Bacterial Cell Cultures in a Lab-on-a-Disc: A Simple and Versatile Tool for Quantification of Antibiotic Treatment Efficacy.

Pathogenic bacterial biofilms can be life-threatening, greatly decrease patient's quality of life, and are a substantial burden on the healthcare system. Current methods for evaluation of antibacterial treatments in clinics and in vitro systems used in drug development and screening either do not facilitate biofilm formation or are cumbersome to operate, need large reagent volumes, and are costly, limiting their usability. To address these issues, this work presents the development of a robust in vitro cell culture platform compatible with confocal microscopy. The platform shaped as a compact disc facilitates long-term bacterial culture without external pumps and tubing and can be operated for several days without additional liquid handling. As an example, Pseudomonas aeruginosa biofilm is grown from single cells, and it is shown that (1) the platform delivers reproducible and reliable results; (2) growth is dependent on flow rate and growth medium composition; and (3) efficacy of antibiotic treatment depends on the formed biofilm. This platform enables biofilm growth, quantification, and treatment as in a conventional flow setup while decreasing the application barrier of lab-on-chip systems. It provides an easy-to-use, affordable option for end users working with cell culturing in relation to, e.g., diagnostics and drug screening.

Critical review on biofilm methods.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

BACKGROUND: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. RESULTS: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids. CONCLUSION: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Secreted single-stranded DNA is involved in the initial phase of biofilm formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an obligate human pathogen that colonizes the genital tract and causes gonorrhoea. Neisseria gonorrhoeae can form biofilms during natural cervical infections, on glass and in continuous flow-chamber systems. These biofilms contain large amounts of extracellular DNA, which plays an important role in biofilm formation. Many clinical isolates contain a gonococcal genetic island that encodes a type IV secretion system (T4SS). The T4SS of N. gonorrhoeae strain MS11 secretes ssDNA directly into the medium. Biofilm formation, studied in continuous flow-chamber systems by confocal laser scanning microscopy (CLSM), was strongly reduced, especially in the initial phases of biofilm formation, in the presence of Exonuclease I, which specifically degrades ssDNA or in a ΔtraB strain that does not secrete ssDNA. To specifically detect ssDNA in biofilms using CLSM, a novel method was developed in which thermostable fluorescently labelled ssDNA- and ss/dsDNA-binding proteins were used to visualize ssDNA and total DNA in biofilms and planktonic cultures. Remarkably, mainly dsDNA was detected in biofilms of the ssDNA secreting strain. We conclude that the secreted ssDNA facilitates initial biofilm formation, but that the secreted ssDNA is not retained in mature biofilms.

Biosynthesis enhancement of tropodithietic acid (TDA) antibacterial compound through biofilm formation by marine bacteria Phaeobacter inhibens on micro-structured polymer surfaces.

Although aquaculture is a major player in current and future food production, the routine use of antibiotics provides ample ground for development of antibiotic resistance. An alternative route to disease control is the use of probiotic bacteria such as the marine bacteria Phaeobacter inhibens which produces tropodithietic acid (TDA) that inhibit pathogens without affecting the fish. Improving conditions for the formation of biofilm and TDA-synthesis is a promising avenue for biocontrol in aquaculture. In this study, the biosynthesis of TDA by Phaeobacter inhibens grown on micro-structured polymeric surfaces in micro-fluidic flow-cells is investigated. The formation of biofilms on three surface topographies; hexagonal micro-pit-arrays, hexagonal micro-pillar-arrays, and planar references is investigated. The biomass on these surfaces is measured by a non-invasive confocal microscopy 3D imaging technique, and the corresponding TDA production is monitored by liquid chromatography mass spectrometry (LC-MS) in samples collected from the outlets of the microfluidic channels. Although all surfaces support growth of P. inhibens, biomass appears to be decoupled from total TDA biosynthesis as the micro-pit-arrays generate the largest biomass while the micro-pillar-arrays produce significantly higher amounts of TDA. The findings highlight the potential for optimized micro-structured surfaces to maintain biofilms of probiotic bacteria for sustainable aquacultures.

Microbial biofilms in biorefinery - Towards a sustainable production of low-value bulk chemicals and fuels.

Harnessing the potential of biocatalytic conversion of renewable biomass into value-added products is still hampered by unfavorable process economics. This has promoted the use of biofilms as an alternative to overcome the limitations of traditional planktonic systems. In this paper, the benefits and challenges of biofilm fermentations are reviewed with a focus on the production of low-value bulk chemicals and fuels from waste biomass. Our study demonstrates that biofilm fermentations can potentially improve productivities and product yields by increasing biomass retention and allowing for continuous operation at high dilution rates. Furthermore, we show that biofilms can tolerate hazardous environments, which improve the conversion of crude biomass under substrate and product inhibitory conditions. Additionally, we present examples for the improved conversion of pure and crude substrates into bulk chemicals by mixed microbial biofilms, which can benefit from microenvironments in biofilms for synergistic multi-species reactions, and improved resistance to contaminants. Finally, we suggest the use of mathematical models as useful tools to supplement experimental insights related to the effects of physico-chemical and biological phenomena on the process. Major challenges for biofilm fermentations arise from inconsistent fermentation performance, slow reactor start-up, biofilm carrier costs and carrier clogging, insufficient biofilm monitoring and process control, challenges in reactor sterilization and scale-up, and issues in recovering dilute products. The key to a successful commercialization of the technology is likely going to be an interdisciplinary approach. Crucial research areas might include genetic engineering combined with the development of specialized biofilm reactors, biofilm carrier development, in-situ biofilm monitoring, model-based process control, mixed microbial biofilm technology, development of suitable biofilm reactor scale-up criteria, and in-situ product recovery.

Microfluidic dissolved oxygen gradient generator biochip as a useful tool in bacterial biofilm studies.

A microfluidic chip for generation of gradients of dissolved oxygen was designed, fabricated and tested. The novel way of active oxygen depletion through a gas permeable membrane was applied. Numerical simulations for generation of O(2) gradients were correlated with measured oxygen concentrations. The developed microsystem was used to study growth patterns of the bacterium Pseudomonas aeruginosa in medium with different oxygen concentrations. The results showed that attachment of Pseudomonas aeruginosa to the substrate changed with oxygen concentration. This demonstrates that the device can be used for studies requiring controlled oxygen levels and for future studies of microaerobic and anaerobic conditions.

Evaluation of enoyl-acyl carrier protein reductase inhibitors as Pseudomonas aeruginosa quorum-quenching reagents.

Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent. EGCG treatment was further shown to be able to attenuate the production of virulence factors and biofilm formation of P. aeruginosa.

Synthesis of carbon quantum dot-poly lactic-co-glycolic acid hybrid nanoparticles for chemo-photothermal therapy against bacterial biofilms.

Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the resistance to antibiotics. Poly lactic-co-glycolic acid (PLGA)-based nanoparticle delivery systems have been intensively investigated to combat the bacterial biofilms-associated infections. However, some drawbacks associated with current PLGA-based nanoformulations (e.g. the relatively low drug loading capability, premature burst release and/or incapability of on-demand release of cargos at the site of action) restrict the transition from the lab research to the clinical applications. One potent strategy to overcome the above-mentioned limitations is exploiting the unique properties of carbon quantum dots (CQDs) and combining CQDs with the conventional PLGA nanoparticles. In the present study, the CQDs were innovatively incorporated into PLGA nanoparticles by using a microfluidic method. The resulting CQD-PLGA hybrid nanoparticles presented good loading capability of azithromycin (a macrolide antibiotic, AZI) and tobramycin (an aminoglycoside antibiotic, TOB), and stimuli-responsive release of the cargos upon laser irradiation. Consequently, AZI-loaded CQD-PLGA hybrid nanoparticles showed chemo-photothermally synergistic anti-biofilm effects against P. aeruginosa biofilms. Additionally, the CQD-PLGA hybrid nanoparticles demonstrated good biocompatibility with the eukaryotic cells. Overall, the proof-of-concept of CQD-PLGA hybrid nanoparticles may open a new possibility in chemo-photothermal therapy against bacterial biofilms.

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans. RESULTS: Deletion of the genes encoding AnAA13A that possesses a CBM20 starch-binding module, AnAA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking AnAA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the AnAA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the AnAA13B-deficient strain as compared to the reference. CONCLUSIONS: This study shows that the loss of AA13, especially the starch-binding AnAA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions.

Ultrasmall TPGS-PLGA Hybrid Nanoparticles for Site-Specific Delivery of Antibiotics into Pseudomonas aeruginosa Biofilms in Lungs.

Inhaled antibiotic treatment of cystic fibrosis-related bacterial biofilm infections is challenging because of the pathological environment of the lungs. Here, we present an "environment-adaptive" nanoparticle composed of a solid poly lactic-co-glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for the site-specific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultrasmall size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g., quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa. In conclusion, the ultrasmall TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics in a sustained manner in the vicinity of the biofilm-forming bacteria.

Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy.

Biofilms are agglomerates of microorganisms surrounded by a self-produced extracellular matrix. During the last 10 years, there has been an increasing recognition of biofilms as a highly significant topic in microbiology with relevance for a variety of areas in our society including the environment, industry, and human health. Accordingly a number of biofilm model systems, molecular tools, microscopic techniques, and image analysis programs have been employed for the study of biofilms under controlled and reproducible conditions. Studies using confocal laser scanning microscopy (CLSM) of biofilms formed in flow-chamber experimental systems by genetically color-coded bacteria have provided detailed knowledge about biofilm developmental processes, cell differentiations, spatial organization, and function of laboratory-grown biofilms, in some cases down to the single cell level. In addition, the molecular mechanisms underlying the increased tolerance that biofilm cells often display towards antibiotic treatment are beginning to be unravelled.

Utilizing nanoparticles for improving anti-biofilm effects of azithromycin: A head-to-head comparison of modified hyaluronic acid nanogels and coated poly (lactic-co-glycolic acid) nanoparticles.

HYPOTHESIS: The widespread resistance of bacteria to traditional antibiotic treatments has expedited the search for novel therapies against these pathogens. The hypothesis of this work is that two distinctively different polymeric delivery systems, specifically D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles and octenyl succinic anhydride-modified low molecular weight hyaluronic acid (OSA-HA) nanogels may be used to substantially improve the properties of azithromycin, allowing its use for effective treatment of Pseudomonas aeruginosa biofilm infections. EXPERIMENTS: Azithromycin was encapsulated in both delivery systems and the physicochemical properties of the loaded delivery systems, including size, surface charge and drug loading were evaluated. Additionally, particle interaction with a mucin layer, penetration into a bacterial biofilm, prevention of biofilm formation and eradication of pre-formed biofilms, the influence on production of virulence factors and bacterial motility as well as cytotoxicity towards hepatocytes and lung epithelial cells were compared head-to-head. FINDINGS: The TPGS-PLGA nanoparticles noticeably improved the antimicrobial activity and the biofilm prevention activity of azithromycin whereas the OSA-HA nanogels showed reduced mucin interactions together with improved reduction of pre-formed biofilms and maintained the low eukaryotic cell cytotoxicity of azithromycin.

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections.

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

Application of RNA-seq and Bioimaging Methods to Study Microbe-Microbe Interactions and Their Effects on Biofilm Formation and Gene Expression.

Complex interactions between pathogenic bacteria, the microbiota, and the host can modify pathogen physiology and behavior. We describe two different experimental approaches to study microbe-microbe interactions in in vitro systems containing surface-associated microbial populations. One method is the application of RNA sequencing (RNA-seq) to determine the transcriptional changes in pathogenic bacteria in response to microbial interspecies interactions. The other method combines flow cell devices for bacterial cultivation and growth with high-resolution bioimaging to analyze the microscale structural organization of interacting microbial populations within mixed-species biofilms.

Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4-5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.

Immunomodulating potential of supplementation with probiotics: a dose-response study in healthy young adults.

Certain probiotic microorganisms have been found beneficial in the treatment of immune-related diseases and may also affect immune function in healthy people. Intervention studies of probiotics in healthy humans are urgently required. Here, the immunomodulating potential of Bifidobacterium animalis ssp. lactis (BB-12) and Lactobacillus paracasei ssp. paracasei (CRL-431) was studied in a double-blind placebo-controlled parallel dose-response trial (n=71) based on five randomly assigned groups of young healthy adults supplemented for 3 weeks with 0, 10(8), 10(9), 10(10) and 10(11) CFU day(-1), respectively, of a mixture of BB-12 and CRL-431. No statistically significant dose-dependent effect was found for phagocytic activity in blood leukocytes, fecal immunoglobulin A (IgA) concentrations or production of interferon-gamma and interleukin-10 in blood cells. When evaluating data according to the amount of viable BB-12 recovered from faeces, the interferon-gamma production in blood cells was significantly reduced. In conclusion, no solid effect on the immune function of young healthy adults supplemented with even high doses of B. animalis ssp. lactis BB-12 and L. paracasei ssp. paracasei CRL-431 was demonstrated in this study.

Utilization and control of ecological interactions in polymicrobial infections and community-based microbial cell factories.

Microbial activities are most often shaped by interactions between co-existing microbes within mixed-species communities. Dissection of the molecular mechanisms of species interactions within communities is a central issue in microbial ecology, and our ability to engineer and control microbial communities depends, to a large extent, on our knowledge of these interactions. This review highlights the recent advances regarding molecular characterization of microbe-microbe interactions that modulate community structure, activity, and stability, and aims to illustrate how these findings have helped us reach an engineering-level understanding of microbial communities in relation to both human health and industrial biotechnology.

Physiological responses of Pseudomonas putida KT2442 to phosphate starvation.

The physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology, qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA), cellular ATP and ribosome content, and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions, the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However, after this decline, viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced, and ATP levels dropped to very low values after the onset of starvation; later, however, there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow, possibly utilizing nutrients derived from dead cells, and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system, we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus, long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.

Methods for dynamic investigations of surface-attached in vitro bacterial and fungal biofilms.

Three dynamic models for the investigation of in vitro biofilm formation are described in this chapter. In the 6-well plate assay presented here, the placing of the plate on a rotating platform provides shear, thereby making the system dynamic with respect to the static microtiter assay.The second reported model, especially suitable for harvesting high amounts of cells for transcriptomic or proteomic investigations, is based on numerous glass beads placed in a flask incubated with shaking on a rotating platform, thus increasing the surface area for biofilm formation. Finally, the flow-cell system, that is the driving model for elucidating the biofilm-forming process in vitro as well as the biofilm tolerance towards antibiotics and host defense components, is illustrated here.