Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner’s 2A (R2A) for 10 days, and were subsequently stored at ?70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10⁵ CFU cm^?2 and 10–14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.     

On-demand release of Candida albicans biofilms from urinary catheters by mechanical surface deformation

Abstract

Candida albicans is a leading cause of catheter-associated urinary tract infections and elimination of these biofilm-based infections without antifungal agents would constitute a significant medical advance. A novel urinary catheter prototype that utilizes on-demand surface deformation is effective at eliminating bacterial biofilms and here the broader applicability of this prototype to remove fungal biofilms has been demonstrated. C. albicans biofilms were debonded from prototypes by selectively inflating four additional intralumens surrounding the main lumen of the catheters to provide the necessary surface strain to remove the adhered biofilm. Deformable catheters eliminated significantly more biofilm than the controls (>90% eliminated vs 10% control; p < 0.001). Mechanical testing revealed that fungal biofilms have an elastic modulus of 45 ± 6.7 kPa with a fracture energy of 0.4–2 J m^?2. This study underscores the potential of mechanical disruption as a materials design strategy to combat fungal device-associated infections.     

Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm

Abstract

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48?h biofilm was reduced by 2.10 log cfu cm^?2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ～2.5 log cfu cm^?2 of viable cells remaining after 105?min cleaning by water at 65?°C, compared to 4.5 log cfu cm^?2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste     

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm^?2) and 3.5?×?10⁵ CFU cm^?2, meaning between 7.0?×?10⁴ and 1.1?×?10⁷ cells cm^?2 have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

Quantifying Salmonella Population Dynamics in Water and Biofilms

Members of the bacterial genus Salmonella are recognized worldwide as major zoonotic pathogens often found to persist in non-enteric environments including heterogeneous aquatic biofilms. In this study, Salmonella isolates that had been detected repeatedly over time in aquatic biofilms at different sites in Spring Lake, San Marcos, Texas, were identified as serovars Give, Thompson, Newport and -:z10:z39. Pathogenicity results from feeding studies with the nematode Caenorhabditis elegans as host confirmed that these strains were pathogenic, with Salmonella-fed C. elegans dying faster (mean survival time between 3 and 4 days) than controls, i.e., Escherichia coli-fed C. elegans (mean survival time of 9.5 days). Cells of these isolates inoculated into water at a density of up to 10⁶?ml^?1 water declined numerically by 3 orders of magnitude within 2 days, reaching the detection limit of our quantitative polymerase chain reaction (qPCR)-based quantification technique (i.e., 10³ cells ml^?1). Similar patterns were obtained for cells in heterogeneous aquatic biofilms developed on tiles and originally free of Salmonella that were kept in the inoculated water. Cell numbers increased during the first days to more than 10⁷ cells cm^?2, and then declined over time. Ten-fold higher cell numbers of Salmonella inoculated into water or into biofilm resulted in similar patterns of population dynamics, though cells in biofilms remained detectable with numbers around 10⁴ cells cm^?2 after 4 weeks. Independent of detectability by qPCR, samples of all treatments harbored viable salmonellae that resembled the inoculated isolates after 4 weeks of incubation. These results demonstrate that pathogenic salmonellae were isolated from heterogeneous aquatic biofilms and that they could persist and stay viable in such biofilms in high numbers for some time.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml^?1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.     

Pyrrolomycins as potential anti-staphylococcal biofilms agents

With the goal of discovering new anti-infective agents active against microbial biofilms, this investigation focused on some natural pyrrolomycins, a family of halogenated pyrrole antibiotics. In this study the anti-staphylococcal biofilm activity of pyrrolomycins C, D, F1, F2a, F2b, F3 and of the synthesized related compounds I, II, III were investigated. The susceptibility of six staphylococcal biofilms was determined by methyltiazotetrazolium staining. Most of the compounds were active at concentrations of 1.5 μg ml^?1 with significant inhibition percentages. A few of the compounds were active at the lowest screening concentration of 0.045 μg ml^?1. The population log reduction of activity against the two best biofilm forming Staphylococcus aureus strains as determined by viable plate counts is also reported. In order to adequately assess the utility of these compounds, their toxicity against human cells was evaluated. It is concluded that pyrrolomycins and synthetic derivatives are promising compounds for developing novel effective chemical countermeasures against staphylococcal biofilms.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l^?1, while the minimum biofilm elimination concentration (MBEC) was 780–3,120 mg l^?1. Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.     

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 μm^–3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 μm^–3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

Thermal dependence of clearance and metabolic rates in slow- and fast-growing spats of manila clam Ruditapes philippinarum

Thermal dependence of clearance rate (CR: l h^?1), standard (SMR: J h^?1) and routine metabolic rates (RMR: J h^?1), were analyzed in fast (F)- and slow (S)-growing juveniles of the clam Ruditapes philippinarum. Physiological rates were measured at the maintenance temperature (17 °C), and compared with measurements performed at 10 and 24 °C after 16 h and 14 days to analyze acute and acclimated responses, respectively. Metabolic rates (both RMR and SMR) differed significantly between F and S seeds, irrespective of temperature. Mass-specific CRs were not different for F and S seeds but were significantly higher in F clams for rates standardized according to allometric size-scaling rules. Acute thermal dependency of CR was equal for F and S clams: mean Q ₁₀ were ≈3 and 2 in temperature ranges of 10–17 and 17–24 °C, respectively. CR did not change after 2 weeks of acclimation to temperatures. Acute thermal effects on SMR were similar in both groups (Q ₁₀ ≈ 1 and 1.6 in temperature ranges of 10–17 and 17–24 °C, respectively). Large differences between groups were found in the acute thermal dependence of RMR: Q ₁₀ in F clams (≈1.2 and 1.9 at temperature ranges of 10–17 and 17–24 °C, respectively) were similar to those found for SMR (Q ₁₀ = 1.0 and 1.7). In contrast, RMR of S clams exhibited maximum thermal dependence (Q ₁₀ = 3.1) at 10–17 °C and become depressed at higher temperatures (Q ₁₀ = 0.9 at 17–24 °C). A recovery of RMR in S clams was recorded upon acclimation to 24 °C. Contrasting metabolic patterns between fast and slow growers are interpreted as a consequence of differential thermal sensitivity of the fraction of metabolism associated to food processing and assimilation.     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Determination of biofilm mechanical properties from tensile tests performed using a micro-cantilever method

Recently, a micro-cantilever method was introduced for measuring the ultimate tensile strength of intact bacterial biofilms. Herein, is reported the analysis of the video files from the testing of a 4-day-old Staphylococcus epidermidis biofilm to determine the elastic modulus, toughness, and failure strain. Elastic modulus (1270±280 Pa) was within the range of previously reported values (17–6000 Pa). The high failure strains (240±16%) indicate the substantial ductility of bacterial biofilms. In addition, the toughness of the biofilm sample was determined from the area under the stress–strain plot (2.8±0.44 kJ m^?3). Thus, it was demonstrated that the micro-cantilever test video files can be used for the determination of other mechanical property parameters besides ultimate tensile strength.     

Tobermolite effects on methane removal activity and microbial community of a lab-scale soil biocover

Three identical lab-scale biocovers were packed with an engineered soil (BC 1), tobermolite only (BC 2), and a mixture of the soil and tobermolite (BC 3), and were operated at an inlet load of 338–400 g-CH₄ m^?2 d^?1 and a space velocity of 0.12 h^?1. The methane removal capacity was 293 ± 47 g-CH₄ m^?2 d^?1 in steady state in the BC 3, which was significantly higher than those in the BC 1 and BC 2 (106 ± 24 and 114 ± 48 g-CH₄ m^?2 d^?1, respectively). Quantitative PCR indicated that bacterial and methanotrophic densities (6.62–6.78 × 10⁷ 16S rDNA gene copy number g-dry sample^?1 and 1.37–2.23 × 10⁷ pmoA gene copy number g-dry sample^?1 in the BC 1 and BC 3, respectively) were significantly higher than those in the BC 2. Ribosomal tag pyrosequencing showed that methanotrophs comprised approximately 60 % of the bacterial community in the BC 2 and BC 3, while they only comprised 43 % in the BC 1. The engineered soil favored the growth of total bacteria including methanotrophs, while the presence of tobermolite enhanced the relative abundance of methanotrophs, resulting in an improved habitat for methanotrophs as well as greater methane mitigation performance in the mixture. Moreover, a batch experiment indicated that the soil and tobermolite mixture could display a stable methane oxidation level over wide temperature (20–40 °C, at least 38 μmol g-dry sample^?1 h^?1) and pH (5–8, at least 61 μmol g-dry sample^?1 h^?1) ranges. In conclusion, the soil and tobermolite mixture is promising for methane mitigation.     

Biofilm formation by the yeast Rhodotorula mucilaginosa: process,repeatability and cell attachment in a continuous biofilm reactor

Yeast biofilms contribute to quality impairment of industrial processes and also play an important role in clinical infections. Little is known about biofilm formation and their treatment. The aim of this study was to establish a multi-layer yeast biofilm model using a modified 3.7 l bench-top bioreactor operated in continuous mode (D = 0.12 h^?1). The repeatability of biofilm formation was tested by comparing five bioprocesses with Rhodotorula mucilaginosa, a strain isolated from washing machines. The amount of biofilm formed after 6 days post inoculation was 83 μg cm^?2 protein, 197 μg cm^?2 polysaccharide and 6.9 × 10⁶ CFU cm^?2 on smooth polypropylene surfaces. Roughening the surface doubled the amount of biofilm but also increased its spatial variability. Plasma modification of polypropylene significantly reduced the hydrophobicity but did not enhance cell attachment. The biofilm formed on polypropylene coupons could be used for sanitation studies.     

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m^?2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m^?2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.     

Exploiting the antivirulence efficacy of an ajoene-ciprofloxacin combination against Pseudomonas aeruginosa biofilm associated murine acute pyelonephritis

The study investigated the in vitro, ex vivo and in vivo efficacy of ajoene and ciprofloxacin (CIP) alone and in combination against Pseudomonas aeruginosa biofilms and biofilm-associated murine acute pyelonephritis. The ajoene–CIP combination exhibited significant greater (p < 0.05) antimotility and biofilm inhibitory effects than those obtained when they were applied individually. The combined action of the agents resulted in a significant increase in serum sensitivity and phagocytic uptake and killing of P. aeruginosa (p < 0.001) compared to the untreated control. Mice groups treated with an ajoene (25 mg kg^?1) and CIP (30 mg kg^?1 or 15 mg kg^?1) combination showed a significantly (p < 0.001) reduced bacterial load in the kidney and bladder as compared to that of infected controls and mice treated with solo agents on the fifth day post-infection. The decreased levels of biomarkers and photomicrographs of the kidney tissue of the treated mice showed a reduced severity of damage. Hence, the study highlights the antivirulent and therapeutic efficacy of the ajoene-CIP combination at the minimal dosage of CIP.     

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

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