Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC₅₀ = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.     

Study of the major essential oil compounds of Coriandrum sativum against Acinetobacter baumannii and the effect of linalool on adhesion,biofilms and quorum sensing

Acinetobacter baumannii is a pathogen that has the ability to adhere to surfaces in the hospital environment and to form biofilms which are increasingly resistant to antimicrobial agents. The aim of this work was to study the antimicrobial activity of the major oil compounds of Coriandrum sativum against A. baumannii. The effect of linalool on planktonic cells and biofilms of A. baumannii on different surfaces, as well as its effect on adhesion and quorum sensing was evaluated. From all the compounds evaluated, linalool was the compound with the best antibacterial activity, with minimum inhibitory concentration values between 2 and 8 μl ml^?1. Linalool also inhibited biofilm formation and dispersed established biofilms of A. baumannii, changed the adhesion of A. baumannii to surfaces and interfered with the quorum- sensing system. Thus, linalool could be a promising antimicrobial agent for controlling planktonic cells and biofilms of A. baumannii.     

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.     

A bioremediation approach using natural transformation in pure-culture and mixed-population biofilms

Bacterial transformation by naked DNA is thought to contribute to gene transfer and microbial evolution within natural environments. In nature many microbial communities exist as complex assemblages known as biofilms where genetic exchange is facilitated. It may be possible to take advantage of natural transformation processes to modify the phenotypes of biofilm communities giving them specific and desirable functions. Work described here shows that biofilms composed of either pure cultures or mixed populations can be transformed with specific catabolic genes such that the communities acquire the ability to degrade a particular xenobiotic compound. Biofilms were transformed by plasmids bearing genes encoding green fluorescent protein (mut2) and/or atrazine chlorohydrolase (atzA). Confocal microscopy was used to quantify the number of transformants expressing mut2 in the biofilms. Degradation of atrazine by expressed atzA was quantified by tandem mass spectrometry. PCR analysis was performed to confirm the presence of atzA in transformed biofilms. These results indicate that it should be possible to use natural transformation to enhance bioremediation processes performed by biofilms.     

Outer Membrane Vesicles of Helicobacter pylori TK1402 are Involved in Biofilm Formation

Background  

Helicobacter pylori forms biofilms on glass surfaces at the air-liquid interface in in vitro batch cultures; however, biofilms of H. pylori have not been well characterized. In the present study, we analyzed the ability of H. pylori strains to form biofilms and characterized the underlying mechanisms of H. pylori biofilm formation.     

Damage of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilms by carolacton,a secondary metabolite from the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis>

Background  

Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans.     

Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression

Candida glabrata has emerged as the second most prevalent fungal pathogen and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms present a high tolerance to antifungal agents used in fungal infection treatment. The mechanisms of biofilm tolerance to antifungal agents remain poorly understood. Thus, the aim of this study was to evaluate the effects of fluconazole (FLU) on the formation and control of C. glabrata biofilms and its relation with the expression of genes encoding for ABC transporters, CDR1, SNQ2, and PDR1. For that, minimal inhibitory concentration values for seven C. glabrata strains were determined and the effect of FLU against C. glabrata biofilms was evaluated by total biomass quantification and viable cell enumeration. Matrices from biofilms were analyzed in terms of protein, carbohydrate and DNA content. ABC transporter gene expression was analyzed for quantitative real-time PCR. In addition to the high amounts of proteins and carbohydrates detected in the extracellular matrices in the presence of FLU, this work showed that the overexpression of efflux pumps is a possible mechanism of biofilm tolerance to FLU and this phenomenon alters the structure of C. glabrata biofilms by creating cell clusters.     

The architecture of a mixed fungal–bacterial biofilm is modulated by quorum-sensing signals

Interkingdom communication is of particular relevance in polymicrobial biofilms. In this work, the ability of the fungus Ophiostoma piceae to form biofilms individually and in consortium with the bacterium Pseudomonas putida, as well as the effect of fungal and bacterial signal molecules on the architecture of the biofilms was evaluated. Pseudomonas putida KT2440 is able to form biofilms through the secretion of exopolysaccharides and two large extracellular adhesion proteins, LapA and LapF. It has two intercellular signalling systems, one mediated by dodecanoic acid and an orphan LuxR receptor that could participate in the response to AHL-type quorum sensing molecules (QSMs). Furthermore, the dimorphic fungus O. piceae uses farnesol as QSM to control its yeast to hyphae morphological transition. Results show for the first time the ability of this fungus to form biofilms alone and in mixed cultures with the bacterium. Biofilms were induced by bacterial and fungal QSMs. The essential role of LapA-LapF proteins in the architecture of biofilms was corroborated, LapA was induced by farnesol and dodecanol, while LapF by 3-oxo-C6-HSL and 3-oxo-C12-HSL. Our results indicate that fungal signals can induce a transient rise in the levels of the secondary messenger c-di-GMP, which control biofilm formation and architecture.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

Involvement of curli fimbriae in the biofilm formation of Enterobacter cloacae

In this study, we examined the biofilm forming ability, the mRNA expression of curli genes and the morphologies of curli fimbriae and biofilms in clinical isolates of Enterobacter cloacae. The csgBA operon was found in 11 (78.6%) of the 14 isolates. The ability of E. cloacae isolates to form biofilms was significantly correlated with the mRNA expression level of the csgA and csgD genes. The curli protein fimbriae appeared as tangled fibers and the curli-proficient strain formed mature biofilms. Our data suggest that the expression of the curli fimbriae play an important role in biofilm formation in E. cloacae.     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

Anti-biofilm properties of the antimicrobial peptide temporin 1Tb and its ability,in combination with EDTA,to eradicate Staphylococcus epidermidis biofilms on silicone catheters

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2–4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.     

Degradation of methicillin-resistant Staphylococcus aureus biofilms using a chimeric lysin

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a large number of chronic infections due to its ability to form robust biofilms. Herein, the authors evaluated the anti-biofilm activity of a Staphylococcus specific chimeric lysin ClyH on MRSA biofilms. ClyH is known to be active against planktonic MRSA cells in vitro and in vivo. The minimum concentrations for biofilm eradication (MCBE) of ClyH were 6.2–50?mg?l^?1, much lower than those of antibiotics. Scanning electron microscope (SEM) analysis revealed that ClyH eliminated MRSA biofilms through cell lytic activity in a time-dependent manner. Viable plate counts and kinetic analysis demonstrated that biofilms of different ages displayed varying susceptibility to ClyH. Together with previously demonstrated in vivo efficacy of ClyH against MRSA, the degradation efficacy against biofilms of different ages indicates that ClyH could be used to remove MRSA biofilms in vivo.     

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84–7.25 CFU cm^?2 at 192?h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.     

The activity of a small lytic peptide PTP-7 on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

One of the most important features of bacterial biofilms is their resistance to antibiotics and to the host immune system. In this study, we have found that a small lytic peptide, PTP-7, is very potent to Gram-positive bacteria and is able to kill antibiotic sensitive and resistant Staphylococcus aureus indiscriminately. Further studies have revealed that despite being a cationic peptide, the antibacterial activity of PTP-7 was not affected by the negatively charged extracellular polymeric substance (EPS) of biofilms. PTP-7 could diffuse into the deep layer of S. aureus biofilms to kill bacteria inside biofilms efficiently and effectively. Neither the high concentrations of metal ions nor the acidic pH in biofilms affected the activity of peptide PTP-7. It seems that the unique sequence/structure together with the resistant bacteria killing ability of peptide PTP-7 confers its anti-biofilm activity. This study sheds new light on the treatment of bacterial biofilms, especially various biofilm related infections.     

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ_168 h 3.2 log₁₀ cfu cm^?2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.     

The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state

Listeria monocytogenes is a typical foodborne pathogen that causes hard-to-treat bacterial infections, mainly due to its ability to form biofilm and enter into a viable but non-culturable state (VBNC). In this study, we investigated the removal effects of four antimicrobial agents on L. monocytogenes biofilms formed at 32°C and 10°C, analysed the resistances of the mature biofilms to antimicrobial agents, and explored the VBNC state of cells in mature biofilms induced by lavender essential oil (LEO). The results showed that the growth of L. monocytogenes was completely inhibited when 1·6% (v/v) of the LEO was added. Meanwhile, the results of the crystal violet staining and XTT reduction method indicated that different concentrations of LEO significantly reduced L. monocytogenes biofilms biomass and metabolic activities, followed by sodium hypochlorite, lactic acid, and hydrogen peroxide. Moreover, the confocal laser scanning microscopy (CLSM) images confirmed that the treated biofilms became thinner, the structure was sparse, and the appearance was blurry. More interestingly, L. monocytogenes biofilms developed at 10°C were less susceptible to the sanitizers than those formed at 32°C. In addition, LEO presented a more significant dispersing effect on the biofilm cells, and 1/2 MIC to 4 MIC of LEO could induce fewer VBNC state cells in biofilm and plankton compared with sodium hypochlorite. This study indicated that the LEO could be considered as an ideal antibiofilm agent for controlling L. monocytogenes. But we should pay attention to the resistance of the biofilms developed at low temperatures.     

Fungal Biofilms in the Clinical Lab Setting

Device-related infections are often associated with biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) formed by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from infections associated with catheters and dentures, and both Candida and Fusarium are commonly isolated from contact lens–related infections such as fungal keratitis. These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Drug resistance in fungal biofilms is multifactorial and phase-dependent; for example, efflux pumps mediate resistance in biofilms during early phase, whereas altered membrane sterol composition contributes to resistance in mature phase. Both substrate type and surface coatings play an important role in the pathogenesis of device-related fungal biofilms. Host immune cells influence the ability of Candida to form biofilms in vitro. This review summarizes recent advances in research on fungal biofilms and discusses their clinical relevance.     

Biofilm Formation and Biocide Susceptibility Testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC™ assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state. Received: 15 February 2002 / Accepted: 28 March 2002     

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage φIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, φIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.