Towards a nondestructive chemical characterization of biofilm matrix by Raman microscopy

In this study, the applicability of Raman microscopy (RM) for nondestructive chemical analysis of biofilm matrix, including microbial constituents and extracellular polymeric substances (EPS), has been assessed. The examination of a wide range of reference samples such as biofilm-specific polysaccharides, proteins, microorganisms, and encapsulated bacteria revealed characteristic frequency regions and specific marker bands for different biofilm constituents. Based on received data, the assignment of Raman bands in spectra of multispecies biofilms was performed. The study of different multispecies biofilms showed that RM can correlate various structural appearances within the biofilm to variations in their chemical composition and provide chemical information about a complex biofilm matrix. The results of RM analysis of biofilms are in good agreement with data obtained by confocal laser scanning microscopy (CLSM). Thus, RM is a promising tool for a label-free chemical characterization of different biofilm constituents. Moreover, the combination of RM with CLSM analysis for the study of biofilms grown under different environmental conditions can provide new insights into the complex structure/function correlations in biofilms.     

Microfluidic approaches to bacterial biofilm formation

Bacterial biofilms-aggregations of bacterial cells and extracellular polymeric substrates (EPS)-are an important subject of research in the fields of biology and medical science. Under aquatic conditions, bacterial cells form biofilms as a mechanism for improving survival and dispersion. In this review, we discuss bacterial biofilm development as a structurally and dynamically complex biological system and propose microfluidic approaches for the study of bacterial biofilms. Biofilms develop through a series of steps as bacteria interact with their environment. Gene expression and environmental conditions, including surface properties, hydrodynamic conditions, quorum sensing signals, and the characteristics of the medium, can have positive or negative influences on bacterial biofilm formation. The influences of each factor and the combined effects of multiple factors may be addressed using microfluidic approaches, which provide a promising means for controlling the hydrodynamic conditions, establishing stable chemical gradients, performing measurement in a high-throughput manner, providing real-time monitoring, and providing in vivo-like in vitro culture devices. An increased understanding of biofilms derived from microfluidic approaches may be relevant to improving our understanding of the contributions of determinants to bacterial biofilm development.     

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.     

Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.     

Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis

Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity.     

Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate,sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging

Understanding the chemical composition of biofilm matrices is vital in different fields of biology such as surgery, dental medicine, synthetic grafts and bioremediation. The knowledge of biofilm development, composition, active reduction sites and remediation efficacy will help in the development of effective solutions and evaluation of remediating approaches prior to implementation. Surface-enhanced Raman spectroscopy (SERS) based imaging is an invaluable tool to obtain an understanding of the remediating efficacy of microorganisms and its role in the formation of organic and inorganic compounds in biofilms. We demonstrate for the first time, the presence of chromate, sulfate, nitrate and reduced trivalent chromium in soil biofilms. In addition, we demonstrate that SERS imaging was able to validate two observations made by previous studies on chromate/sulfate and chromate/nitrate interactions in Shewanella oneidensis MR-1 biofilms. Additionally, we show a detailed Raman mapping based evidence of the existence of chromate–sulfate competition for cellular entry. Subsequently, we use Raman mapping to study the effect of nitrate on chromate reduction. The findings presented in this paper are among the first to report – detection of multiple metallic ions in bacterial biofilms using intracellular SERS substrates. Such a detailed characterization of biofilms using gold nanoislands based SERS mapping substrate can be extended to study cellular localization of other metallic ions and chemical species of biological and toxicological significance and their effect on reduction reactions in bacterial biofilms.     

Testing antimicrobials against biofilm bacteria

Standard laboratory methods are needed to assess the efficacy of antimicrobial agents that are applied to biofilm bacteria. Existing standard suspension tests and dried surface tests show much greater efficacy than antimicrobial agents applied to biofilms. The greater resistance of biofilm bacteria to antimicrobial agents can be attributed to a number of interacting factors, including reaction and diffusion processes that limit an agent's accessibility to bacteria, phenotypic changes in biofilm bacteria caused by stress, and adaptation of the bacteria. Because biofilm systems are so diverse, a variety of new biofilm tests with features that differ in important ways from existing tests will ultimately be required. For example, the biofilm test apparatus may include a pump and a continuous-flow stirred tank reactor. This report provides an overview of biofilm testing and suggests a strategy for creating standard test methods.     

Analysis of changes in attenuated total reflection FTIR fingerprints of Pseudomonas fluorescens from planktonic state to nascent biofilm state

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a useful method for monitoring biofilm in situ, non-destructively, in real time, and under fully hydrated conditions. In this work we focused on changes in Pseudomonas fluorescens ATR-FTIR fingerprint accompanying the very early stages of biofilm formation: initial bacterial adhesion and the very beginning of biofilm development in the presence of nutrients. To help interpreting variations in the ATR-FTIR fingerprint of sessile bacteria, ATR-FTIR spectra of planktonic bacteria in different growth phases were also examined, and the average surface coverage and spatial arrangement of bacteria on the ATR crystal were determined by epifluorescence microscopy. The proteins, nucleic acids and polysaccharides ATR-FTIR spectral data recorded during growth of sessile bacteria were shown to be linked to changes in the physiological state of the bacteria, possibly accompanied by extracellular polymeric substances production. This work clearly showed by spectroscopic method how bacteria change drastically their metabolism during the first hours of biofilm formation.     

Anti-fouling chemistry of chiral monolayers: enhancing biofilm resistance on racemic surface

This work reports the resistance to protein adsorption and bacterial biofilm formation by chiral monolayers of polyol-terminated alkanethiols surrounding micrometer-sized patterns of methyl-terminated alkanethiols on gold films. We discover that patterned surfaces surrounded by chiral polyol monolayers can distinguish different stages of biofilm formation. After inoculation on the surfaces, bacteria first reversibly attached on the chiral polyol monolayers. Over time, the bacteria detached from the polyol surfaces, and attached on the hydrophobic micropatterns to form biofilms. Interestingly, while both enantiomers of gulitol- and mannonamide-terminated monolayer resisted adsorption of proteins (bovine serum albumin, lysozyme, and fibrinogen) and confined biofilms formed on the micropatterns, the monolayers formed by the racemic mixture of either pair of enantiomers exhibited stronger antifouling chemistry against both protein adsorption and biofilm formation than monolayers formed by one enantiomer alone. These results reveal the different chemistries that separate the different stages of biofilm formation, and the stereochemical influence on resisting biofoulings at a molecular-level.     

Improved procedure for wastewater biofilm removal and analysis

Fixed-film processes for wastewater treatment are becoming widely used. Their efficiency is usually estimated only from substrate removal rate measurements. A better understanding of fixed culture composition and activity is needed to optimize processes in which they are involved. These analyses often require that biofilms are removed from their substrata, but this procedure is one of the most limiting steps in biofilm investigations, especially when cell counts are involved.

The main objective of the study was to develop an optimal removal procedure based on sonication for analysis of biofilm parameters such as total and active bacterial counts by epifluorescent microscopy (4',6-diamidino-2-phenylindole (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) stainings) and total organic content as estimated by total proteins determination and chemical oxygen demand measurements. Experiments were carried out on nitrifying biofilms developed on a plastic granular substratum.

Results confirmed that the removal step is crucial in biofilm analysis. The repeatability of bacterial enumeration was evaluated as well as the efficiency of sonication treatment and optimal conditions for attached cell removal. Within the studied range of sonication conditions, the sonication time and the duty cycle improved the removal efficiency of active bacteria, whereas the sonication power had the opposite effect.     

微生物燃料电池中生物膜成长对电池电化学性能的影响

以大肠杆菌为接种体,葡萄糖为基质,在1 000 Ω恒外阻下生成电活性生物膜,研究了生物膜的形成对电池电化学行为的影响。应用循环伏安、阻抗测试、极化分析、输出功率和阳极电势来考察其电化学表现。研究结果表明,随着生物膜完全成熟,阳极极化电阻减小66.5%,阳极电势逐渐降低,最大输出功率密度增加260%。     

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.     

Small molecule control of bacterial biofilms

Bacterial biofilms are defined as a surface attached community of bacteria embedded in a matrix of extracellular polymeric substances that they have produced. When in the biofilm state, bacteria are more resistant to antibiotics and the host immune response than are their planktonic counterparts. Biofilms are increasingly recognized as being significant in human disease, accounting for 80% of bacterial infections in the body and diseases associated with bacterial biofilms include: lung infections of cystic fibrosis patients, colitis, urethritis, conjunctivitis, otitis, endocarditis and periodontitis. Additionally, biofilm infections of indwelling medical devices are of particular concern, as once the device is colonized infection is virtually impossible to eradicate. Given the prominence of biofilms in infectious diseases, there has been an increased effort toward the development of small molecules that will modulate bacterial biofilm development and maintenance. In this review, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms through non-microbicidal mechanisms. The review discuses the numerous approaches that have been applied to the discovery of lead small molecules that mediate biofilm development. These approaches are grouped into: (1) the identification and development of small molecules that target one of the bacterial signaling pathways involved in biofilm regulation, (2) chemical library screening for compounds with anti-biofilm activity, and (3) the identification of natural products that possess anti-biofilm activity, and the chemical manipulation of these natural products to obtain analogues with increased activity.     

Discrimination of biofilm samples using pattern recognition techniques

Biofilms are complex aggregates formed by microorganisms such as bacteria, fungi and algae, which grow at the interfaces between water and natural or artificial materials. They are actively involved in processes of sorption and desorption of metal ions in water and reflect the environmental conditions in the recent past. Therefore, biofilms can be used as bioindicators of water quality. The goal of this study was to determine whether the biofilms, developed in different aquatic systems, could be successfully discriminated using data on their elemental compositions. Biofilms were grown on natural or polycarbonate materials in flowing water, standing water and seawater bodies. Using an unsupervised technique such as principal component analysis (PCA) and several supervised methods like classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination–DPLS (UVE-DPLS), we could confirm the uniqueness of sea biofilms and make a distinction between flowing water and standing water biofilms. The CART, DPLS and UVE-DPLS discriminant models were validated with an independent test set selected either by the Kennard and Stone method or the duplex algorithm. The best model was obtained from CART with 100% correct classification rate for the test set designed by the Kennard and Stone algorithm. With CART, one variable describing the Mg content in the biofilm water phase was found to be important for the discrimination of flowing water and standing water biofilms.     

Effects of disordered hemispherical micropatterns on Staphylococcus epidermidis biofilm formation

Surfaces which have physical patterns in the scale of bacteria cells have been shown to influence the microorganism's adhesion and biofilm formation characteristics. Layer-by-layer self-assembly was utilized to create disordered hemispherical patterns on poly(dimethylsiloxane) with a feature size of 0.5 μm, 1.0 μm and 2.0 μm. The effects of pattern size on the retention and biofilm formation of Staphylococcus epidermidis were examined as a function of culture time. The 1.0 μm pattern significantly reduced biofilm surface coverage by ～30% after 5 h of culture in comparison to that on an unpatterned surface while the effect of the 0.5 and 2.0 μm patterns was negligible. On the 1.0 μm surface, bacteria initially adhered on the unpatterned areas of the disordered surface and subsequently developed into biofilms by spreading across the unpatterned areas while avoiding those covered by the pattern. The results suggest that the size of surface patterns is an important factor in altering bacteria adhesion and biofilm formation characteristics.     

Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy

Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes.     

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.     

A Multinuclear Metal Complex Based DNase‐Mimetic Artificial Enzyme: Matrix Cleavage for Combating Bacterial Biofilms

Extracellular DNA (eDNA) is an essential structural component during biofilm formation, including initial bacterial adhesion, subsequent development, and final maturation. Herein, the construction of a DNase‐mimetic artificial enzyme (DMAE) for anti‐biofilm applications is described. By confining passivated gold nanoparticles with multiple cerium(IV) complexes on the surface of colloidal magnetic Fe₃O₄ /SiO₂ core/shell particles, a robust and recoverable artificial enzyme with DNase‐like activity was obtained, which exhibited high cleavage ability towards both model substrates and eDNA. Compared to the high environmental sensitivity of natural DNase in anti‐biofilm applications, DMAE exhibited a much better operational stability and easier recoverability. When DMAE was coated on substratum surfaces, biofilm formation was inhibited for prolonged periods of time, and the DMAE excelled in the dispersion of established biofilms of various ages. Finally, the presence of DMAE remarkably potentiated the efficiency of traditional antibiotics to kill biofilm‐encased bacteria and eradiate biofilms.     

Winning the fight against biofilms: the first six-month study showing no biofilm formation on zwitterionic polyurethanes

Biofilms have been a long-standing challenge for healthcare, water transport, and many other industries. They lead to bacterial growth and infections in animals, food products, and humans, cause premature removal of the implanted materials or devices from patients, and facilitate fouling and corrosion of metals. Despite some published and patented methods on minimizing the effects of biofilms for a short period (less than two weeks), there exists no successful means to mitigate or prevent the long-term formation of biofilms. It is even more challenging to integrate critical anti-fouling properties with other needed physical and chemical properties for a range of applications. In this study, we developed a novel approach for combining incompatible, highly polar anti-fouling groups with less polar, mechanically modifying groups into one material. A multifunctional carboxybetaine precursor was designed and introduced into polyurethane. The carboxybetaine precursors undergo rapid, self-catalyzed hydrolysis at the water/material interface and provide critical anti-fouling properties that lead to undetectable bacterial attachment and zero biofilm formation after six months of constant exposure to Pseudomonas aeruginosa and Staphylococcus epidermidis under the static condition in a nutrient-rich medium. This zwitterionic polyurethane is the first material to demonstrate both critical anti-biofilm properties and tunable mechanical properties and directly validates the unproven anti-fouling strategy and hypothesis for biofilm formation prevention. This approach of designing ‘multitasking materials’ will be useful for the development of next generation anti-fouling materials for a variety of applications.

To prevent biofilms and biofoulings, a versatile zwitterionic polyurethane material platform was invented with an unmatched anti-fouling potency, as shown by a 6-month study where no bacterial attachment or biofilm formation was observed.