Evaluation of antimicrobial effects of novel implant materials by testing the prevention of biofilm formation using a simple small scale medium-throughput growth inhibition assay

Staphylococcal colonization of implants is a serious complication of orthopaedic surgery. Anti-infectious modification of implant surfaces may serve to prevent bacterial colonization. The authors set out to develop an in vitro test system for the analysis of prevention of biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus on implant materials. Biofilm growth was monitored over 10 days on titanium disks in order to develop appropriate test parameters. Bacterial cell counts following ultrasonic treatment of the colonized samples were compared with scanning electron microscope images of the specimens. Copper ion containing surfaces (ie copper [Cu] and inter-metallic Ti-Cu films) were used for growth inhibition assays: copper ion releasing specimens led to reduced bacterial numbers in biofilms and decreased bacterial persistence in the model used. The assay used represents an inexpensive and quick in vitro screen for the antibacterial effects of novel implant surface materials.     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

Bacterial-protozoa interactions; an update on the role these phenomena play towards human illness

The usage of water with poor microbiological quality increases the risk of human illness. This review discusses and updates current thinking on the nature of the interaction between a range of human bacterial pathogens and waterborne protozoa. The importance of protozoa acting as protective environments for pathogenic bacteria from disinfection and of promoting extended survival in otherwise hostile environments is highlighted. The significance of biofilms in water systems, and new relationships between Salmonella and Campylobacter and water-borne protozoa are also discussed. The protection of pathogenic bacteria from disinfection within protozoa and/or biofilms has important implications for water safety.     

柱[5]芳烃衍生物对细菌抗菌活性及生物被膜抑制的研究进展北大核心

病原体的耐药性很强,其生物被膜(biofilm,BF)的形成是导致耐药性的主要原因之一。生物被膜一旦形成,根除难度很大,会导致患者持久性感染,引发多种慢性疾病,并给全球医疗体系带来沉重负担。柱芳烃(pillararenes)是一类具有独特柱状结构的新型大环化合物,由于其在构建功能化和生物活性材料开发中的潜在应用引起人们广泛的关注。此外,它们在预防和控制抗生素耐药性(antimicrobial resistance,AMR)方面具有广阔的应用前景。本文综述了柱[5]芳烃衍生物对细菌病原菌的抗菌活性,并进一步揭示其在抗菌活性中的抑菌机制,尤其是对生物被膜的抑制作用。在此基础上,探索新的抑菌杀菌策略,用非传统药物以解决抗生素耐药性问题,以期为开发新的抗菌剂防控生物被膜或治疗细菌感染提供理论依据。     

Inactivation of Pseudomonas aeruginosa PA01 biofilms by hyperthermia using superparamagnetic nanoparticles

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL⁻¹ SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.     

Appreciating the role of carbon nanotube composites in preventing biofouling and promoting biofilms on material surfaces in environmental engineering: A review

The ability of carbon nanotubes (CNTs) to undergo surface modification allows them to form nanocomposites (NCs) with materials such as polymers, metal nanoparticles, biomolecules, and metal oxides. The biocidal nature, protein fouling resistance, and fouling release properties of CNT-NCs render them the perfect material for biofouling prevention. At the same time, the cytotoxicity of CNT-NCs can be reduced before applying them as substrates to promote biofilm formation in environmental biotechnology applications. This paper reviews the potential prospects of CNT-NCs to accomplish two widely varying objectives in environmental engineering applications: (i) preventing biofouling, and (ii) promoting the formation of desirable biofilms on materials surface. This paper addresses practical issues such as costs, risks to human health, and ecological impacts that are associated with the application, development and commercialization of CNT-NC technology.     

Synthesis and Evaluation of Water‐Soluble 2‐Aryl‐1‐Sulfonylpyrrolidine Derivatives as Bacterial Biofilm Formation Inhibitors

The approach to the novel 1‐[(2‐aminoethyl)sulfonyl]‐2‐arylpyrrolidines via unique intramolecular cyclization/aza‐Michael reactions of N‐(4,4‐diethoxybutyl)ethenesulfonamide have been developed, which benefits from high yields of target compounds, mild reaction conditions, usage of inexpensive and low‐toxic reagents, and allows for wide variability in both amine and aryl moieties. Biotesting with whole‐cell luminescent bacterial biosensors responding to DNA damage showed that all tested compounds are not genotoxic. Tested compounds differently affect the formation of biofilms by Vibrio aquamarinus DSM 26054. Some of the tested compounds were found to suppress the bacterial biofilms growth and thus are promising candidates for further studies.     

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.     

In vitro anaerobic biofilms of human colonic microbiota

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.     

The silver lining: towards the responsible and limited usage of silver

Silver has attracted a lot of attention as a powerful, broad spectrum and natural antimicrobial agent since the ancient times because of its nontoxic nature to the human body at low concentrations. It has been used in treatment of various infections and ulcers, storage of water and prevention of bacterial growth on the surfaces and within materials. However, there are numerous medical and health benefits of colloidal or nanosilver apart from its microbicidal ability which as yet has not been fully embraced by the medical community. These include antiplatelet activity, antioxidant effect, anticancer activity, wound healing and bone regeneration, enhancement of immunity, and increase in antibiotic efficiency. Additionally silver also provides protection against alcohol toxicity, upper respiratory tract infections and stomach ailments. Although nanosilver has been proposed for various topical applications, its usage by ingestion and inhalation remains controversial due to the lack of detailed and precise toxicity information. These beneficial properties of silver can be utilized by using silver at very low concentrations which are not harmful to the human body and environment. The following review discusses the diverse medical applications of silver and further recommends human clinical studies for its in vivo usage.     

A Comparison of Effects of Broad-Spectrum Antibiotics and Biosurfactants on Established Bacterial Biofilms

Current antibiofilm solutions based on planktonic bacterial physiology have limited efficacy in clinical and occasionally environmental settings. This has prompted a search for suitable alternatives to conventional therapies. This study compares the inhibitory properties of two biological surfactants (rhamnolipids and a plant-derived surfactant) against a selection of broad-spectrum antibiotics (ampicillin, chloramphenicol and kanamycin). Testing was carried out on a range of bacterial physiologies from planktonic and mixed bacterial biofilms. Rhamnolipids (Rhs) have been extensively characterised for their role in the development of biofilms and inhibition of planktonic bacteria. However, there are limited direct comparisons with antimicrobial substances on established biofilms comprising single or mixed bacterial strains. Baseline measurements of inhibitory activity using planktonic bacterial assays established that broad-spectrum antibiotics were 500 times more effective at inhibiting bacterial growth than either Rhs or plant surfactants. Conversely, Rhs and plant biosurfactants reduced biofilm biomass of established single bacterial biofilms by 74–88 and 74–98 %, respectively. Only kanamycin showed activity against biofilms of Bacillus subtilis and Staphylococcus aureus. Broad-spectrum antibiotics were also ineffective against a complex biofilm of marine bacteria; however, Rhs and plant biosurfactants reduced biofilm biomass by 69 and 42 %, respectively. These data suggest that Rhs and plant-derived surfactants may have an important role in the inhibition of complex biofilms.     

What drives bacteria to produce a biofilm?

Nearly 40 years ago, Dr. R.J. Gibbons made the first reports of the clinical relevance of what we now know as bacterial biofilms when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans [Sci. Am. 238 (1978) 86]. As the clinical relevance of bacterial biofilm formation became increasingly apparent, interest in the phenomenon exploded. Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fundamental questions remain. The intent of this review is to consider the reasons why bacteria switch from a free-floating to a biofilm mode of growth. The currently available wealth of data pertaining to the molecular genetics of biofilm formation in commonly studied, clinically relevant, single-species biofilms will be discussed in an effort to decipher the motivation behind the transition from planktonic to sessile growth in the human body. Four potential incentives behind the formation of biofilms by bacteria during infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area (colonization), (3) utilization of cooperative benefits (community), (4) biofilms normally grow as biofilms and planktonic cultures are an in vitro artifact (biofilms as the default mode of growth).     

The role of bacterial biofilms in ocular infections

There is increasing evidence that bacterial biofilms play a role in a variety of ocular infections. Bacterial growth is characterized as a biofilm when bacteria attach to a surface and/or to each other. This is distinguished from a planktonic or free-living mode of bacterial growth where these interactions are not present. Biofilm formation is a genetically controlled process in the life cycle of bacteria resulting in numerous changes in the cellular physiology of the organism, often including increased antibiotic resistance compared to growth under planktonic conditions. The presence of bacterial biofilms has been demonstrated on many medical devices including intravenous catheters, as well as materials relevant to the eye such as contact lenses, scleral buckles, suture material, and intraocular lenses. Many ocular infections often occur when such prosthetic devices come in contact with or are implanted in the eye. For instance, 56% of corneal ulcers in the United States are associated with contact lens wear. Bacterial biofilms may participate in ocular infections by allowing bacteria to persist on abiotic surfaces that come in contact with, or are implanted in the eye, and by direct biofilm formation on the biotic surfaces of the eye. An understanding of the role of bacterial biofilm formation in ocular infections may aid in the development of future antimicrobial strategies in ophthalmology. We review the current literature and concepts relating to biofilm formation and infections of the eye.     

Anti-biofouling property of vanillin on Aeromonas hydrophila initial biofilm on various membrane surfaces

Biofouling is a serious problem on filter membranes of water purification systems due to formation of bacterial biofilms, which can be detrimental to the membrane performance. Biofouling occurs on membrane surface and therefore greatly influences the physical and chemical aspects of the surface. Several membranes including microfiltration, ultrafiltration, and reverse osmosis (RO) membranes were used to learn about the anti-biofouling properties of vanillin affecting the membrane performances. Vanillin has been recognized as a potential quorum quenching compound for Aeromonas hydrophila biofilms. The initial attachment and dynamics of biofilm growth were monitored using scanning electron microscopy and confocal laser scanning microscopy. Biofilm quantities were measured using a plate count method and total protein determinations. Vanillin addition was effective in the prevention of biofilm formation on the tested membrane surfaces. Among the membranes, RO membranes made with cellulose acetate showed the most substantial reduction of biofilm formation by addition of vanillin. The biofilm reduction was confirmed by the results of surface coverage, biomass and protein accumulation. The HPLC spectrum of the spent culture with vanillin addition showed that vanillin may interfere with quorum sensing molecules and thus prevent the formation of the biofilms.     

Osteopontin reduces biofilm formation in a multi-species model of dental biofilm

Background

Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.

Methodology/Principal Findings

Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.

Conclusions/Significance

OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.     

Mini-review: efficacy of lytic bacteriophages on multispecies biofilms

There is potential for phages to prevent and control bacterial biofilms, but few studies have examined the effect of phages on the multispecies biofilms that characterize most bacterial infections. This paper reviews the mechanism of action of phages, the evidence supporting the view that phage therapy will be effective against bacterial targets and the opposite viewpoint, phage application approaches, and the comparative advantage of phage therapy in multispecies biofilms. The few reports measuring the actions of lytic phages against multispecies biofilms are also reviewed. The authors are cautiously optimistic about the application of phages against their targets when in multispecies biofilms because some lysis mechanisms do not require species specificity.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

用气溶胶法和摇床法建立铜绿假单胞菌生物膜体外模型

目的模拟体内环境,体外建立细菌生物膜模型,为进一步深入研究细菌生物膜生物学特点提供基础。方法将粘附载体置于气溶胶法和摇床法模拟体内细菌生物膜形成的微环境中,将铜绿假单胞菌株培养3d后,取出标本分别进行通过FITC—ConA染色及SYT09／PI染色,然后分别进行荧光显微镜检测及激光共聚焦检测,观察细菌生物膜的形成情况;进行电子显微镜扫描观察形成的细菌生物膜的形态特点。结果在气溶胶的微环境下,FITC—ConA染色后在荧光显微镜观察到明亮成片状的细菌生物膜;SYT09／PI染色后在激光共聚焦检测,观察到片状,层叠如积云状,棉絮样的细菌生物膜;在电子显微镜扫描观察到大量细菌成团聚集,团状丛生突出表面,具有立体结构的细菌生物膜。在摇床法的微环境下,用3种检测方法都观察到成流线状的细菌生物膜。结论运用气溶胶法、摇床法可成功建立分别模拟体内呼吸系统及循环、泌尿系统的微环境下生物膜形成模型。     

Tat transport of linker-containing proteins in Escherichia coli

Little is known about the dynamics of cellular growth, death, and evolution within bacterial biofilms. Here we show evidence of evolution within single-species biofilms in real time. Escherichia coli harvested from 22-day-old biofilms express a competitive advantage over cells incubated in biofilms for shorter periods of time. This advantage is manifested as the ability of aged cells to outcompete younger cells in the presence of a pre-existing biofilm, even though cells from older biofilms do not express an increased ability to form initial biofilms on a fresh, unoccupied surface. This phenomenon is similar to the growth advantage in stationary phase, or GASP, phenotype exhibited by planktonically grown cells when incubated under competitive conditions. The ability of bacteria in biofilms to show rapid heritable change has implications for our understanding of the adaptive abilities of biofilms in a wide variety of natural and man-made environments.     

Biofilms as a Mode of Existence of Bacteria in External Environment and Host Body: The Phenomenon,Genetic Control,and Regulation Systems of Development

Studies of the last decade have shown that most bacteria exist in natural ecosystems as specifically organized, attached to substrates biofilms rather than as freely floating plankton cells. The formation of these biofilms is a complex and highly regulated process. The development of biofilm communities is a primary strategy of bacterial survival not only in the external environment but also in the bodies of infected macroorganisms. In these organisms, bacteria are joined by complicated cell–cell associations, which makes them functionally similar to multicellular organisms. In the present review, we consider the structural organization of biofilms, factors affecting initiation of the biofilm formation, differential expression of bacterial genes at various stages of the biofilm development and their regulation. The significance of studies in this field for medicine, in particular, for prevention and protection against pathogenic bacteria, is discussed.