功能的银/四氧化三铁/还原氧化石墨烯(Ag/Fe_3O_4/rGO)纳米复合抗菌材料.采用X射线粉末衍射仪(XRD)、X射线光电子能谱仪(XPS)、透射电子显微镜(TEM)等对复合材料进行了表征.结果显示,Fe_3O_4和Ag纳米颗粒均匀分布在rGO片层上.复合材料的饱和磁化率(Ms)为40.5 A·m~2·kg·(-1),表明其具有较强的磁性,将其与菌液混合后,在磁场作用下10 min即可吸附沉降完成磁分离.以大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)为实验菌株,通过琼脂扩散法评价了复合材料的抗菌性能.结果表明,该复合材料具有良好的抗菌效果,对E.coli和S.aureus的抑菌圈直径分别为18 mm和13 mm,最低抑菌浓度值(MIC)分别为50 mg/L和80 mg/L,最低杀菌浓度值(MBC)分别为30 mg/L和50 mg/L.