Biofilms最新文献

Drinking water distribution systems (DWDS) are an engineered system designed to protect water quality during delivery from treatment works to consumers’ taps. Biofilms form on the vast internal surfaces of DWDS, impacting water quality by their activity and/or mobilisation into the bulk-water. Disinfection-residuals are often maintained in drinking water to mitigate planktonic microbial contamination (and associated water quality/health risks). However, the impact of residual-disinfection upon biofilms, and the subsequent unintended risk they may present to water quality, is unclear.

To address this, an internationally-unique, temperature-controlled, full-scale DWDS test facility, fed with water from the local DWDS, was used to grow biofilms (for 28 days). The facility enables three simultaneous conditions to be run in replicate pipe loops (each ~200m long, 79mm internal diameter, PE100 pipe). Conditions studied were Low-, Medium- and High-chlorine regimes. Various water quality parameters were monitored throughout, biofilms were sampled every two weeks (n=5). Physical, chemical and molecular analyses were applied to characterise the matrix (structure and composition) and microbial communities (via analysis of bacterial 16S rRNA and fungal ITS genes) of biofilms developed under the different chlorine regimes. After growth, a “mobilisation” test was conducted simulating hydraulic changes that occur in DWDS. Biofilms from each chlorine regime were exposed to increasing shear stresses to determine any water quality degradation as a consequence of biofilm mobilisation.

High-chlorine residual concentration during development reduced biofilm bacterial concentrations but increased inorganics and selected for unique bacterial and fungal communities. Ultimately the biofilms developed under a High-chlorine residual resulted in the greatest decrease in water quality, in response to mobilisation, and the Low-chlorine regime resulted in biofilms which had the lowest impact on water quality. These unanticipated findings suggest chlorine-boosting should be considered carefully and may actually exacerbate water quality issues. The derived understanding could impact the long-term management of DWDS water quality and biofilm, whilst challenging the current mind-set of continuous residual-disinfection control strategies.

In the oil and gas industry, internal corrosion represents one of the major threats to asset lifetime and integrity. Of the types of internal corrosion, microbiologically influenced corrosion (MIC) is the most difficult to predict and monitor due to the unpredictable nature of microbial growth and the minimal metal loss resulting in through wall failure (pitting). MIC results from biofilm communities interacting directly and indirectly with the metal. Due to the structure and nature of these pipelines, directly monitoring sessile growth is impossible. As a result, most MIC monitoring is done through planktonic cells retrieved from fluid samples as a proxy for sessile populations.

Growth curves are one of the most fundamental methods of quantitatively assessing microbial growth. In the lab, pure cultures are measured using optical densities, biomass staining, direct microscopic counting and counting colony forming units (CFU) on specialized media while more advanced techniques involve quantitative PCR (qPCR) of key genes. While PCR technologies are more easily transferred from the field to the lab, CFU counts are impossible in the field. Alternatives to the CFU are colorimetric activity assays such as “bug bottles” or biological activity reaction test (BART) bottles but aren’t sensitive and require long incubation times. More sensitive assays such as ATP measurements are also used but can be misleading as high metabolically active samples will give higher cell count equivalents than a metabolically slow community of an identical size.

To systematically evaluate a best practice, we conducted growth curves in a lab scenario using six pure cultures and techniques predominantly used in the field to determine how these techniques compare and accurately measure microbial growth. The six species used are Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. The techniques used are optical density at 600 nm, ATP activity measurements using a luciferase-based assay, DNA concentration and 16S rRNA copy numbers.

It was found that most lines of data follow the expected sigmoidal growth curve to varying degrees for all species. OD₆₀₀ readings follow the expected sigmoidal curves, exhibiting a lag phase, log growth phase and a stationary phase. ATP peaks during mid log phase and quickly declines, never showing a distinct stationary phase, while DNA concentrations closely follow the OD₆₀₀ readings but decline to death phase more rapidly. qPCR of the 16S rRNA genes revealed this data followed the same trends but was less susceptible to fluctuations.

Assessing microbial biofilms in the environment and on anthropogenic industrial infrastructure is extremely challenging given sampling, storage and transportation to the lab.  This work begins to establish best practices for growth of environmental communities

Introduction: The ability of bacteria to colonize implant surfaces and tissues as a biofilm plays a relevant role in medical-device-associated infections. Staphylococcus aureus strains can produce a biofilm matrix made of the poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide and/or proteins. PIA/PNAG is synthesised by enzymes encoded by the icaADBC operon whose expression is repressed by the transcriptional regulator IcaR, while the protein-dependent biofilm is commonly associated to fibronectin-biding proteins, FnBPA and FnBPB, encoded by fnbA and fnbB genes. The aim of this work was to identify common genetic features in the regulatory regions of biofilm-related genes among clinical S. aureus strains derived from periprosthetic joint infections (PJI).  

Material and Methods: Genomes of 45 S. aureus strains from PJI were sequenced. Firstly, the sequence comprising the entire icaADBC regulatory region (5’UTR of icaADBC and icaR, the icaR coding sequence and its 3’UTR region) and secondly, the sequence of the promoter region of fnbAB were compared to those of S. aureus MW2 strain. Regulatory regions containing distinctive features were identified, fused to a reporter gene and introduced in a reference strain to analyze differences in gene expression.

Results: In the case of the icaADBC operon, single nucleotide polymorphisms (SNPs) in the icaADBC regulatory region allowed clustering of the strains in five groups from which a representative strain was chosen for further studies: S. aureus MIC 6924 (20% of isolates), MIC 6934 (13%), MIC 6936 (7%), MIC 6948 (2%) and MIC 7018 (4%). Of note, MICs 6948 and 7018 contained mutations in the icaR coding sequence. In this respect, a single nucleotide mutation in icaR (Val176Glu) caused a significant increase in icaADBC transcription and thus, in PIA/PNAG production and biofilm formation. In contrast, none of the rest of the SNPs found in the icaADBC regulatory region modified the transcription levels of the reporter gene. With respect to fnBPA and fnBPB genes and in agreement with previous studies, 100% of the strains contained the fnbA gene whereas only 69% contained the fnbB gene. The promoter region of fnbA was found to be highly conserved. SNPs in the promoter region of fnbB allowed clustering the strains in five groups. From these, the most frequently identified pattern was represented by S. aureus MIC 6948 (53%) and correlated with a lower level of reporter expression, whereas the group containing SNPs in the LexA binding sites was represented by MIC 7014 (4%) and correlated with higher expression levels.

Conclusion:  Our results suggest that S. aureus isolates from

The membrane aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that can greatly decrease energy requirements for wastewater treatment. It consists of cassettes of air-supplying, hollow-fiber membranes that can retrofit existing activated sludge processes. MABR behavior differs from conventional biofilm processes due to the counter-diffusion of the electron donor (ammonia) and acceptor (oxygen).

Partial nitrification (PN), or partial nitrification Anammox (PNA), can further improve MABR energy efficiency and cost effectiveness.  To achieve this, ammonia oxidizing bacteria (AOB) must outcompete nitrite-oxidizing bacteria (NOB).  High temperatures favor AOB, but it is not feasible to heat the wastewater influent.  However, high-temperature compressed air can be supplied to the membrane lumen, increasing temperatures inside the biofilm without increasing the bulk temperatures. No previous research has addressed temperature gradients in biofilms, which can lead to gradients in  biodegradation kinetics, diffusivities, and O₂ solubility.

The objective of this research was to explore the effect of temperature gradients in MABR biofilms, especially with respect to PN. We used a one-dimensional multi-species biofilm model, which considers the MABR physical and biochemical behavior, especially with respect to temperature. The model was implemented using COMSOL Multiphysics. We also used bench-scale experiments to explore the effect of biofilm temperature gradients on MABR nitrification and PN performances and microbial community structure.

Model simulations showed that MABR biofilms exposed to a temperature gradient from 20 ºC (biofilm interior) to 10 ºC (bulk liquid) had a 60% increase in nitrification rates compared with biofilms at 10 ºC. More importantly, the model predicted a complete out competition of NOBs within the biofilm.

Preliminary experimental results confirm a significant (105%) increase in nitrification fluxes with a temperature of 30ºC compared to ambient temperatures (20ºC). Future experiments will validate the model predicted effects of biofilm temperature gradients on nitrification fluxes and microbial community structure.

Escherichia coli biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.

In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from E. coli biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of E. coli JM109(DE3) transformed with a plasmid containing a T7 promoter.

It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].

It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.

References:

[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in Escherichia coli biofilms: A non-conventional form of high cell density cultivation. Process Biochemistry, 57, 1-8. https://doi.org/10.1016/j.procbio.2017.03.018

[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576

Background:

The adhesion of planktonic bacteria to a surface (biotic or abiotic), and their subsequent ability to aggregate into multicellular communities called biofilms, is a major driving force of failing antibiotic therapy and persistence in chronic infections caused by a variety of pathogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) and plaguing healthcare systems worldwide. Biofilms are estimated to be involved in over 80% of all microbial infections in humans, and commonly exhibit extreme resistance to conventional antimicrobial treatments. Consequently, there is an urgent need for novel antimicrobial agents, which target biofilm residing cells. Here, we present the development and evaluation of a new generation of dual-acting nitroxide functionalised antibiotics with potent biofilm eradication activity.

Methodology:

Synthetic organic chemistry was utilised to produce a new generation of nitroxide functionalised antibiotics with targeted biofilm eradication capabilities. These compounds were tested for biofilm eradication and/or dispersal of several bacterial species using the MBEC^TM device, a reproducible high-throughput static biofilm formation system. Mature biofilms were treated with serial dilutions of the specific test agent(s) and recovered bacterial numbers were quantified by absorbance spectroscopy at 600 nm or plating for viable cell counts. Treated biofilms were also stained with Live/Dead (SYTO-9/PI) bacterial viability kit and analysed by fluorescence and confocal laser scanning microscopy.

Results:

Nitroxide functionalised antibiotics exhibit potent biofilm-eradication activity against a variety of medically important pathogens, including P. aeruginosa, uropathogenic E. coli, and S. aureus. In Minimal Biofilm Eradication Concentration (MBEC) assays nitroxide functionalised antibiotics were 64-fold more potent against S. aureus biofilms, and at least 2-fold more potent against uropathogenic E. coli biofilms than the parent antibiotic ciprofloxacin.

Conclusions:

Currently, antibiotics are often entirely ineffective against biofilm infections. Nitroxide functionalised antibiotics represent a promising new strategy, which could circumvent the resistance of Gram-positive and Gram-negative biofilms to conventional treatments.