Implications of Growth and Starvation Conditions in Bacterial Adhesion and Transport

Biofouling is synonymous with unwanted biofilms and leads to problems ranging from efficiency and resource loss to health risks. While a number of bacterial properties including biomass concentration and hydrophobicity are considered critical to biofilm development and bacterial adhesion, the variations in these properties under growth and starvation conditions are not very well known. Here, we describe the trends in these properties for four Gram-negative bacteria under growth and extended starvation conditions. A convenient and frequently-used laboratory assay, the microbial adhesion to hydrocarbons (MATH) test, was used to determine the microbial hydrophobicity based on the partitioning of cells at an aqueous-hydrocarbon interface. The bacteria tested exhibited a plateau in hydrophobicity values during the stationary growth phase and longer starvation durations (≥10 days). Starved cultures had higher hydrophobicity and lower cell sizes than growth cultures. Interestingly, hydrocarbon exposure led to an increase in cell size for starved cells as compared to control cultures, while cells under growth conditions did not show significant size changes due to hydrocarbon presence. Cells starved for short durations (up to 7–10 days) exhibited significant variations in microbial hydrophobicity, cell size, and biomass concentration (total proteins and optical density). These results show the importance of studying the bacterial properties as a function of growth and starvation phase for cell adhesion in the context of biofilm formation and biofouling.     

Electroactive biofilms of sulphate reducing bacteria

Biofilms formed from a pure strain of Desulfovibrio desulfuricans 27774 on stainless steel and graphite polarised surfaces were studied. The polarisation conditions applied were −0.4 V vs. SCE for different times. A cathodic current related with the biofilms growth was observed with a maximum intensity of −270 mA m⁻² that remained stable for several days using graphite electrodes. These sulphate reducing bacteria biofilms present electrocatalytic activity towards hydrogen and oxygen reduction reactions. Electrode polarisation has a selective effect on the catalytic activity. The biofilms were also observed by scanning electronic microscopy revealing the formation of homogeneous films on the surfaces.     

(+)-Dehydroabietic Acid,an Abietane-Type Diterpene,Inhibits Staphylococcus aureus Biofilms in Vitro

Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1), (+)-dehydroabietic acid (2) and (+)-dehydroabietylamine (3) that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+)-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values) and it was best tolerated by three different mammalian cell lines. Since (+)-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates.     

Novel Strategies for the Prevention and Treatment of Biofilm Related Infections

Biofilm formation by human bacterial pathogens on implanted medical devices causes major morbidity and mortality among patients, and leads to billions of dollars in healthcare cost. Biofilm is a complex bacterial community that is highly resistant to antibiotics and human immunity. As a result, novel therapeutic solutions other than the conventional antibiotic therapies are in urgent need. In this review, we will discuss the recent research in discovery of alternative approaches to prevent or treat biofilms. Current anti-biofilm technologies could be divided into two groups. The first group focuses on targeting the biofilm forming process of bacteria based on our understanding of the molecular mechanism of biofilm formation. Small molecules and enzymes have been developed to inhibit or disrupt biofilm formation. Another group of anti-biofilm technologies focuses on modifying the biomaterials used in medical devices to make them resistant to biofilm formation. While these novel anti-biofilm approaches are still in nascent phases of development, efforts devoted to these technologies could eventually lead to anti-biofilm therapies that are superior to the current antibiotic treatment.     

Second-Generation Meridianin Analogues Inhibit the Formation of Mycobacterium smegmatis Biofilms and Sensitize Polymyxin-Resistant Gram-Negative Bacteria to Colistin

Drug-resistant bacteria are rapidly becoming a significant problem across the globe. One element that factors into this crisis is the role played by bacterial biofilms in the recalcitrance of some infections to the effects of conventional antibiotics. Bacteria within a biofilm are highly tolerant of both antibiotic treatment and host immune responses. Biofilms are implicated in many chronic infections, including tuberculosis, in which they can act as bacterial reservoirs, requiring an arduous antibiotic regimen to eradicate the infection. A separate, compounding problem is that antibiotics once seen as last-resort drugs, such as the polymyxin colistin, are now seeing more frequent usage as resistance to front-line drugs in Gram-negative bacteria becomes more prevalent. The increased use of such antibiotics inevitably leads to an increased frequency of resistance. Drugs that inhibit biofilms and/or act as adjuvants to overcome resistance to existing antibiotics will potentially be an important component of future approaches to antibacterial treatment. We have previously demonstrated that analogues of the meridianin natural product family possess adjuvant and antibiofilm activities. In this study, we explore structural variation of the lead molecule from previous studies, and identify compounds showing both improved biofilm inhibition potency and synergy with colistin.     

New evidences on the catalase mechanism of microbial corrosion

Changes on the oxygen reduction rate induced on aluminium brass by cell-free bacterial cultures of an isolate belonging to the genus Pseudomonas were studied in relation to the bacteria phase of growth and to the surface oxide layer composition after various electrochemical pre-treatments of the metal samples. Cultures isolated from the stationary phase of growth strongly influenced the oxygen reduction kinetics. Cathodic currents increased throughout the potential range tested when Cu₂O and CuO were present simultaneously in the surface film (so-called aged surfaces). In this case, the maximum increment (35%) was observed within the oxygen reduction limiting current region. On pre-oxidised surfaces, when the oxide film was composed mainly by CuO, the effect induced by stationary phase cultures was even higher, with the limiting current density increasing by almost 60%. On pre-reduced surfaces on the other hand, when only a submonolayer of Cu₂O was covering the surface, there was no effect as current density values were similar to those obtained in control experiments. Exponential phase cell-free cultures did not modify the limiting current values in any of the surfaces investigated. Results were in agreement with the participation of catalase as a bacterial catalyst for the oxygen reduction process. The normalised catalase activity from different stationary phase cell-free cultures ranged from 0.88 to 4.02 mg ml⁻¹ U⁻¹, while there was no observable activity in exponential phase cultures. The incidence of the catalase mechanism in microbiologically influenced corrosion processes induced by aerobic biofilms is highlighted on the basis of the results obtained using metabolites from planktonic cells and their agreement with most of the experimental evidences so far reported by other authors.     

Eradicating Biofilms of Carbapenem-Resistant Enterobacteriaceae by Simultaneously Dispersing the Biomass and Killing Planktonic Bacteria with PEGylated Branched Polyethyleneimine

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging pathogens that cause variety of severe infections. CRE evade antibiotic treatments because these bacteria produce enzymes that degrade a wide range of antibiotics including carbapenems and β-lactams. The formation of biofilms aggravates CRE infections, especially in a wound environment. These difficulties lead to persistent infection and non-healing wounds. This creates the need for new compounds to overcome CRE antimicrobial resistance and disrupt biofilms. Recent studies in our lab show that 600 Da branched polyethyleneimine (BPEI) and its derivative PEG350-BPEI can overcome antimicrobial resistance and eradicate biofilms in methicillin-resistant S. aureus, methicillin-resistant S. epidermidis, P. aeruginosa, and E. coli. In this study, the ability of 600 Da BPEI and PEG350-BPEI to eradicate carbapenem-resistant Enterobacteriaceae bacteria and their biofilms is demonstrated. We show that both BPEI and PEG350-BPEI have anti-biofilm efficacy against CRE strains expressing Klebsiella pneumoniae carbapenemases (KPCs) and metallo-β-lactamases (MBLs), such as New Delhi MBL (NDM-1). Furthermore, our results illustrate that BPEI affects planktonic CRE bacteria by increasing bacterial length and width from the inability to proceed with normal cell division processes. These data demonstrate the multi-functional properties of 600 Da BPEI and PEG350-BPEI to reduce biofilm formation and mitigate virulence in carbapenem-resistant Enterobacteriaceae.     

蒸汽在含有不可溶核和可溶无机盐的细颗粒物表面的核化特性

鉴于生物质直接燃烧和生物质与煤混合燃烧发电过程排放细颗粒物表面通常含有一定量的可溶无机盐,基于经典异质核化理论,综合考虑晶核生长的表面扩散和直接沉积机制建立了改进的蒸汽在包含球形不可溶核和可溶无机盐的细颗粒物表面的异质核化模型,利用数值模拟方法,对4种组分颗粒(不可溶颗粒以及3种含可溶无机盐的颗粒)的异质核化特性进行对比分析。结果表明,在中等接触角条件下,不可溶颗粒的临界晶核形成自由能和临界晶核半径最大,含KCl颗粒次之,含NaCl颗粒再次之,含CaCl₂颗粒最小;临界晶核条件下,表面扩散机制与直接沉积机制引起的水分子添加速率之比随颗粒半径的增大先略有增加而后保持不变,随接触角的增大而单调下降。研究还发现,当接触角较小时,含可溶无机盐颗粒的成核临界饱和度低于不可溶颗粒;当接触角较大时,含KCl和NaCl颗粒的成核临界饱和度先后超过不可溶颗粒。     

蒸汽在含有不可溶核和可溶无机盐的细颗粒物表面的核化特性

鉴于生物质直接燃烧和生物质与煤混合燃烧发电过程排放细颗粒物表面通常含有一定量的可溶无机盐,基于经典异质核化理论,综合考虑晶核生长的表面扩散和直接沉积机制建立了改进的蒸汽在包含球形不可溶核和可溶无机盐的细颗粒物表面的异质核化模型,利用数值模拟方法,对4种组分颗粒(不可溶颗粒以及3种含可溶无机盐的颗粒)的异质核化特性进行对比分析。结果表明,在中等接触角条件下,不可溶颗粒的临界晶核形成自由能和临界晶核半径最大,含KCl颗粒次之,含NaCl颗粒再次之,含CaCl₂颗粒最小;临界晶核条件下,表面扩散机制与直接沉积机制引起的水分子添加速率之比随颗粒半径的增大先略有增加而后保持不变,随接触角的增大而单调下降。研究还发现,当接触角较小时,含可溶无机盐颗粒的成核临界饱和度低于不可溶颗粒;当接触角较大时,含KCl和NaCl颗粒的成核临界饱和度先后超过不可溶颗粒。     

Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa

Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms.     

12‐crown‐4–ether and tri(ethylene glycol) dimethyl–ether plasma‐coated stainless steel surfaces and their ability to reduce bacterial biofilm deposition

It has been demonstrated that surfaces coated with poly(ethylene glycol) (PEG) are capable of reducing protein adsorption, bacterial attachment, and biofilm formation. In this communication cold‐plasma–enhanced processes were employed for the deposition of PEG‐like structures onto stainless steel surfaces. Stainless steel samples were coated under 1,4,7,10‐tetraoxacyclododecane (12‐crown‐4)–ether and tri(ethylene glycol) dimethyl ether (triglyme)–radio frequency (RF)–plasma conditions. The chemistry and characteristics of plasma‐coated samples and biofilms were investigated using electron spectroscopy for chemical analysis (ESCA), atomic force microscopy (AFM), and water contact angle analysis. ESCA analysis indicated that the plasma modification resulted in the deposition of PEG‐like structures, built up mainly of –CH₂? CH₂? O– linkages. Plasma‐coated stainless steel surfaces were more hydrophilic and had lower surface roughness values compared to those of unmodified substrates. Compared to the unmodified surfaces, they not only significantly reduced bacterial attachment and biofilm formation in the presence of a mixed culture of Salmonella typhimurium, Staphylococcus epidermidis, and Pseudomonas fluorescens but also influenced the chemical characteristics of the biofilm. Thus, plasma deposition of PEG‐like structures will be of use to the food‐processing and medical industries searching for new technologies to reduce bacterial contamination. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 3425–3438, 2001     

5-Benzylidene-4-Oxazolidinones Are Synergistic with Antibiotics for the Treatment of Staphylococcus aureus Biofilms

The failure of frontline antibiotics in the clinic is one of the most serious threats to human health and requires a multitude of novel therapeutics and innovative approaches to treatment so as to curtail the growing crisis. In addition to traditional resistance mechanisms resulting in the lack of efficacy of many antibiotics, most chronic and recurring infections are further made tolerant to antibiotic action by the presence of biofilms. Herein, we report an expanded set of 5-benzylidene-4-oxazolidinones that are able to inhibit the formation of Staphylococcus aureus biofilms, disperse preformed biofilms, and, in combination with common antibiotics, are able to significantly reduce the bacterial load in a robust collagen-matrix model of biofilm infection.     

Phenazine Antibiotic-Inspired Discovery of Bacterial Biofilm-Eradicating Agents

Bacterial biofilms are surface-attached communities of slow-growing and non-replicating persister cells that demonstrate high levels of antibiotic tolerance. Biofilms occur in nearly 80 % of infections and present unique challenges to our current arsenal of antibiotic therapies, all of which were initially discovered for their abilities to target rapidly dividing, free-floating planktonic bacteria. Bacterial biofilms are credited as the underlying cause of chronic and recurring bacterial infections. Innovative approaches are required to identify new small molecules that operate through bacterial growth-independent mechanisms to effectively eradicate biofilms. One source of inspiration comes from within the lungs of young cystic fibrosis (CF) patients, who often endure persistent Staphylococcus aureus infections. As these CF patients age, Pseudomonas aeruginosa co-infects the lungs and utilizes phenazine antibiotics to eradicate the established S. aureus infection. Our group has taken a special interest in this microbial competition strategy and we are investigating the potential of phenazine antibiotic-inspired compounds and synthetic analogues thereof to eradicate persistent bacterial biofilms. To discover new biofilm-eradicating agents, we have established an interdisciplinary research program involving synthetic medicinal chemistry, microbiology and molecular biology. From these efforts, we have identified a series of halogenated phenazines (HPs) that potently eradicate bacterial biofilms, and future work aims to translate these preliminary findings into ground-breaking clinical advances for the treatment of persistent biofilm infections.     

The Antimicrobial Peptide Gad-1 Clears Pseudomonas aeruginosa Biofilms under Cystic Fibrosis Conditions

Bacterial infections in cystic fibrosis (CF) patients are an emerging health issue and lead to a premature death. CF is a hereditary disease that creates a thick mucus in the lungs that is prone to bacterial biofilm formation, specifically Pseudomonas aeruginosa biofilms. These biofilms are very difficult to treat because many of them have antibiotic resistance that is worsened by the presence of extracellular DNA (eDNA). eDNA helps to stabilize biofilms and can bind antimicrobial compounds to lessen their effects. The metallo-antimicrobial peptide Gaduscidin-1 (Gad-1) eradicates established P. aeruginosa biofilms through a combination of modes of action that includes nuclease activity that can cleave eDNA in biofilms. In addition, Gad-1 exhibits synergistic activity when used with the antibiotics kanamycin and ciprofloxacin, thus making Gad-1 a new lead compound for the potential treatment of bacterial biofilms in CF patients.     

Environmental Stimuli Shape Biofilm Formation and the Virulence of Periodontal Pathogens

Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.     

Removal of biofilms using carbon dioxide aerosols

The formation of biofilm (bacterial film) has been serious concerns in a wide variety of applications, because it is involved in many human and device-associated infections. We present a novel method of effectively and rapidly removing Escherichia coli (XL1-blue) biofilm from a silicon chip, using carbon dioxide aerosols. The aerosols were generated by adiabatic expansion of a high-pressure CO₂ gas through a nozzle and they were applied to biofilms that had been grown for 24 h on silicon chips. We measured the percentage area cover of the bacteria from the scanning electron micrographs taken before and after applying the aerosols. The decrease in the percentage area cover, caused by the aerosols, was measured as several parameters such as the distance between the nozzle and the chip, the angle of the nozzle axis relative to the horizontal, CO₂ stagnation pressure, rinsing solution, the aerosol exposure time, and drying time were varied. Nearly 100% of the biofilms were removed within 90 s whether the chip surfaces were very humid (no-drying) or dry (7 h-drying) immediately before applying the aerosols. This method has potential application to cleaning of a wide variety of bio-contaminated surfaces.     

Metal and Metal Oxide Nanoparticle as a Novel Antibiotic Carrier for the Direct Delivery of Antibiotics

In addition to the benefits, increasing the constant need for antibiotics has resulted in the development of antibiotic bacterial resistance over time. Antibiotic tolerance mainly evolves in these bacteria through efflux pumps and biofilms. Leading to its modern and profitable uses, emerging nanotechnology is a significant field of research that is considered as the most important scientific breakthrough in recent years. Metal nanoparticles as nanocarriers are currently attracting a lot of interest from scientists, because of their wide range of applications and higher compatibility with bioactive components. As a consequence of their ability to inhibit the growth of bacteria, nanoparticles have been shown to have significant antibacterial, antifungal, antiviral, and antiparasitic efficacy in the battle against antibiotic resistance in microorganisms. As a result, this study covers bacterial tolerance to antibiotics, the antibacterial properties of various metal nanoparticles, their mechanisms, and the use of various metal and metal oxide nanoparticles as novel antibiotic carriers for direct antibiotic delivery.     

Therapeutic Strategies To Counteract Antibiotic Resistance in MRSA Biofilm-Associated Infections

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the leading causes of persistent human infections. This pathogen is widespread and is able to colonize asymptomatically about a third of the population, causing moderate to severe infections. It is currently considered the most common cause of nosocomial infections and one of the main causes of death in hospitalized patients. Due to its high morbidity and mortality rate and its ability to resist most antibiotics on the market, it has been termed a “superbug”. Its ability to form biofilms on biotic and abiotic surfaces seems to be the primarily means of MRSA antibiotic resistance and pervasiveness. Importantly, more than 80 % of bacterial infections are biofilm-mediated. Biofilm formation on indwelling catheters, prosthetic devices and implants is recognized as the cause of serious chronic infections in hospital environments. In this review we discuss the most relevant literature of the last five years concerning the development of synthetic small molecules able to inhibit biofilm formation or to eradicate or disperse pre-formed biofilms in the fight against MRSA diseases. The aim is to provide guidelines for the development of new anti-virulence strategies based on the knowledge so far acquired, and, to identify the main flaws of this research field, which have hindered the generation of new market-approved anti-MRSA drugs that are able to act against biofilm-associated infections     

Spatial Patterns of Microbial Retention on Polymer Surfaces

Microbial adhesion and retention on surfaces are complex phenomena, critical to the formation and development of biofilms. Recently, the focus of research has been more and more on the importance of retention of bacteria under fluctuating high shear forces in biofilm formation. The aim of the present work was to carry out a comparative study of the retention process of different bacterial and yeast species using: (1) a range of surfaces with different surface free energy properties and (2) a number of different bacterial cell physiological states. It was found for the first time that once a threshold cell number is retained on the surface, microbial retention patterns are formed following a power law, i.e., not stochastic. Our results demonstrated that the overall spatial patterns of microbial retention observed for the different substrates are similar for the all investigated cell types and that the drastic modification of the surface free energy does not affect this spatial organization. On the other hand, the microbial retention patterns appear to be significantly affected by the physiological state of the cells. Finally, the experimental retention patterns have been well simulated by a general agent-based model, confirming that the typical fractal distribution of retained cells is the result of a self-organization process.     

CO2 plasma modification of high-modulus carbon fibers and their adhesion to epoxy resins

Interfacial bond strength is often a performance-limiting factor of carbon-fiber-reinforced composites. This limitation is most prevalent when higher-modulus fibers or relatively unreactive matrix resins, such as engineering thermoplastics or high-temperature thermoset resin systems, are used. Radio-frequency (RF) glow discharge plasmas are an effective means of modifying carbon-fiber surface chemical characteristics to promote adhesion. It has been previously shown that oxidizing plasmas are especially effective compared with electro-oxidative treatments for treating carbon fiber surfaces as revealed by titrations, electron spectroscopy, wetting, and inverse gas chromatography measurements. This study evaluated the effectiveness of CO₂ plasmas on two experimental high-modulus carbon/graphite fibers and correlated the plasma surface modification with interfacial adhesion in an epoxy matrix composite system. The results show that CO₂ plasma treatment increased the surface oxygen content by nearly a factor of 2 over typical electro-oxidation treatments. The increased oxygen is mainly in the form of hydroxyl, ketone, and carboxyl-like moieties. Unidirectional composites were prepared from as-received and plasma-modified versions of each type of experimental fiber. The composites containing plasma-modified filaments exhibited 1.5-3.0 times the strength of composites fabricated with untreated or electro-oxidized filaments in transverse-flexural tests. Short-beam shear strength increased by two times over those with as-produced filaments and is equivalent to that of composites containing electro-oxidized filaments.