Resistance Of Bacterial Biofilms Research Articles

Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.

BackgroundNon-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either “light” (L) isoleucine or “heavy” (H) 13C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.ResultsComparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.ConclusionsThe present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0329-9) contains supplementary material, which is available to authorized users.

Abstract 473: The Association of Bacterial Biofilms With Carotid Atherosclerotic Disease: A Study in Progress

Background: There is evidence to support that bacteria present in atherosclerotic plaque may play a role in disease pathogenesis. Several studies utilizing antibiotics to inhibit progression of atherosclerotic disease have been unsuccessful. Biofilms are complex microenvironments of bacterial colonies encased within a matrix that firmly adheres to surfaces. They exhibit extensive genotypic and phenotypic diversity and are 100 to 1000 times more resistant to antibiotics than planktonic cultures. We hypothesized that bacterial biofilms are present in carotid atheroma. Methods: The IBIS Biosensor- Florescent in situ hybridization (FISH) pipeline for microbial discovery was applied to atherosclerotic samples obtained from 8 patients who underwent open CEA at Allegheny General Hospital. The extracted nucleic acid from CEA samples were aliquoted into wells of a microtiter plate containing broad range primers for PCR. The products were desalted in a 96-well plate format, sequentially electrosprayed into a mass spectrometer and the spectral signals processed to determine the masses of each of the PCR products present. The base composition of each amplicon was therefore unambiguously deduced. Using combined base compositions from multiple PCRs; the identities of the pathogens and their relative concentrations in the starting sample were determined. FISH using fluorescent probes targeted to the 16s rRNA, designed for each sample based on the result from the IBIS, was then performed. Confocal microscopy of the FISH stained samples allowed visualization of the specific bacterial species. Results: One or more bacterial species were present in four of the eight CEA samples analyzed (50%). Species present were Staphylococcus capitis/ caprae, Staphylococcus warneri, Propionibacterium acnes, and Staphylococcus epidermidis. Further analyses in an attempt to correlate these findings with oral microbiota, dental and clinical history is currently ongoing. Conclusion: In our ongoing study 50% of analyzed carotid atherosclerotic samples contained the highly antibiotic resistant bacterial biofilms. Mechanisms of biofilm formation in atheroma need to be explored.

Peering into the matrix: A look at biofilms and their inherent antibiotic resistance

Biofilms are increasingly being regarded as the predominant form of bacterial growth in natural settings. These structures consist of bacterial cells immobilized at a surface and encased in a self-produced matrix of extracellular polymeric substances. In clinical settings, biofilms are the cause of persistent infections that are difficult to clear through the action of the host immune system. Biofilm-encased cells are also associated with increased levels of antibiotic resistance compared to their planktonic counterparts. The result is increased morbidity and mortality when biofilms are associated with disease. In this review, the focus of discussion will be the various mechanisms of antibiotic resistance common to biofilms and the role these mechanisms play in the pathogenesis of major clinically-relevant microorganisms. Antibiotic penetration, altered microenvironments, phenotypic variation, and adaptive resistance mechanisms are all key players in the development of antibiotic resistance in bacterial biofilms. Though the relative significance of each individual mechanism varies, when combined they confer extensive protection to the biofilm’s cellular populations.
 
 Keywords: biofilms; antibiotic resistance (mechanisms of); clinical applications; disease; antibiotic penetration; altered microenvironments; phenotypic variation; adaptive resistance; review

Role of the extracellular polymer matrix in resistance of bacterial biofilms to extreme environmental factors

Biofilms of a number of gram-positive and gram-negative bacteria (both environmental strains from the stratal waters of oil fields and collection strains) were found to exhibit higher resistance to extreme physicochemical factors (unfavorable temperature, pH, and salt concentration) than planktonic cultures. The extracellular polymers forming the structure of the biofilm matrix were shown to contribute significantly to this resistance, since suppression of matrix formation by subbacteriostatic concentrations of azithromycin (for Pseudomonas acephalitica) or mutation in the cvil gene encoding N-hexanoyl homoserine lactone synthetase (for Chromobacterium violaceum CV026) resulted in the resistance of biofilms being decreased almost to the level of planktonic cultures. The role of the biofilm matrix for bacterial survival under extreme conditions is discussed.

Persistent middle ear effusion presumably biofilm-related in a paediatric patient with common variable immunodeficiency

Bacterial biofilms play a role in upper respiratory tract diseases, including acute and chronic middle ear diseases, and are involved in chronic infections and resistance to antibiotic treatment. In particular, the nasopharynx and the surrounding tissues act as important reservoirs of resistant bacterial biofilms, which have been detected in biopsies taken from adenoid and/or middle ear mucosa of children with chronic middle ear effusion. Here we describe the management of a child with congenital immunodeficiency and a chronic middle ear effusion, resistant to traditional medical treatment and presumably due to nasopharyngeal colonization by bacterial biofilms, which has been successfully treated by means of medicated nasal douches delivering antibiotic and a biofilm-destroying compound.

Seasonal and Successional Influences on Bacterial Community Composition Exceed That of Protozoan Grazing in River Biofilms

The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.

Analysis of Fluid Flow and Wall Shear Stress Patterns Inside Partially Filled Agitated Culture Well Plates

The appearance of highly resistant bacterial biofilms in both community and hospitals environments is a major challenge in modern clinical medicine. The biofilm structural morphology, believed to be an important factor affecting the behavioral properties of these "super bugs", is strongly influenced by the local hydrodynamics over the microcolonies. Despite the common use of agitated well plates in the biology community, they have been used rather blindly without knowing the flow characteristics and influence of the rotational speed and fluid volume in these containers. The main purpose of this study is to characterize the flow in these high-throughput devices to link local hydrodynamics to observed behavior in cell cultures. In this work, the flow and wall shear stress distribution in six-well culture plates under planar orbital translation is simulated using Computational Fluid Dynamics (CFD). Free surface, flow pattern and wall shear stress for two shaker speeds (100 and 200 rpm) and two volumes of fluid (2 and 4 mL) were investigated. Measurements with a non-intrusive optical shear stress sensor and High Frame-rate Particle Imaging Velocimetry (HFPIV) are used to validate CFD predictions. An analytical model to predict the free surface shape is proposed. Results show a complex three-dimensional flow pattern, varying in both time and space. The distribution of wall shear stress in these culture plates has been related to the topology of flow. This understanding helps explain observed endothelial cell orientation and bacterial biofilm distributions observed in culture dishes. The results suggest that the mean surface stress field is insufficient to capture the underlying dynamics mitigating biological processes.

Resistance of bacterial biofilms to disinfectants: a review

A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extrapolymeric substances. It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a multifactorial process involving the spatial organization of the biofilm. This review will discuss the mechanisms identified as playing a role in biofilm resistance to disinfectants, as well as novel anti-biofilm strategies that have recently been explored.

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.

Cell density and mobility protect swarming bacteria against antibiotics

Swarming bacteria move in multicellular groups and exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. We find that high densities promote bacterial survival, even in a nonswarming state, but that the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials. We find no evidence of induced resistance pathways or quorum-sensing mechanisms controlling this group resistance, which occurs at a cost to cells directly exposed to the antibiotic. This work has relevance to the adaptive antibiotic resistance of bacterial biofilms.

Antibiotic resistance of bacterial biofilms

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the body's defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms. Conventional resistance mechanisms such as chromosomal β-lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase) as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.

Resistance of Bacterial Biofilms Formed on Stainless Steel Surface to disinfecting Agent

The natural ability of microorganisms for adhesion and biofilm formation on various surfaces is one of the factors causing the inefficiency of a disinfection agent, despite its proven activity in vitro. The aim of the study was to determine the effectiveness of disinfecting substances on bacterial biofilms formed on stainless steel surface. A universally applied disinfecting agent was used in the tests. Bacterial strains: Listeria innocua, Pseudomonas putida, Micrococcus luteus, Staphylococcus hominis strains, were isolated from food contact surfaces, after a cleaning and disinfection process. The disinfecting agent was a commercially available acid specimen based on hydrogen peroxide and peroxyacetic acid, the substance that was designed for food industry usage. Model tests were carried out on biofilm formed on stainless steel (type 304, no 4 finish). Biofilms were recorded by electron scanning microscope. The disinfecting agent in usable concentration, 0.5% and during 10 minutes was ineffective for biofilms. The reduction of cells in biofilms was only 1-2 logarithmic cycles. The use of the agent in higher concentration--1% for 30 minutes caused reduction of cell number by around 5 logarithmic cycles only in the case of one microorganism, M. luteus. For other types: L. innocua, P. putida, S. hominis, the requirements placed on disinfecting agents were not fulfilled. The results of experiments proved that bacterial biofilms are resistant to the disinfectant applied in its operational parameters. Disinfecting effectiveness was achieved after twofold increase of the agent's concentration.

Antimicrobial resistance in bacterial biofilms

Bacteria can grow by forming a matrix-enclosed biofilm which adheres to a surface, providing some major advantages over the planktonic form of the organism. The ability to colonize different surfaces is utilized by some bacterial species, leading to significant infections. These range from minor skin complaints to life-threatening systemic infections. Bacteria adhering to implanted medical devices may be the cause of recurrent infection; bacterial persistence and the increasing use of indwelling medical devices has led to a steady increase in device-related bacterial infections. These bacteria are very resistant to antimicrobial therapy and the concentration of drugs required to achieve bactericidal activity may be at a higher order of magnitude than for planktonic bacteria. Different control strategies have been designed to inhibit biofilm formation on medical devices; however, it seems that biomaterial composition may play a role in the development of antimicrobial resistance, depending on the composition of the biomaterial, the bacterial species and the drugs. In this review, we discuss the most important mechanisms related to biofilm resistance to antimicrobial agents and bacterial interaction with medical devices, and their consequences for treatment failure.

Modeling physiological resistance in bacterial biofilms

A mathematical model of the action of antimicrobial agents on bacterial biofilms is presented. The model includes the fluid dynamics in and around the biofilm, advective and diffusive transport of two chemical constituents and the mechanism of physiological resistance. Although the mathematical model applies in three dimensions, we present two-dimensional simulations for arbitrary biofilm domains and various dosing strategies. The model allows the prediction of the spatial evolution of bacterial population and chemical constituents as well as different dosing strategies based on the fluid motion. We find that the interaction between the nutrient and the antimicrobial agent can reproduce survival curves which are comparable to other model predictions as well as experimental results. The model predicts that exposing the biofilm to low concentration doses of antimicrobial agent for longer time is more effective than short time dosing with high antimicrobial agent concentration. The effects of flow reversal and the roughness of the fluid/biofilm are also investigated. We find that reversing the flow increases the effectiveness of dosing. In addition, we show that overall survival decreases with increasing surface roughness.

Interactions between biocide cationic agents and bacterial biofilms.

The resistance of bacterial biofilms to physical and chemical agents is attributed in the literature to various interconnected processes. The limitation of mass transfer alters the growth rate, and physiological changes in the bacteria in the film also appear. The present work describes an approach to determination of the mechanisms involved in the resistance of bacteria to quaternary ammonium compounds (benzalkonium chloride) according to the C-chain lengths of those compounds. For Pseudomonas aeruginosa CIP A 22, the level of resistance of the bacteria in the biofilm relative to that of planktonic bacteria increased with the C-chain length. For cells within the biofilm, the exopolysaccharide induced a characteristic increase in surface hydrophilicity. However, this hydrophilicity was eliminated by simple resuspension and washing. The sensitivity to quaternary ammonium compounds was restored to over 90%. Staphylococcus aureus CIP 53 154 had a very high level of resistance when it was in the biofilm form. A characteristic of bacteria from the biofilm was a reduction in the percent hydrophobicity, but the essential point is that this hydrophobicity was retained after the biofilm bacteria were resuspended and washed. The recovery of sensitivity was thus only partial. These results indicate that the factors involved in biofilm resistance to quaternary ammonium compounds vary according to the bacterial modifications induced by the formation of a biofilm. In the case of P. aeruginosa, we have underlined the involvement of the exopolysaccharide and particularly the three-dimensional structure (water channels). In the case of S. aureus, the role of the three-dimensional structure is limited and drastic physiological changes in the biofilm cells are more highly implicated in resistance.

Mechanisms of antibiotic resistance in bacterial biofilms

AbstractBacteria that attach to a surface and grow as a biofilm are protected from killing by antibiotics. Reduced antibiotic susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to current treatments.

Riddle of biofilm resistance.

A biofilm is a population of cells growing on a surface and enclosed in an exopolysaccharide matrix. Biofilms are notoriously difficult to eradicate and are a source of many recalcitrant infections. The nature of bacterial biofilm resistance to antimicrobials is the subject of the present minireview. Pathogenic yeast such as Candida albicans also form recalcitrant biofilms, and this topic has recently been reviewed (5). Resistance is an ability of a microorganism to grow in the presence of an elevated level of an antimicrobial. In short, a strain for which the MIC is increased is resistant. By this conventional criterion, biofilm cells do not necessarily show increased resistance. With some exceptions, biofilm cells do not grow better than planktonic cells in the presence of a broad range of antimicrobials. This is evident from examination of susceptibility data in the biofilm literature (33). However, in most biofilm susceptibility studies, only survival of cells in a preformed biofilm rather than the ability of a biofilm to grow is recorded. Accordingly, the reported “resistance” describes an increased resistance of cells to killing. This is indeed what biofilms are good at: they are not easily eradicated by cidal antimicrobials. The ability of antimicrobials to inhibit biofilm growth indicates that they are able to diffuse through the biofilm and act normally against their targets. Why, then, do biofilm cells not die? This is the crux of the problem and the riddle that needs to be solved.

A comparative in vitro evaluation of different therapeutic protocols for vascular graft infections

Grafting techniques in vascular surgery are frequently complicated by severe infections, associated with mortality and limb-loss rates of 25--40%, 14 that occur both early after implantation of grafts and even much later. The severity of these infections is a consequence of the extremely high resistance of bacterial biofilms growing on graft surfaces, due to the production of esopolymeric substances. 5-7 Several approaches have been attempted to overcome this problem, including improvement in aseptic surgical techniques, development of new biomaterials intended to be less suitable for bacterial colonisation, bonding of antibacterial agents to grafts by different means, 8ql and research for new systemic agents able to disrupt biofilms. 12'13 Each different approach resulted in some improvement in therapeutic outcomes of grafting procedures, but none proved to be a definitive solution. The most threatening feature of biofilm growth with regard to resistance to antibacterials and host defences is certainly the production of glycocalyx. This was the target of several studies devoted to the development of new therapeutic strategies for prosthetic infections. Recently the results of two studies disclosed two potentially useful strategies to enhance the activity of antibiotics on bacterial biofilms, and namely the use of proteolytic enzymes 1B and the use of macrolides ~a both in association with antibiotics. The present work aimed at evaluating in vitro the effect of both proteolytic enzymes and macrolides, in association with different antibiotics and in conjunction, on bacterial biofilms formed on different biomaterials commonly used in vascular surgery.

Comparative Efficacies of Soft Contact Lens Disinfectant Solutions Against Microbial Films in Lens Cases

Biofilms of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus epidermidis, Streptococcus pyogenes, and Candida albicans, established in the wells of a polyethylene contact lens case, retained viability to certain soft contact lens disinfectant solutions after exposure for the manufacturer's minimum recommended disinfection times. The relative order of resistance of bacterial biofilms was as follows: S marcescens was greater than P aeruginosa, which was greater than S epidermidis, which was greater than S pyogenes. Air drying of biofilms for 10 hours increased the efficacy of the disinfectant solutions, but drying was not enough to decrease the incidence of recovery to 0% for all solutions. Hydrogen peroxide was more effective against biofilms than disinfectant solutions formulated with chlorhexidine gluconate or polyquaternium-1 or polyaminopropyl biguanide. We recommend that determination of efficacy of contact lens disinfectant solutions should include challenges against biofilms.

Resistance of bacterial biofilms to antibiotics: a growth-rate related effect?

Resistance of bacterial biofilms to antibiotics: a growth-rate related effect?