Polycarbonate Coupons Research Articles

Mechanical behavior of recyclable polymeric specimens made by additive manufacturing

Additive Manufacturing (AM) has seen a massive growth in engineering applications in recent years. Fused Filament Fabrication (FFF), probably the most widespread technology, is nowadays capable of producing polymeric components for all kinds of need: from common use to advanced engineering applications, thanks to the usage of a wide range of polymer materials. Some of them (such as PEEK, PEI and others so-called ultra-polymers) offer very good mechanical properties and sometimes can be even capable of replacing metals. In addition, they are stable at relatively high temperatures and thermally re-processable and recyclable, showing good biocompatibility in most of cases. The distinct advantages of AM have facilitated the rapid development of polymer products with complex customized structures and functionalities, thereby enhancing their applications in various fields. In this work, the mechanical behaviors of FFF manufactured polycarbonate (PC) and Polymethyl methacrylate (PMMA) coupons, both suitable for building construction applications thanks to their good mechanical strength, thermal insulation capability and resistance to ultraviolet (UV) rays, were preliminarily investigated. The aim was to assess the durability of such materials in order to possibly exploit their potential in substitution to conventional ones, in light of their recyclability, so as to eventually promote circularity.

Evaluation of Listeria monocytogenes biofilms attachment and formation on different surfaces using a CDC biofilm reactor

Listeria monocytogenes can adapt, persist, and form biofilms on food premises surfaces, representing a challenge for food safety, since they led to disease transmission, food contamination and spoilage during production. Physical interventions (scrubbing and wiping) can help controlling formation, nevertheless when biofilms are formed, they are usually very resistant to current control strategies used in the food industry. Biofilm attachment and formation is influenced by environment characteristics, substrate properties and microbial motility. The purpose of this study was to evaluate the ability of L. monocytogenes to attach and form biofilms on different surfaces (wood, nylon, and polycarbonate) representative of the materials used during produce harvesting and storage. Multi-strain L. monocytogenes biofilms were grown in a CDC Biofilm reactor at 20 ± 2 °C up to 96-h and characterized for: a) attachment strength by enumerating cells after rinsing; b) hydrophobicity and interfacial tension by contact angle measurements; c) biofilm architecture by Laser Scanning Confocal Microscopy. All experiments were done in triplicate. Material, incubation, and solvent significantly affected the hydrophobicity and wetting properties of L. monocytogenes biofilms (P < 0.05). The type of material and incubation time significantly influenced hydrophobicity and wetting properties of L. monocytogenes biofilms (P < 0.05). Highest contact angle and lowest interfacial tension were observed on polycarbonate coupons. The data presented contributes to understanding Listeria biofilms grow on different surfaces commonly used in produce harvesting and storage. The data obtained in this study can be used when evaluating intervention strategies to control this pathogen in food premises.

Combined Effects of Sanitizers and UV-C Light on Listeria monocytogenes Biofilm Growth and Survivability on Produce-Harvesting Materials Used in the Tree Fruit Production Industry

Listeria monocytogenes is an aggressive biofilm former that can establish and persist in food processing environments. Commonly associated with ready-to-eat and dairy products, this pathogenic bacterium has recently been increasingly linked to fresh produce outbreaks. Equipment used during harvesting and handling of produce can provide a niche environment for biofilm growth and persistence. Based on a survey conducted among stakeholders in the tree fruit production industry, three favored materials for storing and harvesting produce were identified: nylon, wood, and plastic. The purpose of this study was to investigate the application of the generally recognized as safe sanitizers lactic acid, thymol, and silver citric acid (SDC) and UV-C light alone or in combination for 2 or 5 min on different food-contact surfaces used during tree fruit harvesting and storing. Multistrain L. monocytogenes biofilms were grown in a Centers for Disease Control and Prevention biofilm reactor for 96 h on wood, nylon, and polycarbonate coupons at 20 ± 2°C. After each treatment, coupons were neutralized and the remaining cells were enumerated. Results showed that the most effective treatment was the simultaneous use of UV-C light and SDC (4-log reduction) and that the least effective treatment was UV-C light alone (P &lt; 0.05). The type of material was found to play a significant role in the efficacy of the sanitizers (P &lt; 0.05). This study demonstrates the ability of L. monocytogenes to grow and form biofilms on different surfaces and contributes to an understanding of the response of this food safety threat against antimicrobial intervention strategies.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Mild Positive Pressure Improves the Efficacy of Benzalkonium Chloride against Staphylococcus aureus Biofilm.

Current protocols using liquid disinfectants to disinfect heat-sensitive hospital items frequently fail, as evidenced by the continued isolation of bacteria following decontamination. The contamination is, in part, due to biofilm formation. We hypothesize that mild positive pressure (PP) will disrupt this biofilm structure and improve liquid disinfectant/detergent penetration to biofilm bacteria for improved killing. Staphylococcus aureus biofilm, grown on polycarbonate coupons in the biofilm reactor under shear at 35 °C for 3 days, was treated for 10 min and 60 min with various dilutions of benzalkonium chloride without PP at 1 atmosphere (atm), and with PP at 3, 5, 7, and 10 atm. The effect on biofilm and residual bacterial viability was determined by standard plate counts, confocal laser scanning microscopy, and scanning electron microscopy. Combined use of benzalkonium chloride and PP up to 10 atm significantly increased biofilm killing up to 4.27 logs, as compared to the treatment using disinfectant alone. Microscopy results were consistent with the viability plate count results. PP improved disinfectant efficacy against bacterial biofilm. The use of mild PP is possible in many flow situations or if equipment/contaminated surfaces can be placed in a pressure chamber.

Role of Simulated Body Fluid in Biofilm Formation and Growth on a Polycarbonate Material

Bacteria form biofilms to facilitate colonization, and biofilm formation on polymeric medical devices is a common cause of hospital-acquired infection. Simulated body fluid (SBF) is a supersaturated calcium-phosphate solution with ionic composition nearly equal to that of human blood plasma, and has been used to test the bioactivity of materials. The purpose of this work was to understand whether SBF influenced surface structure on bacterial adhesion and biofilm formation on polycarbonate, a polymer commonly used in medical devices. In this study, polycarbonate coupons were immersed in a SBF solution at 37 oC for 7 and 14 days and air-dried for 30 minutes, and compared with deionized water immersion. Colony biofilms of P. aeruginosa were then investigated by growing bacteria on top surface of immersed coupons for 24 and 48 hours, and observed by the quantitative assay (areal cell density) and visualized using a field emission scanning electron microscopy (FESEM). Results stated that a prolonged immersion time of coupons in deionized water enhanced biofilm growth. Immersion in SBF for a period of 14 days showed a significant reduction in the viability following 24 hours of incubation compared to that in deionized water incubated for 48 hours. FESEM further demonstrated that P. aeruginosa had a tendency to form biofilm on a polycarbonate substrate, and was able to develop biofilms on both the SBF and deionized water. Significant cell clusters and bacterial adhesion was observed at 48-hour incubation. These insights can potentially assist in the establishment of infection and colonization of this opportunistic pathogen, and will aid the development of strategies to prevent biofilm formation.

Evaluation of the Pathogenic-Mixed Biofilm Formation of Pseudomonas aeruginosa/Staphylococcus aureus and Treatment with Limonene on Three Different Materials by a Dynamic Model.

Background: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. Methods: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). Results: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. Conclusions: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Impact of colour on the bioreceptivity of granite to the green alga Apatococcus lobatus: Laboratory and field testing

Recent work has demonstrated that surface colour affects the formation of cyanobacterial subaerial biofilms on polycarbonate coupons and, in turn, influences their bioreceptivity. To explore whether colour is important on other substrates, the influence of colour on the primary bioreceptivity of granite to the terrestrial green alga Apatococcus lobatus (Trebouxiophyceae, Chlorophyta) has been assessed. Two granitoids (Grissal and Rosa Porriño) with the same texture, and very similar chemical composition, open porosity and surface roughness, but different coloration related to feldspars (i.e. greyish and reddish) were used to conduct bioreceptivity studies in parallel field and laboratory tests. Light microscopy, colour spectrophotometry, PAM fluorometry, and optical profilometry were used to evaluate colonisation and its impacts. Short-term results (after 7 and 10 weeks of colonisation by a mono-species biofilm) from both lab and field trials, showed significantly more algae growth on reddish granite (Rosa Porriño) than on greyish granite (Grissal). Also, optical profilometry and light microscopy demonstrated that on both granites algal aggregates developed in hollows. We attribute the roughly double levels of A. lobatus growth on reddish vs greyish granite to differences in the amount of radiant energy absorbed and the higher levels of red wavelength light (known to encourage algal growth) reflected from the reddish surface.

Antibiofilm Efficacy of Peptide 1018 against Listeria monocytogenes and Shiga Toxigenic Escherichia coli on Equipment Surfaces

Listeria monocytogenes and Shiga toxigenic Escherichia coli (STEC) are important foodborne bacterial pathogens that can form biofilms on equipment surfaces at food processing facilities. Pathogens in biofilms are resistant to conventional antimicrobials and require higher antimicrobial concentrations to be inactivated. In this study, the efficacy of a synthetic innate defense regulator peptide 1018 (peptide 1018) for inactivating L. monocytogenes and STEC (O26, O111, O145, O157) biofilms on stainless steel and polycarbonate surfaces was investigated. Stainless steel and polycarbonate coupons (12 mm in diameter) were used in a Centers for Disease Control and Prevention biofilm reactor containing 400 mL of 10% tryptic soy broth (TSB) that had been inoculated with an individual strain of L. monocytogenes or STEC to obtain 6 log CFU/mL populations. The reactor was set with a constant flow rate at 50 mL/h of 10% TSB for 48 h. After 48 h, coupons were treated with peptide 1018 at 0, 10, 20, or 50 μg/mL in phosphate buffer saline (PBS) for 24 h. Surviving bacterial populations were determined by scraping off the coupons and spiral plating on selective media. Significantly higher levels of pathogens in biofilms formed by certain bacterial strains, including L. monocytogenes F6854, E. coli O157:H7 RM4407 and NADC5713, and non-O157 E. coli NADC3629, were recovered on polycarbonate surfaces than on stainless steel. Antibiofilm efficacy of peptide 1018 against pathogens was concentration-dependent and varied with the type of pathogen and material surfaces. Peptide 1018 at 50 μg/mL significantly inactivated all tested bacterial biofilms on both surfaces compared with the PBS control (P < 0.05). L. monocytogenes was the bacterium most sensitive to peptide 1018; on stainless steel surfaces treated with 50 μg/mL peptide 1018, there was a 3.7- to 4.6-log CFU/cm2 reduction in Listeria populations compared with a 1.0- to 3.5-log CFU/cm2 reduction of STEC. Results suggest that peptide 1018 may be used to inactivate L. monocytogenes and STEC biofilms on equipment surfaces.

Microscale and molecular analyses of river biofilm communities treated with microgram levels of cerium oxide nanoparticles indicate limited but significant effects

Cerium oxide (CeO2) nanoparticles are used as in-fuel catalysts and in manufacturing processes, creating a potential for release to aquatic environments. Exposures at 1 and 10 μg/L CeO2-nanoparticles were made to assess effects during the development of river biofilm communities. Scanning transmission x-ray microscopy (STXM) indicated extensive sorption of nanoparticles to the community and co-localization with lipid moieties. Following 8 weeks of development, polycarbonate coupons were removed from the reactors and used for molecular analyses, denaturing gradient gel electrophoresis analysis (DGGE-16S rRNA) and 16S rRNA amplicon sequencing. Microscopic imaging of the biofilm communities (bacterial, photosynthetic biomass, exopolymer composition, thickness, protozoan numbers), as well as carbon substrate utilization fingerprinting was performed. There was a trend toward reduced photosynthetic biomass, but no significant effects of CeO2 exposure were found on photosynthetic and bacterial biomass or biofilm thickness. Sole carbon source utilization analyses indicated increased utilization of 10 carbon sources in the carbohydrate, carboxylic acid and amino acids categories related to CeO2 exposures; however, predominantly, no significant effects (p < 0.05) were detected. Measures of microbial diversity, lectin binding affinities of exopolymeric substances and results of DGGE analyses, indicated significant changes to community composition (p < 0.05) with CeO2 exposure. Increased binding of the lectin Canavalia ensiformis was observed, consistent with changes in bacterial-associated polymers. Whereas, no significant changes were observed in binding to residues associated with algal and cyanobacterial exopolymers. 16S rRNA amplicon sequencing of community DNA indicated changes in diversity and shifts in community composition; however, these did not trend with increasing CeO2 exposure. Counting of protozoans in the biofilm communities indicated no significant effects on this trophic level. Thus, based on biomass and functional measures, CeO2 nanoparticles did not appear to have significant effects; however, there was evidence of selection pressure resulting in significant changes in microbial community composition.

Growth substrate may influence biofilm susceptibility to antibiotics.

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

Transfer of dry surface biofilm in the healthcare environment: the role of healthcare workers' hands as vehicles

Dry surface biofilms (DSBs) persist for extended periods in hospital, and may play a significant role in transmission of healthcare-associated infections. To determine whether DSBs may be transferred from hospital surfaces to healthcare workers' hands. Twelve-day Staphylococcus aureus DSB was grown on polycarbonate and glass coupons in a CDC Biofilm Reactor®. A total of 1.8×106 and 8.8×105 bacteria grew on the polycarbonate and glass coupons respectively. Transmission was tested by lifting the coupon with forefinger and thumb of ungloved hands to a height of 30 cm, then touching horse blood agar (HBA) plates 19 sequential times. Transferred bacterial number was determined by colony-forming units. The effect of DSB wetting on biofilm transfer was tested with 5% neutral detergent treatment for 5 s. Between 5.5 and 6.6% of the DSB bacteria were transferred to hands with one touch and ∼20% were then transferred to HBA with one touch, giving an overall transfer rate of 1.26% and 1.04% for polycarbonate and glass coupons, respectively. Detergent treatment had little effect on bacterial removal from coupons, but, for biofilm grown on polycarbonate, significantly increased transferral to HBA (P<0.001) to 5.2%. Large numbers of bacteria were transferred by bare hands to multiple fomites. One-third of polycarbonate coupons transferred >1000 colonies during the first five sequential touches. Sufficient bacteria to cause infection were transmitted up to 19 times following one touch of the DSB. DSB bacteria are transferred by hands from one fomite to multiple fomites, suggesting that DSB may serve as a persistent environmental source of pathogens.

The Effect of Negative Pressure Wound Therapy with and without Instillation on Mature Biofilms In Vitro.

Background: To investigate the effect of negative pressure wound therapy (NPWT) with and without instillation (NPWTi) on in vitro mature biofilm. Methods: Mature biofilms of Pseudomonas aeruginosa and Staphylococcus aureus were grown under shear (130 rpm) on polycarbonate coupons in a CDC biofilm reactor for 3 days. Coupons containing biofilms were placed in a sterile petri dish and sealed using NPWT or NPWTi. Coupons were exposed to treatment for 24 h with NPWT alone or with instillation of: Povidone iodine solution (PVP-I) (10% w/v equivalent to 1% w/v available iodine, BETADINE®, Mundipharma, Singapore), surfactant based antimicrobial solution with polyhexamethylene biguanide (SBPHMB) (Prontosan®, B Braun Medical, Melsungen, Germany), Gentamicin 1 µg/mL (GM) (G1264 Sigma-Aldrich Pty Ltd., Castle Hill, Australia) Rifampicin 24 µg/mL (RF) (R3501 Sigma-Aldrich Pty Ltd., Castle Hill, Australia) and NaCl 0.9% (Baxter, Deerfield, IL, USA). Bacterial cell viability and biofilm architecture pre-and post-treatment were assessed using colony forming units (cfu), Live/Dead viability staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Results: Significant reductions were obtained in S. aureus biofilm thickness (65%) and mass (47%) when treated with NPWTi as compared to NPWT only. NPWTi with instillation of SBPHMB, PVP-I and RF achieved between 2 and 8 log10 reductions against S. aureus biofilm (p < 0.05–0.001). Conversely, PVP-I and SBMO achieved a 3.5 log10 reduction against P. aeruginosa (p < 0.05). Conclusions: NPWT alters biofilm architecture by reducing biofilm thickness and mass, but this does not affect bacterial cell viability. NPWT with instillation of certain antimicrobials solutions may provide a further synergistic effect in reducing the number of viable biofilm microorganisms. Our in vitro model may be used for screening the effectiveness of antimicrobials used under instillation prior to animal or human studies.

Impact of Zinc Orthophosphate on Simulated Drinking Water Biofilms Influenced by Lead and Copper

AbstractPhosphate, a key nutrient for bacterial growth, is also a key component of many corrosion-control programs to manage lead and copper corrosion in premise plumbing. Bench-scale stagnant water galvanic macrocells with lead and copper components were fed with drinking water containing three levels of zinc orthophosphate [0 (control), 1, and 3 mg l−1-PO4]. Suspended polycarbonate coupons, representing benign downstream fixtures, were placed in the macrocells, thus enabling biofilm formation on this material. Community profiling using denaturing gradient gel electrophoresis (16S rDNA PCR-DGGE) revealed that phosphate dose (primarily) and metal type (to a lesser extent) influenced biofilm community diversity. Generally, community diversity increased with increasing heterotrophic plate counts that in turn rose in response to elevated phosphate. Partial 16s rDNA sequences obtained from DGGE gel bands identified the dominant bacterial taxa as the phyla Verrumicrobia, Firmicutes, Bacteroidetes, and α-Prote...

Effect of High Chloride Concentrations on Microbial Regrowth in Drinking Water Distribution Systems

AbstractThis study was designed to identify the effects of variable concentrations of chloride on microbial regrowth, disinfection efficacy, and metal release in drinking water distribution systems. Annular reactors (ARs) containing cast iron or polycarbonate coupons were operated in this study to simulate distribution systems. Experiments were conducted with test water containing chloride concentrations of 10, 75, and 250 mg/L. Chlorine disinfection was applied to achieve 0.2 and 1.0 mg/L free chlorine residuals. Results showed that the ARs operated with elevated chloride concentrations were not significantly different from the ARs operated with low chloride concentrations in terms of microbial regrowth. However, there was a significant increase in iron concentrations after chlorination in the high chloride ARs. Further, more iron release was observed in these ARs at 1.0 mg/L compared to 0.2 mg/L chlorine residual. Higher chlorine doses were required to achieve goal residuals for those ARs with highe...

A new dry-surface biofilm model: An essential tool for efficacy testing of hospital surface decontamination procedures

The environment has been shown to be a source of pathogens causing infections in hospitalised patients. Incorporation of pathogens into biofilms, contaminating dry hospital surfaces, prolongs their survival and renders them tolerant to normal hospital cleaning and disinfection procedures. Currently there is no standard method for testing efficacy of detergents and disinfectants against biofilm formed on dry surfaces. Aim: The aim of this study was to develop a reproducible method of producing Staphylococcus aureus biofilm with properties similar to those of biofilm obtained from dry hospital clinical surfaces, for use in efficacy testing of decontamination products. The properties (composition, architecture) of model biofilm and biofilm obtained from clinical dry surfaces within an intensive care unit were compared. MethodsThe CDC Biofilm Reactor was adapted to create a dry surface biofilm model. S. aureus ATCC 25923 was grown on polycarbonate coupons. Alternating cycles of dehydration and hydration in tryptone soy broth (TSB) were performed over 12days. Number of biofilm bacteria attached to individual coupons was determined by plate culture and the coefficient of variation (CV%) calculated. The DNA, glycoconjugates and protein content of the biofilm were determined by analysing biofilm stained with SYTO 60, Alexa-488-labelled Aleuria aurantia lectin and SyproOrange respectively using Image J and Imaris software. Biofilm architecture was analysed using live/dead staining and confocal microscopy (CM) and scanning electron microscopy (SEM). Model biofilm was compared to naturally formed biofilm containing S. aureus on dry clinical surfaces. ResultsThe CDC Biofilm reactor reproducibly formed a multi-layered, biofilm containing about 107CFU/coupon embedded in thick extracellular polymeric substances. Within run CV was 9.5% and the between run CV was 10.1%. Protein was the principal component of both the in vitro model biofilm and the biofilms found on clinical surfaces. Continued dehydration and ageing of the model biofilm for 30days increased the % of protein, marginally decreased gylcoconjugate % but reduced extracellular DNA by 2/3. The surface of both model and clinical biofilms was rough reflecting the heterogeneous nature of biofilm formation. The average maximum thickness was 30.74±2.1μm for the in vitro biofilm model and between 24 and 47μm for the clinical biofilms examined. ConclusionThe laboratory developed biofilm was similar to clinical biofilms in architecture and composition. We propose that this method is suitable for evaluating the efficacy of surface cleaners and disinfectants in removing biofilm formed on dry clinical surfaces as both within run and between run variation was low, and the required equipment is easy to use, cheap and readily available.

Use of Fulvic Acid or Sodium Silicate-Based Sanitizers to Inactivate L isteria monocytogenes , S almonella Typhimurium and P seudomonas aeruginosa on Food Contact Surfaces

The objective of this study was to determine the efficacy of five fulvic acid-based and one sodium silicate-based sanitizers against Listeria monocytogenes, Salmonella Typhimurium DT104 and Pseudomonas aeruginosa on food contact surfaces. Minimum inhibitory concentration assays were conducted to determine the lowest effective concentration of sanitizers A, B, C, D, E and F against each bacterium. Stainless steel, high-density polyethylene, polyvinyl chloride and polycarbonate coupons were inoculated with the culture of one of the test organisms and dried for 1 h. The coupons were then sprayed with sanitizer A, B, C, E or F, and treated for 2 min. Samples were taken to enumerate the surviving organisms. The sanitizers reduced L. monocytogenes, S. Typhimurium and P. aeruginosa population by 2–6, 2.7–5.8 and 2–6 log cfu/coupon on food contact surfaces, respectively. In general, the tested sanitizers were the most effective on P. aeruginosa, followed by S. Typhimurium and then L. monocytogenes. Practical Applications Microorganisms attached to food contact surfaces can be a source of contamination of foods, leading to spoilage and food safety issues. Chemical disinfection with sanitizers is generally used to reduce the microbial contamination on food contact surfaces. Fulvic acid or sodium silicate-based sanitizers have been developed for use in food processing plants, but studies on the effects of these new sanitizers on food contact surfaces are limited. Our results demonstrated that sodium silicate-based sanitizer A and fulvic acid-based sanitizers B, C, E and F effectively reduced L. monocytogenes, S. Typhimurium and P. aeruginosa populations on stainless steel, high-density polyethylene, polyvinyl chloride and polycarbonate coupon surfaces. These results may help the food industry in selecting alternative sanitizers for cleaning food contact surfaces.

Evaluation of disinfection efficacy of ozone and chlorinated disinfectant against the biofilm of Klebsiella michiganensis and Pseudomonas aeruginosa

Household microorganisms mostly reside in the form of biofilms on wet surfaces in the bathroom and kitchen areas. Microorganisms constituting biofilm communities are less susceptible than their planktonic counterparts to antimicrobial agents and are thus difficult to control. Traditionally, chemical disinfectants have been used as cleaners in the household environment. Recently, incorporation of ozone as a microbial control agent in the consumer product industry has been initiated as an alternative method of disinfection. In this study, antimicrobial efficacy of ozone and a commercial chlorinated disinfectant was evaluated against the biofilm of a commonly occurring Pseudomonas aeruginosa and Klebsiella michiganensis, a newly described species recovered from toothbrush holder. Single species biofilm was grown on borosilicate glass and polycarbonate coupons using the Centers for Disease Control and Prevention (CDC) biofilm reactor. This is the first study demonstrating the biofilm forming capability of K. michiganensis under laboratory conditions using the CDC biofilm reactor. Planktonic cells and mature biofilms were exposed to ozonated water for 2 and 4 min and chlorinated cleaner for 2 min and 10 min. Based on the ozone stability study, the exposure experiments were carried out in two different ways. The type 1 treatment was carried out according to ASTM method E2871-12, with 5 mL of ozonated water as disinfectant in a 50 mL conical centrifuge tube; whereas in type 2 treatment, 14.0 mL of ozonated water was used to avoid any headspace in the capped culture tube. In biofilms, type 1 ozone treatment demonstrated an average log reduction of 0.88 (±0.13) and 0.12 (±0.01) for K. michiganensis and P. aeruginosa, respectively, in 2 min. Similarly, for type 2 treatment, the values were 1.56 (±0.58) and 0.27 (±0.08). In planktonic cells, log reduction was 2.61 (±0.11) in P. aeruginosa and > 4.0 in K. michiganensis, within 2 min. Chlorinated cleaner demonstrated > 4.0 log reduction in all cases. The disinfection efficacy of ozone type 1 treatment varied between bacterial species (p < 0.001) and exposure times (p < 0.04), but not the coupon types (p > 0.95). Within biofilms, log reduction varied significantly between ozone and chorine treatments (p < 0.001). Significant differences in log reduction were also observed between planktonic cells and biofilms (p < 0.001), target organisms (p < 0.05) and the types of ozone treatment (p < 0.05, for K michiganensis only). From the results, it can be concluded that K. michiganensis was more susceptible to the disinfectants compared to P. aeruginosa. Within a shorter contact time, commercial chlorinated cleaner was more effective against both biofilms and planktonic cells than ozonated water.

Effect of water hardness and pipe material on enhanced disinfection with UV light and chlorine

Limited studies have been conducted to evaluate synergistic benefits between disinfectants, although promise has been shown between ultraviolet (UV) light and chlorine-based disinfectants. This research aimed to determine drinking water quality factors that affect potential for enhanced removal of heterotrophic bacteria due to synergy between UV light and free chlorine. An additional goal was to determine the impact of pipe material on the formation of biofilm and the effectiveness of combined disinfection in controlling it. Annular reactors (ARs) containing polycarbonate or cast iron coupons were used to simulate typical drinking water distribution systems. Two experiments were conducted with untreated hard groundwater and groundwater treated with ion exchange to remove hardness, each comparing chorine alone to chlorine with UV pre-treatment. Results show that enhanced removal of heterotrophic plate count (HPC) bacteria existed with hard water when UV preceded chlorine, but synergistic benefits were not consistently observed. Under softened water conditions, no enhanced reduction or synergy was observed. Cast iron ARs supported biofilm HPC bacteria that were unaffected by any disinfection scheme regardless of source water, while bulk HPC bacteria were more resistant to disinfection in polycarbonate than cast iron ARs. Results indicate that disinfection synergy between UV and chlorine is limited in groundwater sources.

Establishment and early succession of bacterial communities in monochloramine-treated drinking water biofilms

Monochloramine is an increasingly used drinking water disinfectant and has been shown to increase nitrifying bacteria and mycobacteria in drinking waters. The potential successions and development of these bacteria were examined by 16S rRNA gene clone libraries generated from various biofilms within a water distribution system simulator. Biofilms were obtained from in-line and off-line devices using borosilicate glass beads, along with polycarbonate coupons from annular reactors incubated for up to 8months in monochloramine-treated drinking water. No significant difference in community structures was observed between biofilm devices and coupon material; however, all biofilm communities that developed on different devices underwent similar successions over time. Early stages of biofilm formation were dominated by Serratia (29%), Cloacibacterium (23%), Diaphorobacter (16%), and Pseudomonas (7%), while Mycobacterium-like phylotypes were the most predominant populations (>27%) in subsequent months. The development of members of the nontuberculous mycobacteria (NTM) after 3months may impact individuals with predisposing conditions, while nitrifiers (related to Nitrospira moscoviensis and Nitrosospira multiformis) could impact water quality. Overall, 90% of the diversity in all the clone library samples was associated with the phyla Proteobacteria, Actinobacteria, and Bacteroidetes. These results provide an ecological insight into biofilm bacterial successions in monochloramine-treated drinking water.