Biofilms最新文献

Introduction

Since antibiotics were discovered, bacteria have demonstrated the ability to develop resistance by many different mechanisms. According to WHO reports from 2014, there has been an alarming increase in the antibiotic resistant bacterial strains in most parts of the world¹. Our previous results showed that a nanoantibiotic (NAB) design created in our laboratory², composed of a cerium oxide core, mesoporous silica shell loaded with capsaicin, and a chitosan coating, are effective against planktonic E. coli. However, most of the pathogenic bacteria form biofilms during infections. That is why the next stage of studying NAB is to determine whether they are effective against biofilms of different species. Moreover, the results of NAB efficiency against planktonic E. coli did not clearly show the contribution of the antibiotic drug component of NAB – capsaicin. Hence, the first step of the current study is to determine whether and to what degree, mesoporous silica nanoparticles (MSN) – serving as NAB model in this case - penetrate biofilms as a function of particle shape and surface coating; as well as finding the efficient concentration of capsaicin against E. coli and S. aureus  to optimize the NAB dosing against biofilms.

Aim

To check in vitro penetration of MSN on S. aureus biofilm and antibacterial activity of NAB and pure capsaicin on E. coli and S. aureus biofilms.

Methods

To investigate NAB efficiency on biofilms MBEC-high-throughput assay³ was performed. Equal biofilms formed on peg-lids were incubated with different concentrations of NAB and capsaicin. After different time point biofilms were sonicated and plated on agar plated to perform CFU counting. To determine the efficient concentration of capsaicin, biofilms were formed in 12 well plates and then incubated with different concentrations of capsaicin. To visualize inhibitory effect, plating for CFU counting and Resazurin assay were applied. To evaluate the penetration of particles, labeled and non-labeled particles were added to fully grown St. aureus biofilms, incubated and visualized with confocal microscopy and structured illumination microscopy.

Results

1. Through two different microscopy techniques penetration of particles into biofilm and their localization next to bacteria cells were observed.
2. In MBEC-high-throughput assay no inhibitory effect of NAB against E. coli biofilms was detected in comparison with untreated bacteria.
3. Resazurin assay and CFU counting method allowed us to determine the most efficient concentration of capsaicin against E. coli and St. aureus biofilms.

Conclusion

1. Use of MSN and NAB in particular to deliver active antiba

Many bacteria use repeats-in-toxin (RTX) adhesins to mediate binding to host cells and facilitate subsequent colonisation and infection by forming biofilms. Vibrio cholerae, the causative agent of cholera, uses a 230-kDa RTX adhesin, FrhA, to facilitate intestinal colonization. FrhA also mediates hemagglutination of red-blood cells (erythrocytes). Here we have demonstrated that the hemagglutination capability of FrhA is localized to a ~ 20-kDa domain near its C terminus. Bioinformatic analyses indicated this erythrocyte-binding domain (VcEBD) is 65% identical to a peptide-binding module found in the 1.5-MDa ice-binding RTX adhesin that helps its Antarctic bacterium, Marinomonas primoryensis, form symbiotic biofilms with diatoms on the underside of sea ice. This suggested that the FrhA binds V. cholerae to proteins present on the cell surface of erythrocytes. X-ray crystallography revealed that VcEBD has an oblong β-sandwich fold with a shallow, Ca²⁺-dependent ligand-binding cavity that can anchor a peptidyl ligand with a free terminal carboxyl group. Using a structure-guided approach, we screened a small library of ~ 60 short peptides and optimized the affinity of VcEBD’s peptidyl ligands by roughly 1,000-fold. Importantly, the high-affinity ligands are effective at blocking V. cholerae from binding to erythrocytes at nano-molar concentrations. Structures of VcEBD in complex with three different peptides further elucidated the molecular basis for their interactions, which sets the stage for the development of ligand-based antagonists that may help disrupt V. cholerae interaction with intestinal cells to prevent or treat cholera. With the spread of antibiotic-resistant pathogenic bacteria, this work sheds light on an anti-adhesion approach for combating bacterial infections without the excessive use of antibiotics.

Biofilms prevention and removal are crucial in many industrial and medical applications. Their complex and cohesive structure provides resistance to cleaning even to strong disinfectants. A key factor for their behavior is the wetting properties of their surfaces.

The main goal of this work is to study the wetting properties of biofilms produced by bacteria Pseudomonas fluorescens. Biofilms are obtained on glass coupons under well controlled flow conditions, using custom-made flow cell devices. Different nutrient concentration and shear flow conditions are investigated.

Biofilm wetting properties are examined under imposed external body forces (forced wetting) through a specialized device, named Kerberos®. Kerberos® is capable of subjecting sessile droplets to varying tilting angles and centrifugal forces while monitoring the variation of the droplet shape in X, Y and Z-directions through three Wi-Fi cameras. Wetting experiments are carried out using water-based solution (dye solution) droplets on biofilm-coated glass coupons. In this work, spreading/sliding behaviour of droplets are investigated only on horizontal substrates (no tilting) under the action of centrifugal forces. Apart from wetting properties, biofilm growth kinetics and surface morphology at different nutrient and shear flow conditions are also assessed.

Results show that, according to the different growth conditions, biofilms present different wetting properties. At lower nutrient concentration and shear flow conditions, spreading and sliding behaviour are similar to that observed in glass coupons in the absence of biofilm. At higher nutrient and shear flow conditions, spontaneous wicking of the biofilm occurs the moment of droplet deposition on the biofilm leading to irregular and jagged shapes of droplets, while on the contrary water droplets look like smooth spherical sections on pure glass. The spontaneous wicking affects the droplet initial shape and so the wetting behaviour during the subsequent rotation tests. In each examined condition, biofilms show hydrophilic properties.

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.

Microbial biofilms are a crucial problem in many areas including the food processing industry, biotechnology, water quality and medical scenarios. The complexity of biofilm formation and subsequent prevention strategies - requires a fundamental understanding of the involved molecular mechanisms and the possibility of long-term monitoring biofilm formation. Infrared attenuated total reflection (IR-ATR) spectroscopy is a versatile analytical technique for monitoring biofilm formation of bacteria isolates in situ, non-destructively, and close to real time as an innovative approach providing molecular insight into biofilm formation [1]. The utility of IR-ATR to investigate microorganism behavior within biofilms derives from the evanescent field penetrating few micrometers into the biofilm formed directly at the interface of a multi-reflection ATR waveguide and the sample. In the present study, isolates from food biogenic amine (BA)-producing bacteria, Lactobacillus parabuchneri DSM 5987 strains formed in cheese are analyzed for developing a deeper understanding on the formation of biofilms, which are significant contributors to the presence of histamine in dairy food products [2]. Infrared spectra were recorded using a custom flow-through ATR assembly for revealing the metabolism of microorganisms within such biofilms along with the effects of the substrate functionality and culture conditions on the extracellular biopolymeric matrices [3,4]. The appearance of key IR bands in the region of 1600-1200 cm^-1 indicates the production of lactic acid or lactate and the presence of amide groups, while most pronounced intensities in 1140-950 cm^-1 correspond to phospholipids, polysaccharides and nucleic acids. In this study, the spectral region between 1700 and 600 cm^-1 was determined to be the representative region for the identification of Lactobacillus parabuchneri biofilms enabling to study bioadhesion mechanisms and physico-chemical property changes during extended periods of biofilm growth. Real time monitoring has led to concrete steps for inhibition and disintegration via suitable antimicrobials by deposition on the IR inactive region of ATR waveguide. Multivariate data evaluation and classification strategies were applied to enable efficient multiparametric analysis for providing molecular information facilitating a better understanding of biofilm formation, maturation and changes in biofilm architecture via IR spectroscopic data. 
 
Keywords: IR-ATR spectroscopy, in situ monitoring, Lactobacillus parabuchneri, biofilm, ATR waveguide, flow-through ATR, lactic acid, multivariate data analysis. 

References: [1] Stenclova P, Freisinger S, et al. Appl. Spectro., 2019; Vol.73 (4) 424-432 [2] Yunda E, Quilès F, et al. Biofouling, 2019; Vol.35 (5) 494-507 [3] D