Biofilm Age Research Articles

Fsr quorum sensing system modulates the temporal development of Enterococcus faecalis biofilm matrix.

Quorum sensing (QS) is a cell-to-cell communication process that regulates major pathogenic attributes in bacteria including biofilm formation, secretion of virulence factors, and antimicrobial resistance. The two-component Fsr-QS system of the nosocomial pathogen Enterococcus faecalis controls the production of extracellular gelatinase that contributes to biofilm development by enhancing the release of nucleic acids into the biofilm matrix. However, the contribution of this system to the deposition of other biofilm matrix components such as polysaccharides and proteins remains unknown. Using wild type and mutant strains, we discovered that biofilm formation was attenuated by inactivation of the Fsr system or its downstream gelatinase production. Inactivation of the Fsr system caused a modest, yet significant reduction in biofilm metabolic activity without affecting cell counts. Inactivation of the QS-signal sensor FsrC and response regulator FsrA resulted in decreased extracellular polysaccharides and proteins in biofilms in a temporal manner. Irrespective of biofilm age, eDNA levels were reduced in the gelatinase mutant strain. Our results collectively suggest that the Fsr system contributes to the temporal deposition of polysaccharides and proteins into the extracellular polymeric matrix (EPS) of E. faecalis biofilm, without affecting bacterial viability. This understanding of the role of the Fsr-QS system in biofilm development may reveal a novel target to develop effective antibiofilm agents to tackle E. faecalis-mediated infections such as in dental root canals, heart valves, and surgical sites.

The self-healing of Bacillus subtilis biofilms.

Self-healing is an intrinsic ability that exists widely in every multicellular biological organism. Our recent experiments have shown that bacterial biofilms also have the ability to self-heal after man-make cuts, but the mechanism of biofilm self-healing have not been studied. We find that the healing process of cuts on the biofilm depends on cut geometries like its location or direction, the biofilm itself like the biofilm age, the growing substrate properties like its hardness, and also the environments such as the competitive growth of multiple biofilms. What is more, the healing rate along the cut is heterogeneous, and the maximum healing rate can reach 260μm/h, which is three times the undestroyed biofilm expansion rate. The cut does not change the rounded shape growth of biofilms. Further study of phenotypic evolution shows that the cut delays bacterial differentiation; motile cells perceive the cut and move to the cut area, while the cut only heals when there are enough matrix-producing cells in the cut area. Our work suggests new ideas for developing self-healing materials.

Biofilms for Turbidity Mitigation in Oil Sands End Pit Lakes.

End pit lakes (EPLs) have been proposed as a method of reclaiming oil sands fluid fine tailings (FFT), which consist primarily of process-affected water and clay- and silt-sized particles. Base Mine Lake (BML) is the first full-scale demonstration EPL and contains thick deposits of FFT capped with water. Because of the fine-grained nature of FFT, turbidity generation and mitigation in BML are issues that may be detrimental to the development of an aquatic ecosystem in the water cap. Laboratory mixing experiments were conducted to investigate the effect of mudline biofilms made up of microbial communities indigenous to FFT on mitigating turbidity in EPLs. Four mixing speeds were tested (80, 120, 160, and 200 rpm), all of which are above the threshold velocity required to initiate erosion of FFT in BML. These mixing speeds were selected to evaluate (i) the effectiveness of biofilms in mitigating turbidity and (ii) the mixing speed required to ‘break’ the biofilms. The impact of biofilm age (10 weeks versus 20 weeks old) on turbidity mitigation was also evaluated. Diverse microbial communities in the biofilms included photoautotrophs, namely cyanobacteria and Chlorophyta (green algae), as well as a number of heterotrophs such as Gammaproteobacteria, Desulfobulbia, and Anaerolineae. Biofilms reduced surface water turbidity by up to 99%, depending on the biofilm age and mixing speed. Lifting and layering in the older biofilms resulted in weaker attachment to the FFT; as such, younger biofilms performed better than older biofilms. However, older biofilms still reduced turbidity by 69% to 95%, depending on the mixing speed. These results indicate that biostabilization is a promising mechanism for turbidity mitigation in EPLs.

Reduced seawater pH alters marine biofilms with impacts for marine polychaete larval settlement

Ocean acidification (OA) can negatively affect early-life stages of marine organisms, with the key processes of larval settlement and metamorphosis potentially vulnerable to reduced seawater pH. Settlement success depends strongly on suitable substrates and environmental cues, with marine biofilms as key settlement inducers for a range of marine invertebrate larvae. This study experimentally investigated (1) how seawater pH determines growth and community composition of marine biofilms, and (2) whether marine biofilms developed under different pH conditions can alter settlement success in the New Zealand serpulid polychaete Galeolaria hystrix. Biofilms were developed under six pH(T) treatments (spanning from 7.0 to 8.1 [ambient]) in a flow-through system for up to 14 months. Biofilms of different ages (7, 10 and 14 months) were used to assay successful settlement of competent G. hystrix larvae reared under ambient conditions. Biofilm microbiomes were characterized through amplicon sequencing of the small subunit ribosomal rRNA gene (16S and 18S). Biofilm community composition was stable over time within each pH treatment and biofilm age did not affect larval settlement selectivity. Seawater pH treatment strongly influenced biofilm community composition, as well as subsequent settlement success when biofilms were presented to competent Galeolaria larvae. Exposure to biofilms incubated under OA-treatments caused a decrease in larval settlement of up to 40% compared to the ambient treatments. We observed a decrease in settlement on biofilms relative to ambient pH for slides incubated at pH 7.9 and 7.7. This trend was reversed at pH 7.4, resulting in high settlement, comparable to ambient biofilms. Settlement decreased on biofilms from pH 7.2, and no settlement was observed on biofilms from pH 7.0. For the first time, we show that long-term incubation of marine biofilms under a wide range of reduced seawater pH treatments can alter marine biofilms in such a way that settlement success in marine invertebrates can be compromised.

The antimicrobial efficacy of remote cold atmospheric plasma effluent against single and mixed bacterial biofilms of varying age

Cold atmospheric plasma (CAP) is a minimal food processing technology of increasing interest in the food industry, as it is mild in nature compared to traditional methods (e.g. pasteurisation) and thus can maintain the food’s desirable qualities. However, due to this mild nature, the potential exists for post-treatment microbial survival and/or stress adaptation. Furthermore, biofilm inactivation by CAP is underexplored and mostly studied on specific foods or on plastic/polymer surfaces. Co-culture effects, biofilm age, and innate biofilm-associated resistance could all impact CAP efficacy, while studies on real foods are limited to the food product investigated without accounting for structural complexity.The effect of a Remote and Enclosed CAP device (Fourth State Medicine Ltd) was investigated on Escherichia coli and Listeria innocua grown as planktonic cells and as single or mixed bacterial biofilms of variable age, on a biphasic viscoelastic food model of controlled rheological and structural complexity. Post-CAP viability was assessed by plate counts, cell sublethal injury was quantified using flow cytometry, and biofilms were characterised and assessed using total protein content and microscopy techniques.A greater impact of CAP on planktonic cells was observed at higher air flow rates, where the ReCAP device operates in a mode more favourable to reactive oxygen species than reactive nitrogen species. Although planktonic E. coli was more susceptible to CAP than planktonic L. innocua, the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore, E. coli produced much higher protein content in both single and mixed biofilms than L. innocua. Consequently, greater survival of L. innocua in mixed biofilms was attributed to a protective effect from E. coli.These results show that biofilm susceptibility to CAP is age and bacteria dependent, and that in mixed biofilms bacteria may become less susceptible to CAP. These findings are of significance to the food industry for the development of effective food decontamination methods using CAP.

ANTIBACTERIAL INFLUENCE OF SILVER NANOPARTICLES ON MULTI-RESISTANT STRAINS OF K. PNEUMONIAE ISOLATED AT HOSPITALS

Introduction. Overuse and misuse of antibiotics in humans, animals, and agriculture has led to the widespread rise of antibiotic resistance and strengthened nosocomial pathogenes' impact. Klebsiella pneumoniae became an increasing threat to public health. Nanomaterials are promising alternatives to conventional antibiotics in the fight against multi-resistant germs. Silver nanoparticles are well-known metallic nanoparticles with antimicrobial activity. Our research aimed to evaluate the spreading of K. pneumonia resistant to antibiotics at hospital and assess the effectiveness of Ag NPs against multi-resistant clinical strains of K. pneumoniae. Material and methods. K. pneumoniae strains were isolated and identified with the use of conventional bacteriological techniques. Susceptibility of the microorganisms was assessed to inhibitors of β-lactamases, carbapenems, macrolides, oxazolidinones, and other groups of antibiotics with use Kirby-Bauer disk diffusion method. The capability of AgNPs to inhibit attachment and multiplication of the K. pneumoniae multi-resistant strains was tested with the use of serial microdilution method, resazurin assay, and SEM. Results. K. pneumoniae was isolated from 13.7% of samples predominantly at the microbial association (97.5%). The microorganisms were resistant to five or more antibiotics in 73.2% of cases. AgNPs possess antimicrobial activity against tested strains at concentrations varied from 1.25 µg/ml to 2.5 µg/ml and kill all germs in 3 hours of incubation. AgNPs inhibited biofilm formation at initial stages and destroyed the mature (2 days) biofilm with Ag NPs treatment at concentrations 20-40 µg/ml. The effectiveness of mature K. pneumoniae biofilm treatment with AgNPs depended on biofilm age. The SEM images of the two-days biofilm reveal lysis of the bacterial cells after the cocultivation with Ag NPs but SEM analysis detected the maintaining of the three-dimensional structure in the case of a five-day biofilm after cocultivation with AgNPs. Conclusions. The distribution of K. pneumonia among patients with laryngeal pathology and its sensitivity to eleven antibiotics were examined. There was revealed the high rate of K. pneumonia multi-resistant strains. Ag NPs have strong antibacterial and anti-biofilm potential against multi-resistant K. pneumoniae. Therefore, our results highlight that the Ag NPs have promising antimicrobial and anti-biofilm abilities against multi-resistant clinical strains of K. pneumoniae.

Colonization of carbapenem-resistant Klebsiella pneumoniae in a sink-drain model biofilm system.

Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella pneumoniae (CPKP). We investigated the colonization of a biofilm consortia by CPKP in a model system simulating a sink-drain P-trap. Centers for Disease Control (CDC) biofilm reactors (CBRs) were inoculated with microbial consortia originally recovered from 2 P-traps collected from separate patient rooms (designated rooms A and B) in a hospital. Biofilms were grown on stainless steel (SS) or polyvinyl chloride (PVC) coupons in autoclaved municipal drinking water (ATW) for 7 or 28 days. Microbial communities in model systems (designated CBR-A or CBR-B) were less diverse than communities in respective P-traps A and B, and they were primarily composed of β and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with either K. pneumoniae ST45 (ie, strain CAV1016) or K. pneumoniae ST258 KPC+ (ie, strain 258), and samples were collected over 21 days. Under most conditions tested (CBR-A: SS, 7-day biofilm; CBR-A: PVC, 28-day biofilm; CBR-B: SS, 7-day and 28-day biofilm; CBR-B: PVC, 28-day biofilm) significantly higher numbers of CAV1016 were observed compared to 258. CAV1016 showed no significant difference in quantity or persistence based on biofilm age (7 days vs 28 days) or substratum type (SS vs PVC). However, counts of 258 were significantly higher on 28-day biofilms and on SS. These results suggest that CPKP persistence in P-trap biofilms may be strain specific or may be related to the type of P-trap material or age of the biofilm.

Impacts of ammonia loading and biofilm age on the prevalence of nitrogen-cycling microorganisms in a full-scale submerged attached-growth reactor.

This study reports the impacts of seasonal ammonia load changes and biofilm age on the quantity of biomass and on the prevalence of ammonia- and nitrite-metabolizing organisms within a submerged attached-growth reactor (SAGR™) following lagoon treatment. Ammonia (NH3 ) loadings (0.12-3.17kg/d) in the primary SAGR were measured over 223days from May to December in 2017. Adjustment of the wastewater flow path on September 1 successfully increased NH3 loading to the primary SAGR, which subsequently caused reactor biomass to increase. The NH3 removal rate in October (0.5kg/d) was greater than rates in June and July (0.3 and 0.2kg/d) despite a water temperature decrease from >24 to 15.6°C. This elevated removal rate in October, and the sustained removal rate in December (0.4kg/d, 5.3°C) were associated with a measured increase in microbial biomass. The relative abundance of the anammox organism C.Brocadia was 5 times greater in the mature biofilm after 686days of growth, and the genus Pseudomonas increased sevenfold. The presence of Pseudomonas, which contains denitrifying species, and anammox suggests a high potential for removal of total nitrogen in SAGRs. PRACTITIONER POINTS: Pseudomonas prevalence and the presence of anammox suggest a high potential for total nitrogen removal in mature SAGR biofilms. The abundance of the anammox microorganism C.Brocadia was greater after 686days of biofilm growth compared with 33days. Simple operational changes can increase biomass in the SAGR to maintain, or even increase, NH3 transformation rates during cold weather.

Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

BackgroundBiofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycin™, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy.ResultsThe commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg L− 1) and concentration-dependent tobramycin (up to 80 mg L− 1), respectively. Two common bacteriological growth media, tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB), were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin T™ removed 72 h and 168 h P. aeruginosa biofilms after 1 day treatment, while few 72 h S. aureus biofilms survived after 2 days treatment with vancomycin containing OSTEOmycin V™.ConclusionsThis study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin™ products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.

The effect of operational conditions on the disinfection by-products formation potential of exopolymeric substances from biofilms in drinking water

Biofilms are ubiquitous in drinking water systems due to their external matrix of exopolymeric substances (EPS) that provide them protection and adaptability. They are even more common in low flow conditions where hydraulics favor their growth. EPS are organic substances (i.e., proteins, carbohydrates and humic substances) that can react with disinfectant, forming disinfection byproducts (DBP), some of which are controlled by water regulation. However, there is little information available on biofilm-disinfectant interaction and the effect of operational conditions such as biofilm age, water velocity, chlorine and pipeline length on the DBP formation potential of EPS (DBPfpEPS). Using experimental setup and studies of two different biofilms: Biofilm 1 (2.6 ± 0.8 mg Cl/L) and Biofilm 2 (0.7 ± 0.2 mg Cl/L), the DBPfpEPS was studied and compared to the DBPfp of filtered water (FW). The DBP studied were trihalomethanes (THM), haloacetic acids (HAA), haloacetonitriles (HAN), chloropropanones (CP) and chloropicrin (CPK). The DBP concentration trend in both EPS and FW was HAA > THM > CP > HAN > CPK. Biofilm age only increased chloroform (CF)fpEPS in Biofilm 1, while other DBPfpEPS decreased. A direct relationship between water velocity and CFfp in Biofilm 1 was found, probably related to higher chlorine diffusion and the production of a more reactive matrix. Chlorine positively affected DBPfpEPS, increasing Cl-HAA, Cl-THM, CPK and Br-HAN. Biofilm 2 produced higher quantities of EPS per meter of pipeline, this constituting a precursor of intermediary DBP 1,1 dichloropropanone (1,1, DCP). The study compared DBP in chlorinated water in contact with biofilm (BCW) and without (CW). Biofilm 1 increased levels of Cl-HAA, Cl-CP and dichloro-acetonitrile, while Biofilm 2 diminished Cl-HAA and Cl-HAN. Biofilm 1 reduced some Br-HAA in BCW, whereas Biofilm 2 promoted Br-HAA and 1,1, DCP in BCW. EPS and biofilms were significant in terms of their effect on DBP formation.

The Efficacy of Photodynamic Inactivation with Laser Diode on Staphylococcus aureus Biofilm with Various Ages of Biofilm

Biofilms are able to cause microorganisms to be 80% more resistant to antibiotics. The extracelullar polymeric substance (EPS) in biofilm functions to protect bacteria, making it difficult for antibiotics to penetrate the biofilm layer. This study aims to determine the effectiveness of photodynamic inactivation with blue diode laser to reduce Staphylococcus aureus biofilm at various ages of biofilms. The light source is a 403 nm blue diode laser with an energy power of about 27.65±0.01 mW. The study was designed with two groups: Group C was the untreated control group with variations in age of biofilms (0; 6; 11; 17; 24; 32; 40 and 48) hours; Group T was a laser treatment group with variations in age of biofilm and energy density (4.23; 8.46; 12.70; 16.93 and 21.16) J/cm2. Biofilm reduction measurement method using ELISA test was performed to calculate OD595 value. The statistical analysis results of variance showed that there was an influence of biofilm age and irradiation energy density of laser on biofilm reduction. Optical density analysis showed the most optimum biofilm reduction happened when biofilm age is perfectly constructed (about 17 hours) and with 91% reduction. The longer biofilm age lived among those biofilms, the greater the reduction. The results of the Scanning Microscope Electron and fluorescent microscope measurement showed destruction site of the EPS biofilm and bacterial cell death. So, the activated photodynamic with 403 nm laser diode is effective to reduce the Staphylococcus aureus biofilm in the maturation phase.

PH-Responsive Antibacterial Resin Adhesives for Secondary Caries Inhibition

Secondary caries caused by dental plaque is one of the major reasons for the high failure rate of resin composite restoration. Although antimicrobial agent–modified dental restoration systems have been researched for years, few reported intelligent anticaries materials could respond to the change of the oral environment and help keep oral eubiosis. Herein, we report tertiary amine (TA)–modified resin adhesives (TA@RAs) with pH-responsive antibacterial effect to reduce the occurrence of secondary caries. Two kinds of newly designed TA monomers were synthesized: DMAEM (dodecylmethylaminoethyl methacrylate) and HMAEM (hexadecylmethylaminoethyl methacrylate). In the minimum inhibitory concentration and minimum bactericidal concentration test against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii, they exhibited antibacterial effect only in acidic medium, which preliminarily verified the acid-activated effect of TAs. Then DMAEM and HMAEM were incorporated into adhesive resin at the mass fraction of 5%, yielding TA@RAs. In vivo and in vitro tests showed that the mechanical properties and biocompatibility of the adhesive were not affected. A S. mutans biofilm model in acidic and neutral medium was used and confirmed that TA@RAs could respond to the critical pH value of de-/remineralization and acquire reversible antibiofilm effect via the protonation and deprotonation of TAs. Meanwhile, the stability of antibacterial effect was confirmed via a 5-d pH-cycling experiment and a saliva-derived biofilm aging model. Furthermore, 16S rRNA gene sequencing showed that TA@RAs could increase the diversity of the saliva-derived biofilms, which implied that the novel materials could help regulate the microbial community to a healthy one. Finally, an in vitro demineralization model and in vivo secondary caries model were applied and demonstrated that TA@RAs could prevent secondary dental caries effectively. In summary, the reversible pH-responsive and non–drug release antibacterial resin adhesives ingeniously overcome the defect of the present materials and hold great promise for clinical application.

Dynamics of Biocorrosion in Copper Pipes under Actual Drinking Water Conditions

Deficient disinfection systems enable bacteria to form in drinking water; these can invade plumbing systems even if the pipes are composed of antibacterial materials such as copper. Severe copper corrosion by microorganisms and their subsequent release into the water system are evidenced by the blue water phenomenon. Proper monitoring and control can reduce such undesirable effects on water quality. However, a lack of data from analysis under actual conditions has limited the development of useful predictive tools and preventive strategies. In this work, an experimental aging system was connected to a drinking water network affected by the blue water phenomenon. The microbially influenced corrosion (MIC) was evaluated by studying the dynamics of the formed bacterial community and its relationship with copper corrosion and the release of copper. The results suggest that the conformation and composition of the biofilm attached to the surface influence the measured parameters. The corrosion rate was variable throughout the sampling time, with the highest value recorded after one year of aging. The composition of biofilms also changed with time; however, the genus Pseudomonas was ubiquitous over the sampling time. No relationship between the corrosion rate and the biofilm age was observed, thereby suggesting that MIC is a dynamic phenomenon that requires further study.

Association between Genetic Polymorphisms in RANK, RANKL and OPG and Peri-Implant Diseases in Patients from the Amazon Region.

The present study evaluated polymorphisms in RANK, RANKL and OPG-encoding genes to assess whether they are associated with mucositis and peri-implantitis in a population from the Brazilian Amazon region. One hundred and fourteen patients with dental implants were included in the study. After clinical and radiographic examination, the sample was categorized into 4 groups, according to the peri-implant status: Healthy (n=71), Mucositis (n=30), Peri-implantitis (n=13) and Diseased (Mucositis + Peri-implantitis, n=43). Genomic DNA was extracted from buccal cells from saliva, and the genetic polymorphism in osteoprotegerin (OPG), Kappa nuclear factor activator receptor (RANKL) and nuclear kappa factor activator receptor (RANK) were genotyped by the real time PCR. Univariate and multivariate statistical analyses were performed to compare clinical variables among groups and to evaluate genotypes and alleles distributions and the established alpha was 5%. Age, peri-implant biotype, diabetes and presence of peri-implant biofilm were associated with mucositis (p<0.05) and peri-implantitis (p<0.05). Smoking, alcoholism, and periodontal biofilms were also associated with the presence of peri-implantitis (p<0.05). Univariate and multivariate analysis did not demonstrate an association of peri-implantitis or mucositis with any genetic polymorphism in RANK (rs3826620), RANKL (rs9594738) and OPG (rs2073618) (p>0.05). The studied genetic polymorphism in RANK, RANKL and OPG were not associated with mucositis and peri-implantitis in a Brazilian population from the Amazon region.

Combined Effect of Cold Atmospheric Plasma and Hydrogen Peroxide Treatment on Mature Listeria monocytogenes and Salmonella Typhimurium Biofilms.

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials.

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials—borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene—at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points—from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.

Dual-Species Model Biofilm Consisting of Listeria monocytogenes and Salmonella Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP).

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature Listeria monocytogenes and S. Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of L. monocytogenes and S. Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0–30 min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25°C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for S. Typhimurium inactivation when this species was part of the dual-species biofilm, while L. monocytogenes inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.

The influence of substrate surface conditioning and biofilm age on the composition of Enterococcus faecalis biofilms.

To investigate the null hypothesis that neither the surface conditioning (collagen, serum, saliva) of hydroxyapatite (HA) discs, nor the biofilm age (3days vs. 21days) has a significant effect on the cellular and matrix composition of biofilms, using Enterococcus faecalis as the model organism. Sterile HA discs were conditioned with collagen, saliva or serum, and inoculated with E.faecalis to form 3-day and 21-day-old biofilms. Unconditioned discs served as controls. The biofilms were analysed using culture-dependent and independent (confocal microscopy and biochemical analysis) methods, to determine the colony-forming units and the biofilm matrix composition (polysaccharides and proteins), respectively. Statistical analyses were performed using appropriate parametric and nonparametric tests (P=0.05). Collagen conditioning significantly increased the number of CFUs in the 21-day biofilms, compared to the 3-day biofilms (P<0.05). Although the biochemical analysis revealed that surface conditioning had no significant effect on the total carbohydrate content in the 21-day biofilms, confocal microscopic analysis revealed that collagen and saliva conditioning selectively increased the polysaccharide content of 21-day biofilms, compared to the 3-day biofilms (P<0.05). The results of this study raise an important methodological concern that the substrate conditioning substances and biofilm age differentially influence the cellular and extracellular matrix components of E.faecalis biofilms.

Comparison of flow regimes on biocorrosion of steel pipe weldments: Community composition and diversity of biofilms

Worldwide, the corrosion of petroleum pipelines is the cause of severe operational failures and damages to the environment, and the majority of them may have been intensified by microbial activities. However, few scientific publications have investigated the influence of seawater with indigenous microorganisms in internal corrosion of welded joints in long-distance pipelines, which has motivated this study. Here, a combination of culture-based and molecular microbiological methods was applied to measure the microbial abundance, diversity and composition of biofilms formed on two corroded weldments (gas tungsten arc - GTAW and shielded metal arc - SMAW) of an API 5L X65 microalloyed steel under laminar and turbulent flow regimes using seawater from Guanabara Bay (Rio de Janeiro, Brazil). Overall, turbulent flow did not reduce the biomass of sessile microorganisms on welded joints compared to laminar flow. Phylogenetic analysis revealed that the Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla in biofilms attached to GTAW and SMAW joints. At the genus level, all samples showed similar microbial composition with dominance of Desulfovibrio. However, analysis at OTU level revealed that there were specific microorganisms in each corrosive sample, even though they were affiliated to sulfate-reducers. Thus, microbial community compositions were influenced by type of weld, flow regime and biofilm age. The results of our study will be of benefit to the further studies of weld biocorrosion and biofilm ecology within pipelines.

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta).

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by 'selection' to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.