Staphylococcus aureus and biofilms:transmission, threats, and promising strategies in animal husbandry

Staphylococcus aureus(S. aureus) is a common pathogenic bacterium in animal husbandry that can cause diseases such as mastitis, skin infections, arthritis, and other ailments. The formation of biofilms threatens and exacerbates S. aureus infection by allowing the bacteria to adhere to pathological areas and livestock product surfaces, thus triggering animal health crises and safety issues with livestock products. To solve this problem, in this review, we provide a brief overview of the harm caused by S. aureus and its biofilms on livestock and animal byproducts(meat and dairy products). We also describe the ways in which S. aureus spreads in animals and the threats it poses to the livestock industry. The processes and molecular mechanisms involved in biofilm formation are then explained. Finally, we discuss strategies for the removal and eradication of S. aureus and biofilms in animal husbandry, including the use of antimicrobial peptides, plant extracts, nanoparticles, phages, and antibodies. These strategies to reduce the spread of S. aureus in animal husbandry help maintain livestock health and improve productivity to ensure the ecologically sustainable development of animal husbandry and the safety of livestock products.

Charge adaptive phytochemical-based nanoparticles for eradication of methicillin-resistant staphylococcus aureus biofilms

The intrinsic resistance of MRSA coupled with biofilm antibiotic tolerance challenges the antibiotic treatment of MRSA biofilm infections.Phytochemical-based nanoplatform is a promising emerging approach for treatment of biofilm infection.However,their therapeutic efficacy was restricted by the low drug loading capacity and lack of selectivity.Herein,we constructed a surface charge adaptive phytochemical-based nanoparticle with high isoliquiritigenin(ISL)loading content for effective treatment of MRSA biofilm.A dimeric ISL prodrug(ISL-G2)bearing a lipase responsive ester bond was synthesized,and then encapsulated into the amphiphilic quaternized oligochitosan.The obtained ISL-G2loaded NPs possessed positively charged surface,which allowed cis-aconityl-D-tyrosine(CA-Tyr)binding via electrostatic interaction to obtain ISL-G2@TMDCOS-Tyr NPs.The NPs maintained their negatively charged surface,thus prolonging the blood circulation time.In response to low pH in the biofilms,the fast removal of CA-Tyr led to a shift in their surface charge from negative to positive,which enhanced the accumulation and penetration of NPs in the biofilms.Sequentially,the pH-triggered release of D-tyrosine dispersed the biofilm and lipase-triggered released of ISL effectively kill biofilm MRSA.An in vivo study was performed on a MRSA biofilm infected wound model.This phytochemical-based system led to~2log CFU(>99%)reduction of biofilm MRSA as compared to untreated wound(P<0.001)with negligible biotoxicity in mice.This phytochemical dimer nanoplatform shows great potential for long-term treatment of resistant bacterial infections.

Temporal characteristics of algae-denitrifying bacteria co-occurrence patterns and denitrifier assembly in epiphytic biofilms on submerged macrophytes in Caohai Lake,SW China

Denitrifying bacteria in epiphytic biofilms play a crucial role in nitrogen cycle in aquatic habitats.However,little is known about the connection between algae and denitrifying bacteria and their assembly processes in epiphytic biofilms.Epiphytic biofilms were collected from submerged macrophytes(Patamogeton lucens and Najas marina L.)in the Caohai Lake,Guizhou,SW China,from July to November 2020 to:(1)investigate the impact of abiotic and biotic variables on denitrifying bacterial communities;(2)investigate the temporal variation of the algae-denitrifying bacteria co-occurrence networks;and(3)determine the contribution of deterministic and stochastic processes to the formation of denitrifying bacterial communities.Abiotic and biotic factors influenced the variation in the denitrifying bacterial community,as shown in the Mantel test.The co-occurrence network analysis unveiled intricate interactions among algae to denitrifying bacteria.Denitrifying bacterial community co-occurrence network complexity(larger average degrees representing stronger network complexity)increased continuously from July to September and decreased in October before increasing in November.The co-occurrence network complexity of the algae and nirS-encoding denitrifying bacteria tended to increase from July to November.The co-occurrence network complexity of the algal and denitrifying bacterial communities was modified by ammonia nitrogen(NH_(4)^(+)-N)and total phosphorus(TP),pH,and water temperature(WT),according to the ordinary least-squares(OLS)model.The modified stochasticity ratio(MST)results reveal that deterministic selection dominated the assembly of denitrifying bacterial communities.The influence of environmental variables to denitrifying bacterial communities,as well as characteristics of algal-bacterial co-occurrence networks and the assembly process of denitrifying bacterial communities,were discovered in epiphytic biofilms in this study.The findings could aid in the appropriate understanding and use of epiphytic biofilms denitrification function,as well as the enhancement of water quality.

Influences of trans-trans farnesol,a membrane-targeting sesquiterpenoid,on Streptococcus mutans physiology and survival within mixed-species oral biofilms

Trans-trans farnesol （tt-farnesol） is a bioactive sesquiterpene alcohol commonly found in propolis （a beehive product） and citrus fruits, which disrupts the ability of Streptococcus mutans （S. mutans） to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria （Streptococcus oralis （S. oralis） 35037 and Actinomyces naeslundii （.4. naeslundil） 12104） was also examined. In general, tt-farnesol （1 mmol-L-1） significantly increased the membrane proton permeability and reduced glycolytie activity of S. mutans in the planktonic state and in biofilms （P〈0.05）. Moreover, topical applications of 1 mmol-L＂l tt-farnesol twice daily （1 min exposure/treatment） reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed-species biofilms after introduction of 1% sucrose. S. oralis （a non-cariogenie organism） became the major species after treatments with tt-farnesol, whereas vehicle-treated biofilms contained mostly S. mutans （〉90% of total bacterial population）. However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt-farnesoi may affect the competi- tiveness of S. mutans in a mixed-species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti-biofilm/anti-caries chemotherapeutic strategies.

Effect of anti-biofilm glass–ionomer cement on Streptococcus mutans biofilms

Dental restorative materials with antimicrobial properties can inhibit bacterial colonization, which may result in a reduction of caries at tooth-filling interaction zones. This study aimed to develop antibacterial glass-ionomer cements （GIC） containing a quaternary ammonium monomer （dimethylaminododecyl methacrylate, DMADDM）, and to investigate their effect on material performance and antibacterial properties. Different mass fractions （0, 1.1% and 2.2%） of DMADDM were incorporated into the GIC. The flexure strength, surface charge density, surface roughness and fluoride release were tested. A Streptococcus mutans biofilm model was used. Exopolysaccharides （EPS） staining was used to analyze the inhibitory effect of DMADDM on the biofilm matrix. In addition, biofilm metabolic activity, lactic acid metabolism and the expression of glucosyltransferase genes g/fB, gtfC and gtfD were measured. GIC containing 1.1% and 2.2% DMADDM had flexural strengths matching those of the commercial control （P〉0.1）. DMADDM was able to increase the surface charge density but reduced surface roughness （P〈0.05）. The incorporation of 1.1% and 2.2% DMADDM elevated the release of fluoride by the GIC in the first 2 days （P〈0.05）. The novel DMADDM-modified GIC significantly reduced biofilm metabolic activity （P〈 0.05） and decreased lactic acid production （P〈 0.05）. The quantitative polymerase chain reaction （qPCR） results showed that the expression of gtfB, g/fC and gtfD decreased when mass fractions of DMADDM increased （P〈0.05）. EPS staining showed that both the bacteria and EPS in biofilm decreased in the DMADDM groups. The incorporation of DMADDM could modify the properties of GIC to influence the development of S. mutans biofilms. In this study, we investigated the interface properties of antibacterial materials for the first time. GIC containing DMADDM can improve material performance and antibacterial properties and may contribute to the better management of secondary caries.

Current understanding of multi-species biofilms

Direct observation of a wide range of natural microorganisms has revealed the fact that the majority of microbes persist as surface-attached communities surrounded by matrix materials, called biofilms. Biofilms can be formed by a single bacterial strain. However, most natural biofilms are actually formed by multiple bacterial species. Conventional methods for bacterial cleaning, such as applications of antibiotics and/or disinfectants are often ineffective for biofilm populations due to their special physiology and physical matrix barrier. It has been estimated that billions of dollars are spent every year worldwide to deal with damage to equipment, contamina- tions of products, energy losses, and infections in human beings resulted from microbial biofilms. Microorganisms compete, cooperate, and communicate with each other in multi-species biofilms. Understanding the mechanisms of multi-species hiofilm formation will facilitate the development of methods for combating bacterial hiofilms in clinical, environmental, industrial, and agricultural areas. The most recent advances in the understanding of multi-species biofilms are summarized and discussed in the review.

The photodynamic therapy on Streptococcus mutans biofilms using erythrosine and dental halogen curing unit

The purpose of our study was to evaluate the effect of photodynamic therapy （PDT）, using erythrosine as a photosensitizing agent and a dental halogen curing unit as a light source, on Streptococcus mutans in a biofilm phase. The S. mutans biofilms were formed in a 24-well cell culture cluster. Test groups consisted of biofilms divided into four groups： group 1： no photosensitizer or light irradiation treatment （control group）; group 2： photosensitizer treatment alone; group 3： light irradiation alone; group 4： photosensitizer treatment and light irradiation. After treatments, the numbers of colony-forming unit （CFU） were counted and samples were examined by confocal laser scanning fluorescence microscopy （CLSM）. Only group 4 （combined treatment） resulted in significant increases in cell death, with rates of 75% and 55% after 8 h of incubation, and 74% and 42% at 12 h, for biofilms formed in brain-heart infusion （BHI） broth supplemented with 0% or O. 1% sucrose, respectively. Therefore, PDT of S. mutans biofilms using a combination of erythrosine and a dental halogen curing unit, both widely used in dental clinics, resulted in a significant increase in cell death. The PDT effects are decreased in biofilms that form in the presence of sucrose.

Community structure and assembly of denitrifying bacteria in epiphytic biofilms in a freshwater lake ecosystem

Denitrifying bacteria are a crucial component of aquatic ecosystem in nitrogen cycle.However,the denitrifying bacterial community dynamics and structure in epiphytic biofilms remain unexplored.The abundance of denitrification gene(nir)and structure of nirS-denitrifying bacterial community in the epiphytic biofilms collected in July and November of 2018 from a typical plateau lake(Caohai Wetland,Guizhou,China)were studied by Real-time Quantitative Polymerase Chain Reaction(qPCR)and highthroughput sequencing.Results show that the gene abundance of nirK was higher than that of nirS(P<0.05),and it was significantly different during the growth period(July)than the decline period(November).The denitrifying bacterial species was similar in the two months and shared 76.18%of OTUs.Proteobacteria(56.55%±22.15%)was the dominant phylum in all the samples.Epiphytic biofilms between growth period and decline period displayed significantly different microbial community structures due to differences in species abundance.Water temperature was the crucial factor that affected the denitrifying microbial community structure in our study.Environmental factors explain only partially the dynamic characteristics of denitrifying microbial communities,implying that the stochastic processes affected the construction of denitrifying microbial communities.As the null model analysis results show,dispersal limitation(stochastic)and undominated processes significantly influenced the assembly of denitrifying microbial communities.This study broadened our understanding of the denitrifying bacterial community structure and its function on epiphytic biofilms in freshwater ecosystems with new information provided.

Disruption of biofilms from sewage pipes under physical and chemical conditioning

Biofilms grown inside two sewage collecting pipes located in industrial and residential areas are studied. Bacterial biomass inside three layers of biofilms was evaluated. Biofilm cohesion under different mixing rate and ionic strength was also investigated. Effects of physical and chemical parameters in the biofilms were evaluated by monitoring turbidity, chemical and biochemical oxygen demands. Extracted organic matter from biofilms was partitioned to polar, aromatic and saturated fractions using activated silica column chromatography. Results revealed that bacterial biomass growth depending on biofilm thickness and stratification. The most loaded stratum in bacteria/biomass was the sewage-biofilm interface stratum that represented 51% of the total bacteria/biomass. Stirring rate and ionic strength of mono- and bivalent salts showed a major influence in biofilm disruption. The stirring time enhanced the exchange dynamic and matter capture between biofilm fragments at the critical stirring rate 90 r/min. Sodium chloride showed the dispersing effect on biofilms in suspension, and decreased the BOD5 （biochemical oxygen demand） beyond the physiological salt concentration.

Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media： Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy（CLSM）-based analyses of the microbial vitality, respiratory activity（5-cyano-2,3-ditolyl tetrazolium chloride, CTC） and production of extracellular polysaccharides（EPS） were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters： culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.

Effects of Cd^(2+) and Pb^(2+) on the substrate biofilms in the integrated vertical-flow constructed wetland

The effects of single Cd^(2+)and Pb^(2+),and combined Cd^(2+)and Pb^(2+)on dehydrogenase activity and polysaccharide content of the substrate biofilms in the integrated vertical-flow constructed wetland(IVCW)were studied.Dehydrogenase activities decreased linearly with the increasing concentrations of Cd^(2+)and Pb^(2+)at different times(6,24,72,and 120 h).The activities at both 6 and 24 h were significantly higher than that at 72 and 120 h in the case of single and combined treatments.The single Cd^(2+)and...

Mass transport model of ions within biofilms under the effect of external field

A mass transport model was developed to predict the transport rate of ions within biofilms, which was experimentally verified using the fluxes of NH4^＋ and Ca^2＋ through the heterotrophic biofilms with the thickness varying from 230 to 1430μm under the effect of external field in the range of-20 V/m to 60 V/m. It is found that the result predicted by the model is in agreement with the experimentally obtained one, with the error less than 5 percent for the thin biofilms. The error increases with the increase of the biofilm thickness. The transport rate of ions caused by electric migration is affected by the charges, field strength, and biofilm thickness and so on.

Biodegradation of Natural Estrogens by Biofilms from Biological Activated Carbon: Effect of Temperature

In order to obtain information on the biodegradation potential of biofilms involved in the removal of natural estrogens by biological activated carbon (BAC) columns, batch degradation of estrone (E1) and 17β-estradiol (E2) at temperature of 5℃, 20℃ and 35℃ by biofilms from four BAC columns (packed with activated carbon of particle size ranging from 0.5 - 0.59 mm and 1.0 - 1.19 mm into two bed depths) was studied. The results indicated that E2 was degraded faster by than E1 at all three temperatures and with the increasing of temperature, the amount of E1 converted from E2 increased. By fitting observed concentration data with a first-order rate expression, the bio-mass based degradation rate constants (kVSS) for E1 and E2 under all experimental conditions were estimated and linear relationship between lnkVSS and 1/T (T = absolute temperature) was demonstrated, resulting that with the increasing of the experimental temperature, degradation rate of biofilms for both E1 and E2 increased, and the increasing rate for E2 was higher than that for E1.

Meeting report: a close look at oral biofilms and microbiomes

The "Biofilms, Microbiomes and Oral Diseases: Challenges and Future Perspectives" symposium jointly organized by Penn Dental Medicine and West China School of Stomatology was held on 30 September 2017 at Penn Wharton China Center(PWCC) in Beijing,China. The topics included the pathogenicity of oral biofilms, novel strategies for the control of biofilm-related diseases, oral microbiome and single-cell approaches, and the link between oral diseases and overall health. Researchers from a number of disciplines, representing institutions from China and Penn Dental Medicine, gathered to discuss advances in our understanding of biofilms, as well as future directions for the control of biofilm-related oral and systemic diseases.

Simulated Solar Microwave Radiation Blocks the Formation of Biofilms

The article presents the results of the experimental study that was devoted to determining the blocking influence of the solar microwave radiation on the process of biofilm formation in Gram-positive and Gram-negative microorganisms. The microwave generator that allows simulating microwave “splashes” of the Sun in the frequency range (4.0 - 4.3 GHz) with the controlled intensity of radiation (from 50 μW/sm2 to 500 μW/sm2) was used for conducting this research. It is found out that the simulated solar radiation of the microwave range blocks the formation of the extracellular matrix by the opportunistic microorganisms. The results of this study confirm the hypothesis of the evolutionary nature of the leading role of the microwave radiation of the Sun in the life processes of organisms. The technology of the exposure on the microorganisms that was used in the experiment opens up the real prospects for reducing the persistent potential of microorganisms and improving the efficiency of the bacterial infections treatment.

A Novel Cellular Autoaggregative Developmentally CRP Regulated Behaviour Generates Massively Chondrule-Like Formations over Surface of Old <i>Escherichia coli</i>K-12 Macrocolony Biofilms

How Escherichia coli bacteria develop a particular colonial, 3-D biofilm morphological pattern is still a poorly understood process. Recently, we reported a new E. coli K-12 morphotype exhibited by old macrocolonies described as volcano-like. The formative developmental process of this morphotype has been presented as a suitable experimental model for the study of 3D patterning in macrocolony biofilms. Here, we report the optical microscopy observations and genetic analysis that have unveiled the existence of a novel autoaggregative behaviour which generates massive lumpiness over the surface of the volcano-like macrocolonies. These lumpy formations are generated by the autoaggregation and strong interaction of tightly packed bacterial cells in structures with a chondrule-like appearance which give the colony’s surface its characteristic microscopic lumpy phenotype. Furthermore, they exhibit different levels of maturation from the edge to the center of the colony. Hence, its generation appears to follow a spatiotemporal program of development during the macrocolony’s morphogenesis. Interestingly, the agar’s hardness influences the morphology exhibited by these formations, with high agar concentration (1.5%, 15 g/L) suppressing its development. This new auto-aggregative E. coli’s behaviour does not require the activity of the biofilm master regulator CsgD, the adhesiveness of flagella, pili type 1, adhesin Ag43, β-1,6-N-acetyl-D-glucosamine polymer-PGA, cellulose or colanic acid, but it is under glucose repression and the control of cAMP receptor protein (CRP). The possible physiological role of these chondrule-like formations in the adaptability of the colony to different stressful environmental conditions is discussed.

Pathological Role of Fungal Biofilms in Fungal Rhinosinusitis: A Case-Control Study

Objective: This case-control study aimed to detect the presence of fungal biofilms in cases of fungal sinusitis, trying to find its role in recurrence of fungal sinus infection and resistance to medical treatment. Patients and Methods: This study was conducted at Ain-Shams University from June-2017 to June-2018 upon 20 patients as the fungal cases are not much in Egypt. All patients were subjected to functional endoscopic sinus surgery and taking specimens. Examination of specimens by scanning electron microscope was made. Results: The prevalence of fungal biofilms was 7 (70%) in cases and 0 (0%) in controls (p-value = 0.001). There is higher risk of fungal biofilms in cases of fungal sinusitis, the odds of biofilms is higher in the study group 2.33 than in the control group 0. The lower is the invasiveness, the higher is the fungal biofilms (OR = 0.94, 95% CI 0.46 - 4.05). Also, the analysis showed that the lower is the recurrence the lower is the fungal biofilms (OR = 2.25, 95% CI 0.15 - 7.93). Conclusion: This study showed the presence of fungal biofilms in different cases of fungal sinusitis whether primary or recurrent, also whether invasive or non-invasive. Studies with large sample size are recommended to get a strong evidence.

Engineering Microbial Biofilms for Improved Productivity of Biochemicals Important in Restoration of Degraded Ecosystems

Biofilms are being engineered in-vitro to produce numerous commodities like biofertilizers, pharmaceuticals, biofuels and electricity, the efficacies of which rely on the biochemicals secreted by the biofilms i.e. extracellular polymeric substances (EPS). It has been shown that once EPS-biochemicals of developed biofilms are applied to an ecosystem, they can restore degraded complex ecosystem networks for improved ecosystem functioning and sustainability. Identification of the EPS biochemicals and understanding their contributions to the network interactions in particular, are at initial stage. In the present study, using Aspergillus niger, Nostoc sp., and gram (-) Stenotrophomonas maltophilia & gram (+) Bacillus subtilis as test fungal (F), cyanobacterial (C), and bacterial (B) counterparts, respectively we analyzed morphology and biochemical parameters of fungal-bacterial (FBBs), fungal-cyanobacterial (FCBs), cyanobacterial-bacterial (CBBs), and fungal-cyanobacterial-bacterial biofilms (FCBBs). Results revealed that the FCBBs produced the highest concentrations of lipids, proteins, and polysaccharides whereas FBBs generated the highest diversity of biochemicals. Bacterial type (i.e. gram + or -) and microbial composition in the biofilm affected the biochemical production. Ecologically and industrially important diverse biochemicals which are used individually as medicines, bioremediating agents and industrial chemicals in human society with certain adverse and beneficial effects were detected in the biofilm-EPS. However, in the nature, simultaneous action of those diverse biochemicals applied as biofertilizers has already shown a huge potential to restore the entire agroecosystems degraded due to farmers’ detrimental practices. This striking difference in utilization of the biochemicals and their enhanced effect when they act simultaneously needs further investigations for their better applications.

The clinical impact of bacterial biofilms

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria （and fungi） are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

Characterization of biofilms in biliary stents and potential factors involved in occlusion

AIM To quantify the components in biofilms and analyze the predisposing factors involved in occlusion of biliary stents. METHODS In a prospective study conducted from April 2011 to March 2014 at a tertiary care hospital, all consecutive patients who required endoscopic biliary stent exchange/removal were included. Etiology of the biliary disease was diagnosed by imaging, cytology and on follow-up. Clinical details of patients with biliary stent retrieval were noted. All extracted stents were collected in sterile containers and immediatelyprocessed for quantification of biofilm proteins and polysaccharides. Molecular identification of commonly known and unknown bacteria was performed by polymerase chain reaction and density gradient gel electrophoresis methods. RESULTS Eighty one patients(41 males) with age range of 20-86 years were studied. The underlying causes for stent insertion were bile duct stones(n = 46; 56.8%) benign stricture(n = 29; 35.8%) and malignancy(n = 6; 7.4%) with cholangitis in 50(61.7%) patients. The retrieved stent sizes were 7 Fr(n = 62; 76.5%) and 10 Fr(n = 19; 23.5%) with 65 days median insertion duration. Polybacterial consortia were detected in 90.1% of the stents. The most common bacteria identified by polymerase chain reaction alone and/or sequencing were Pseudomonas(n = 38), Citrobacter(n = 23), Klebsiella(n = 22), Staphylococcus(n = 20), Serratia(n = 16), Escherichia coli(n = 14), Streptococcus(n = 13), Enterococcus(n = 13), Aeromonas(n = 12), Proteus(n = 10) and Enterobacter(n = 9). Protein concentration according to gender(0.547 ± 0.242 mg/ml vs 0.458 ± 0.259 mg/ml; P = 0.115) as well as age > 60 years and < 60 years(0.468 ± 0.295 mg/ml vs 0.386 ± 0.238 mg/ml; P = 0.205) was non-significant. However, polysaccharide concentration was significant both according to gender(0.052 ± 0.021 mg/ml vs 0.049 ± 0.016 mg/ml; P < 0.0001) and age(0.051 ± 0.026 mg/ml vs 0.038 ± 0.016 mg/ml; P < 0.011). Protein concentration in the biofilm was significantly higher(0.555 ± 0.225 mg/ml vs 0.419 ± 0.276 mg/ml; P = 0.018) in patients with cholangitis, lower(0.356 ± 0.252 mg/ml vs 0.541 ± 0.238 mg/ml; P = 0.005) in the 10 Fr group than the 7 Fr group, and significantly higher(0.609 ± 0.240 mg/ml vs 0.476 ± 0.251 mg/ml; P = 0.060) in stents of ≥ 6 mo of indwelling time. However presence/absence of cholangitis, size of stent, indication of stent insertion and indwelling time did not affect the quantity of polysaccharide concentration.CONCLUSION Plastic stents retrieved from patients with biliary tract disease showed polymicrobial organisms with higher protein content among patients with cholangitis and those with smaller diameter stents. longer indwelling duration had more biofilm formation.