Advanced strategies for combating bacterial biofilms

Biofilms are communities of microorganisms that are formed on and attached to living or nonliving surfaces and are surrounded by an extracellular polymeric material. Biofilm formation enjoys several advantages over the pathogens in the colonization process of medical devices and patients' organs. Unlike planktonic cells, biofilms have high intrinsic resistance to antibiotics and sanitizers, and overcoming them is a significant problematic challenge in the medical and food industries. There are no approved treatments to specifically target biofilms. Thus, it is required to study and present innovative and effective methods to combat a bacterial biofilm. In this review, several strategies have been discussed for combating bacterial biofilms to improve healthcare, food safety, and industrial process.     

生物技术药物的研究开发与产业化现状及前景

本综述了近年来国内外生物技术药物的研究、开发和产业化现状,讨论了我国医药生物技术产业存在的主要问题。提出中国医药生物技术产业发展应遵循自主开发与技术引进相结合及政府引导与市场机制相结合的原则,政府、企业和科技界应发挥各自不同的作用;展望了我国生物药物产业的发展方向和生物技术药物的市场前景。     

Thermal mitigation of Pseudomonas aeruginosa biofilms

Bacterial biofilms infect 2–4% of medical devices upon implantation, resulting in multiple surgeries and increased recovery time due to the very great increase in antibiotic resistance in the biofilm phenotype. This work investigates the feasibility of thermal mitigation of biofilms at physiologically accessible temperatures. Pseudomonas aeruginosa biofilms were cultured to high bacterial density (1.7?×?10⁹ CFU cm^?2) and subjected to thermal shocks ranging from 50°C to 80°C for durations of 1–30 min. The decrease in viable bacteria was closely correlated with an Arrhenius temperature dependence and Weibull-style time dependence, demonstrating up to six orders of magnitude reduction in bacterial load. The bacterial load for films with more conventional initial bacterial densities dropped below quantifiable levels, indicating thermal mitigation as a viable approach to biofilm control.     

Partial characterization of an exopolysaccharide secreted by a marine bacterium,Vibrio neocaledonicus sp. nov., from New Caledonia

Aims

Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS.

Methods and Results

Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA–DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC‐size exclusion chromatography. This EPS exhibits a high N‐acetyl‐hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 × 10³ Da. These data are relevant for possible technological exploitation.

Conclusions

We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer.

Significance and Impact of the Study

This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.     

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy. Extracellular teichoic acid was produced by all strains studied. Some of the clinical strains were shown to produce biofilms with compositions similar to that of the model strain, containing a varying amount of PNAG. The chemical structure of PNAG of the clinical strains was similar to that previously described for the model strains S. epidermidis RP62A and Staphylococcus aureus MN8m, differing only in the amount of charged groups. Biofilms of the strains producing a substantial amount of PNAG were detached by dispersin B, a PNAG-degrading enzyme, while being unsusceptible to proteinase K treatment. On the other hand, some strains produced biofilms without any detectable amount of PNAG. The biofilms of these strains were dispersed by proteinase K, but not by dispersin B.     

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m^?2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m^?2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Update on the challenging role of biofilms in peritoneal dialysis

Biofilms are commonly associated with an increased risk of patient infection. In peritoneal dialysis (PD), catheter associated infection, especially peritonitis, remains a clinically relevant problem. Although the presence of a biofilm is recognized in relapsing, repeat, and catheter-related peritonitis, it remains poorly characterized. In this review, an update on the role of biofilms in PD infections is presented. The emerging concept that host cells and tissue associated biofilms, in addition to the biofilms on the catheters themselves, contribute to the recalcitrance of infections is discussed. Furthermore, the evidence of biofilms on PD catheters, their developmental stages, and the possible influence of the PD environment are reviewed. The focus is given to ex vivo and in vitro studies that contribute to the elucidation of the interplay between host, microbial, and dialysis factors. The key issues that are still to be answered and the challenges to clinical practice are discussed.     

高温油藏生物膜内硫酸盐还原菌的腐蚀机理及防治策略

硫酸盐还原菌(sulfate-reducing bacteria,SRB)广泛分布于高温、高压及高盐的石油油藏中,在油藏硫循环中起主导作用。SRB能在油藏生物膜内生长,有微量低分子有机酸时利用硫酸盐为电子受体并将其还原成硫化氢。硫化氢会腐蚀管道,导致原油泄露等其他安全问题,每年造成的经济损失超过7 000亿元。本文首先总结了油藏生物膜内微生物菌群多样性,分析了生物膜内SRB及其相关菌群的协同腐蚀机理;然后讨论了高温油藏SRB介导的硫氮氢生物地球化学循环过程、胞外电子传递机制及其腐蚀作用,并通过几个高温油藏SRB生物膜内腐蚀的现场案例进一步阐明了SRB的腐蚀机制。在此基础上,提出了应对高温油藏生物膜内SRB腐蚀的生物纳米防治策略,这为高温油藏管道防腐提供了新思路。     

Diffusion of lactose in acidogenic biofilms

Effective diffusivity of lactose in active acidogenic biofilms was measured at 35 degrees C and pH 4.6 with a specially designed diffusion cell. The diffusion cell was designed and operated in such a way that the lactose concentrations on the surface and at the center of a living bacterial aggregate could be measured at steady state. As a model parameter in a widely accepted reaction-diffusion equation which describes lactose distribution in living biofilms, the effective diffusivity of lactose in the biofilms was found to be about 65% of the lactose diffusivity in free solutions. It was experimentally determined that the active biofilms had about 66% void volume made up of channels through which the lactose molecules were transported into the bacterial aggregates. Therefore, the decrease in lactose diffusivity was mainly caused by the biofilm's solid biomass fraction rather than the tortuosity of the channels. (c) 1993 John Wiley & Sons, Inc.     

Disruption of Xylella fastidiosa CVC gumB and gumF genes affects biofilm formation without a detectable influence on exopolysaccharide production

Xylella fastidiosa causes citrus variegated chlorosis (CVC), a destructive disease of citrus. Xylella fastidiosa forms a biofilm inside plants and insect vectors. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix which blocks water and nutrient transport in diseased plants. It is hypothesized that the matrix might be composed of an extracellular polysaccharide (EPS), coded by a cluster of nine genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris. To understand the role of X. fastidiosa gum genes on biofilm formation and EPS biosynthesis, we produced gumB and gumF mutants. Xylella fastidiosa mutants were obtained by insertional duplication mutagenesis and recovered after triply cloning the cells. Xylella fastidiosa gumB and gumF mutants exhibited normal cell characteristics; typical colony morphology and EPS biosynthesis were not altered. It was of note that X. fastidiosa mutants showed a reduced capacity to form biofilm when BCYE was used as the sustaining medium, a difference not observed with PW medium. Unlike X. campestris pv. campestris, the expression of the X. fastidiosa gumB or gumF genes was not regulated by glucose.     

Virulence traits associated with verocytotoxigenic Escherichia coli O157 recovered from freshwater biofilms

AIM: To investigate whether epilithic biofilms in freshwater streams in a mixed UK agricultural river catchment harbour Escherichia coli O157, and if so, whether they demonstrate an association with those excreted by grazing farm animals. METHODS AND RESULTS: Flint shingle, native to the study site, was used as a surface for biofilm development within cages of metal lath set into a stream bed at four locations on a chalkland farm. Shingle was collected from all sites once a month, as were pooled faecal samples from five farm animal populations. Subpopulations of E. coli, including E. coli O157 that demonstrated significant phenotypic and genotypic similarity with animal faecal isolates (t-test, P = 0.05) were isolated. Of 1002 E. coli isolates from biofilms and animal faeces, 48 were confirmed as the O157 strain by latex agglutination. The presence of five virulence traits associated with incidence of human disease was tested using PCR. Stx(2) was the most frequently isolated single gene (30 isolates), while stx(1) was the least frequently recovered (four isolates). CONCLUSION: Escherichia coli O157, expressing up to four virulence factors associated with human disease, reside within freshwater biofilms in this agricultural environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Aquatic biofilms may potentially act as a reservoir for these pathogens, and the implications of the findings for the protection of drinking water resources should be further investigated.     

Quorum sensing inhibition in Pseudomonas aeruginosa biofilms: new insights through network mining

Quorum sensing plays a pivotal role in Pseudomonas aeruginosa’s virulence. This paper reviews experimental results on antimicrobial strategies based on quorum sensing inhibition and discusses current targets in the regulatory network that determines P. aeruginosa biofilm formation and virulence. A bioinformatics framework combining literature mining with information from biomedical ontologies and curated databases was used to create a knowledge network of potential anti-quorum sensing agents for P. aeruginosa. A total of 110 scientific articles, corresponding to 1,004 annotations, were so far included in the network and are analysed in this work. Information on the most studied agents, QS targets and methods is detailed. This knowledge network offers a unique view of existing strategies for quorum sensing inhibition and their main regulatory targets and may be used to readily access otherwise scattered information and to help generate new testable hypotheses. This knowledge network is publicly available at http://pcquorum.org/.     

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air–liquid interface.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Bacteriophages for the treatment of Pseudomonas aeruginosa infections

Bacteriophages were first identified in 1915 and were used as antimicrobial agents from 1919 onwards. Despite apparent successes and widespread application, early users did not understand the nature of these agents and their efficacy remained controversial. As a result, they were replaced in the west by chemical antibiotics once these became available. However, bacteriophages remained a common therapeutic approach in parts of Eastern Europe where they are still in use. Increasing levels of antibiotic-resistant bacterial infections are now driving demand for novel therapeutic approaches. In cases where antibiotic options are limited or nonexistent, the pressure for new agents is greatest. One of the most prominent areas of concern is multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa is a prominent member of this class and is the cause of damaging infections that can be resistant to successful treatment with conventional antibiotics. At the same time, it exhibits a number of properties that make it a suitable target for bacteriophage-based approaches, including growth in biofilms that can hydrolyse following phage infection. Pseudomonas aeruginosa provides a striking example of an infection where clinical need and the availability of a practical therapy coincide.     

A novel in vitro model for haematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life‐threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the process. Using an in vitro catheter infection model, we studied S. aureus biofilm growth, late‐stage dispersal, and reattachment to downstream endothelial cell layers. The ability of the released biofilm material to resist host response and disseminate in vivo was furthermore studied in whole blood and phagocyte survival assays and in a short‐term murine infection model. We found that S. aureus biofilms formed in flow of human plasma release biofilm thromboemboli with embedded bacteria and bacteria‐secreted polysaccharides. The emboli disseminate as antibiotic and immune resistant vehicles that hold the ability to adhere to and initiate colonisation of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth‐grown bacteria but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S. aureus from in vivo‐like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections.