Ultrastructure of Biofilms Formed by Bacteria from Industrial Processes

Microorganisms exist predominantly as sessile multispecies communities in natural habitats. Most bacterial species can form these matrix-enclosed microbial communities called biofilms. Biofilms occur in a wide range of environments, on every surface with sufficient moisture and nutrients, also on surfaces in industrial settings and engineered water systems. This unwanted biofilm formation on equipment surfaces is called biofouling. Biofouling can significantly decrease equipment performance and lifetime and cause contamination and impaired quality of the industrial product. In this thesis we studied bacterial adherence to abiotic surfaces by using coupons of stainless steel coated or not coated with fluoropolymer or diamond like carbon (DLC). As model organisms we used bacterial isolates from paper machines (Meiothermus silvanus, Pseudoxanthomonas taiwanensis and Deinococcus geothermalis) and also well characterised species isolated from medical implants (Staphylococcus epidermidis). We found that coating of steel surface with these materials reduced its tendency towards biofouling: Fluoropolymer and DLC coatings repelled all four biofilm formers on steel. We found great differences between bacterial species in their preference of surfaces to adhere as well as their ultrastructural details, like number and thickness of adhesion organelles they expressed. These details responded differently towards the different surfaces they adhered to. We further found that biofilms of D. geothermalis formed on titanium dioxide coated coupons of glass, steel and titanium, were effectively removed by photocatalytic action in response to irradiation at 360 nm. However, on non-coated glass or steel surfaces irradiation had no detectable effect on the amount of bacterial biomass. We showed that the adhesion organelles of bacteria on illuminated TiO2 coated coupons were complety destroyed whereas on non-coated coupons they looked intact when observed by microscope. Stainless steel is the most widely used material for industrial process equipments and surfaces. The results in this thesis showed that stainless steel is prone to biofouling by phylogenetically distant bacterial species and that coating of the steel may offer a tool for reduced biofouling of industrial equipment. Photocatalysis, on the other hand, is a potential technique for biofilm removal from surfaces in locations where high level of hygiene is required. Our study of natural biofilms on barley kernel surfaces showed that also there the microbes possessed adhesion organelles visible with electronmicroscope both before and after steeping. The microbial community of dry barley kernels turned into a dense biofilm covered with slimy extracellular polymeric substance (EPS) in the kernels after steeping in water. Steeping is the first step in malting. We also presented evidence showing that certain strains of Lactobacillus plantarum and Wickerhamomyces anomalus, when used as starter cultures in the steeping water, could enter the barley kernel and colonise the tissues of the barley kernel. By use of a starter culture it was possible to reduce the extensive production of EPS, which resulted in a faster filtration of the mash.

Estudo de atributos de virulência e resistência a antimicrobianos em amostras de P. aeruginosa

P. aeruginosa é um importante agente de infecções relacionadas à assistência em saúde. Habitualmente, o estabelecimento de infecções agudas é precedido pela colonização das mucosas dos pacientes. Não se sabe, porém, se os processos infecciosos são causados pelas próprias cepas bacterianas colonizadoras ou por outras com que os pacientes entrem em contato, dotadas ou não de maior potencial de virulência ou de resistência a antimicrobianos que as tornem mais eficientes como agentes infecciosos. Assim, este estudo teve como objetivos i) investigar a existência de potenciais diferenças entre amostras de P. aeruginosa que causaram apenas colonização e aquelas responsáveis por infecção, isoladas de um mesmo paciente, quanto a seus fenótipos de virulência e de não susceptibilidade a antimicrobiamos; ii) pesquisar a existência de associação entre características dos paciente, incluindo o tipo de evolução clínica, com as demais variáveis estudadas. No estudo foram incluídos 21 pacientes que desenvolveram infecção por P. aeruginosa durante sua internação no Centro de Terapia Intensiva do Hospital Universitário Clementino Fraga Filho, entre abril de 2007 e abril de 2008. De cada paciente foram selecionadas duas amostras bacterianas: a primeira isolada durante o episódio de infecção e a amostra colonizadora obtida imediatamente antes da ocorrência da infecção. As amostras selecionadas foram estudadas quanto a i) expressão de três mecanismos de virulência (citotoxicidade, aderência a células epiteliais respiratórias humanas e capacidade de formação de biofilme); ii) presença de genes codificadores das proteínas efetoras do sistema de secreção do tipo 3 (SST3 - exoS, exoT, exoU e exoY); iii) perfil de susceptibilidade a antimicrobianos, iv) perfil de fragmentação do DNA cromossômico por eletroforese em gel de campo pulsado (PFGE). As amostras bacterianas obtidas de infecções agudas foram significativamente mais citotóxicas que aquelas obtidas de colonização. Embora sem diferença estatística, a citotoxicidade das amostras que causaram infecção em pacientes que evoluíram para óbito foi superior à citotoxicidade das amostras de pacientes que sobreviveram. O gene que codifica a toxina ExoU foi detectado em 16 amostras (38%), sendo nove de colonização e sete de infecção. Não houve diferença significativa entre as amostras de colonização e infecção quanto à aderência, produção de biofilme, expressão dos genes do SST3 e não-susceptibilidade às diferentes classes de antimicrobianos. Também não foi encontrada associação entre a não-susceptibilidade à quinolona, ou a outras classes de antimicrobianos, e a presença do gene exoU. As 42 amostras de P. aeruginosa estudadas foram incluídas em 20 genótipos. Em 10 deles foi detectado o gene exoU. Amostras de um mesmo genótipo foram uniformes quanto à expressão dos genes do SST3 e a não-susceptibilidade aos antimicrobianos, mas não quanto às outras variáveis estudadas. Em apenas sete pacientes (33,3%), as amostras de colonização e de infecção pertenciam ao mesmo genótipo. Assim, nesse estudo, o estabelecimento do processo infeccioso resultou não da perda do equilíbrio estabelecido entre os mecanismos de agressão das amostras colonizadoras e os de defesa do hospedeiro e sim da introdução de nova cepa bacteriana no organismo hospedeiro, cepa esta dotada de maior potencial citotóxico.

Novel coatings for hard tissue implants

A novel deposition process named CoBlastTM, based on grit blasting technology, has been used to deposit hydroxyapatite (HA) onto titanium (Ti) metal using a dopant/abrasive regime. The various powders (HA powder, apatitic abrasives) and the treated substrates were characterised for chemical composition, coating coverage, crystallinity and topography including surface roughness. The surface roughness of the HA surfaces could be altered using apatitic abrasives of different particle sizes. Compared to the standard plasma spraying process, the CoBlast surface produced excellent coating adhesion, lower dissolution, higher levels of mechanical and chemical stability in stimulated body fluid (SBF). Enhanced viability of osteoblastic cells was also observed on the CoBlast HA surfaces compared to the microblast and untreated Ti as well as the plasma HA coating. CoBlast offers an alternative to the traditional methods of coating HA implants with added versatility. Apatites substituted with antimicrobial metals can also be deposited to add functionality to HA coatings without cytotoxicty. The potential use of these coatings as an infection preventing strategy for application on hard tissue implants was assessed in vitro and also in vivo. Surface physicochemical properties and morphology were determined in addition to surface cytocompatibility assessments using a MG-63 osteoblast cell line. The antibacterial potential of the immobilised metal ion on the surface and the eluted ion to a lesser extent, contributed to the anticolonising behaviour of the surfaces against a standard bacteria strain (S. aureus) as well as a number of clinically relevant strains (MRSA, MSSA and S. epidermis). The results revealed that the surfaces coated with silver substituted apatites (AgA) outperformed the other apatites examined (apatites loaded with Zn, Sr and both Ag and Sr ions). Assessment of bacterial adherence on coated K-wires following subcutaneous implantation in a nude mouse infection model (S. aureus) for two days demonstrated that the 12% wt surface outperformed the 5% wt AgA coating. Lower inflammatory responses were activated with the insertion of the Ag loaded K-wires with a localised infection at the implantation site noted over the two day study period. These results indicated that the AgA coating on the surface of orthopaedic implants demonstrate good biocompatibility whilst inhibiting bacterial adhesion and colonising of the implant surface.

Evaluation of a poly(vinyl pyrollidone)-coated biomaterial for urological use

The associated problems of bacterial biofilm formation and encrustation that may cause obstruction or blockage of urethral catheters and ureteral stents often hinders the effective use of biomaterials within the urinary tract. In this in vitro study, we have investigated the surface properties of a hydrophilic polyvinyl pyrollidone) (PVP)-coating applied to polyurethane and determined its suitability for use as a urinary tract biomaterial by comparing its lubricity and ability to resist bacterial adherence and encrustation with that of uncoated polyurethane and silicone. The PVP-coated polyurethane was significantly more hydrophilic and more lubricious than either uncoated polyurethane or silicone. Adherence of a hydrophilic Escherichia coli isolate to PVP-coated polyurethane and uncoated polyurethane was similar but significantly less than adherence to silicone. Adherence of a hydrophobic Enterococcus faecalis isolate to PVP-coated polyurethane and silicone was similar but was significantly less than adherence to uncoated polyurethane. Struvite encrustation was similar on the PVP-coated polyurethane and silicone but significantly less than on uncoated polyurethane. Furthermore, hydroxyapatite encrustation was significantly less on the PVP-coated polyurethane than on either uncoated polyurethane or silicone. The results suggest that the PVP-coating could be useful in preventing complications caused by bacterial biofilm formation and the deposition of encrustation on biomaterials implanted in the urinary tract and, therefore, warrants further evaluation. (C) 2002 Elsevier Science Ltd. All rights reserved.

Persistence of antimicrobial activity through sustained release of triclosan from pegylated silicone elastomers

Microbial adhesion to silicone elastomer biomaterials is a major problem often resulting in infection and medical device failure. Several strategies have been employed to modulate eukaryotic cell adhesion and to hamper bacterial adherence to polymeric biomaterials. Chemical modification of the surface by grafting of polyethylene glycol (PEG) chains or the incorporation of non-antibiotic antimicrobial agents such as triclosan into the biomaterial matrix may reduce bacterial adhesion. Here, such strategies are simultaneously applied to the preparation of both condensation-cure and addition-cure silicone elastomer systems, seeking a sustained release antimicrobial device biomaterial. The influence of triclosan incorporation and degree of pegylation on antimicrobial release, surface microbial adherence and persistence (Escherichia coli and Staphylococcus epidermidis) were evaluated in vitro. Non-pegylated silicone elastomers provided an increased percentage release of triclosan extending over a relatively short duration (99% release by day 64) compared with their pegylated (4% w/w) counterparts (65% and 72% release by day 64, for condensation and addition-cure systems respectively). Viable E. coli adherence to a non-pegylated silicone elastomer containing 1% w/w triclosan was reduced by over 99% after 24 h compared to the non-pegylated silicone elastomer containing no triclosan. No viable S. epidermidis adhered to any of the triclosan-loaded (>0.1% w/w) formulations other than the control. Persistence of the antimicrobial activity of the triclosan-loaded pegylated silicone elastomers continued for at least 70 days compared to the triclosan-loaded non-pegylated elastomers (at least 49 days). Understanding how PEG affects the release of triclosan from silicone elastomers may prove useful in the development of a biomaterial providing prolonged, effective antimicrobial activity.

Reduction of Staphylococcus aureus and Pseudomonas aeruginosa colonisation on PVC through covalent surface attachment of fluorinated thiols

OBJECTIVES: This study reports the development, characterisation and microbiological testing of surface-modified polyvinylchloride (PVC) films for the purpose of reducing bacterial adherence.

METHODS: Irreversible covalent surface modification was achieved via nucleophilic substitution of fluorinated thiol-terminated compounds onto the polymer backbone. Four fluorinated modifiers, 2,3,5,6-tetrafluorothiophenol (TFTP), 4-(trifluoromethyl)thiophenol (TFMTP), 3,5-bis(trifluoromethyl)benzenethiol (BTFMBT) and 3,3,4,4,5,5,6,6,7, 7,8,8,9,9,10,10,10-heptadecafluoro-decane-1-thiol (HDFDT), were investigated. Modification was confirmed using attenuated total reflectance infrared spectroscopy; Raman mapping demonstrated that modification was homogenous on the macroscopic scale. The influence of fluorination on surface hydrophobicity was studied by contact angle analysis. The effect on microbial adherence was examined using Pseudomonas aeruginosa and Staphylococcus aureus.

KEY FINDINGS: The resultant changes in contact angle relative to control PVC ranged from -4 degrees to +14 degrees . In all cases, adherence of P. aeruginosa and S. aureus was significantly reduced relative to control PVC, with adherence levels ranging from 62% and 51% for TFTP-modified PVC to 32% and 7% for TFMTP-modified PVC.

CONCLUSIONS: These results demonstrate an important method in reducing the incidence of bacterial infection in PVC medical devices without compromising mechanical properties.

In vitro testing of chitosan in gentamicin-loaded bone cement No antimicrobial effect and reduced mechanical performance

Background and purpose Efforts to prevent infection of arthroplasties, including the use of antibiotic-loaded bone cement, are not always successful. We investigated whether the incorporation of chitosan in gentamicin-loaded bone cement increases antibiotic release, and prevents bacterial adherence and biofilm formation by clinical isolates of Staphylococcus spp. In addition, we performed mechanical and degradation tests.

Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

THE EFFECTS OF 3 NON-ANTIBIOTIC, ANTIMICROBIAL AGENTS ON THE SURFACE HYDROPHOBICITY OF CERTAIN MICROORGANISMS EVALUATED BY DIFFERENT METHODS

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.

Photodynamic antimicrobial polymers for infection control

Hospital-acquired infections pose both a major risk to patient wellbeing and an economic burden on global healthcare systems, with the problem compounded by the emergence of multidrug resistant and biocide tolerant bacterial pathogens. Many inanimate surfaces can act as a reservoir for infection, and adequate disinfection is difficult to achieve and requires direct intervention. In this study we demonstrate the preparation and performance of materials with inherent photodynamic, surface-active, persistent antimicrobial properties through the incorporation of photosensitizers into high density poly(ethylene) (HDPE) using hot-melt extrusion, which require no external intervention except a source of visible light. Our aim is to prevent bacterial adherence to these surfaces and eliminate them as reservoirs of nosocomial pathogens, thus presenting a valuable advance in infection control. A two-layer system with one layer comprising photosensitizer-incorporated HDPE, and one layer comprising HDPE alone is also described to demonstrate the versatility of our approach. The photosensitizer-incorporated materials are capable of reducing the adherence of viable bacteria by up to 3.62 Log colony forming units (CFU) per square centimeter of material surface for methicillin resistant Staphylococcus aureus (MRSA), and by up to 1.51 Log CFU/cm2 for Escherichia coli. Potential applications for the technology are in antimicrobial coatings for, or materials comprising objects, such as tubing, collection bags, handrails, finger-plates on hospital doors, or medical equipment found in the healthcare setting.

Semi-interpenetrating polymer networks incorporating polygalacturonic acid for medical device applications

Background Over 20 million people in the US are living with an implantable medical device [ADDIN RW.CITE{{3114 Higgins,DavidM 2009}}1], with similar figures anticipated for Europe. Complications in the use of medical implants include the Foreign Body Response (FBR) characterised by macrophage adherence and fusion, and device-related infection due to bacterial biofilm formationADDIN RW.CITE{{3124 Harding,JacquelineL 2014}}2. Both can have detrimental consequences on the structural and functional integrity of the medical device [ADDIN RW.CITE{{3101 Anderson,JamesM 2008; 3124 Harding,JacquelineL 2014}}2,3], often necessitating removal; a painful and expensive procedure [ADDIN RW.CITE{{3121 Mah,Thien-FahC 2001}}4]. Materials are sought to attenuate both the FBR and device-related infection, leading to medical devices with improved biocompatibility and performance. Objectives The present work involves development of a semi-interpenetrating network (SIPN) hydrogel containing polygalacturonic acid (PGA), a biopolysaccharide similar in structure to hyaluronic acid. We aim to synthesise, characterise and determine the in vitro biocompatibility of the developed SIPN. Results & Discussion We have successfully incorporated PGA into a poly(HEMA) based hydrogel, which shows favourable swelling and wettability. The surface topography appears altered in comparison to the control material, with pronounced micrometer-scale features. In terms of in vitro performance, the SIPN showed increased protein adsorption, and biofilm formation (Staphylococcus epidermidis and Escherichia coli, up to 1 Log CFU/sample greater than control). However the SIPN displayed minimal cytotoxicity towards L929 fibroblasts, and was resistant to the adherence of RAW 264.7 macrophages. Conclusions The PGA incorporated SIPN lacks cytotoxicity and shows reduced macrophage adherence, however the increased biofilm formation highlights a concern regarding possible device related infection in clinical use. Future work will focus on strategies to reduce bacterial adherence, while maintaining biocompatibility.

Caractérisation des souches de Salmonella Typhimurium responsables de septicémies chez le porc

Depuis quelques années, il y a émergence de souches de Salmonella enterica sérovar Typhimurium multirésistantes causant une septicémie et la mort chez le porc. Ceci constitue un problème majeur pour l’industrie porcine et possiblement pour la santé publique. L’objectif de ce projet était de comparer et de caractériser une souche capable de causer une septicémie chez le porc et une souche commensale, en observant l’interaction avec des cellules épithéliales, des macrophages humains et d’identifier des gènes exprimés par les souches septicémiques et les souches commensales. Tout d’abord, l’infection de cellules épithéliales permet d’observer l’adhérence et l’invasion des bactéries, pour ainsi mettre en évidence la capacité des souches à coloniser le tractus gastro-intestinal. La souche commensale possède un pouvoir d’adhésion supérieur à la souche septicémique. Par la suite, l’infection de macrophages permet de caractériser le niveau de phagocytose et de survie. L’importance de la survie dans les macrophages pourrait permettre de faire un lien avec la septicémie. Toutefois, aucune différence n’est observable dans les conditions qui ont été testé. Ensuite, la technique SCOTS (Selective Capture of Transcribed Sequences) est utilisée pour capturer des gènes uniques à la souche septicémique et un autre SCOTS est fait pour capturer les gènes spécifiques à la souche commensale. Finalement, les gènes sont clonés, leur spécificité face aux souches est analysé par dot blot et ils sont identifiés par séquençage suivient d’une analyses bioinformatiques. Les gènes identifiés par SCOTS, lors des captures pour la souche septicémique et la souche commensale, se trouvent à être des gènes communs aux Salmonella. Toutefois, la différence de pathologie causée par les deux souches, n’est peut-être pas l’acquisition de nouveaux gènes, mais plutôt une différence d’expression entre les deux souches.

Évaluation in vitro de la stérilisation au peroxyde d'hydrogène sur les propriétés biologiques de prothèses utilisées lors de stabilisation du genou chez le chien

Objectif—Comparer les effets de la stérilisation au plasma de gaz de peroxyde d’hydrogène (HPGP) à l’oxyde d’éthylène (EO) et à la vapeur (ST) sur les propriétés physico-chimiques et d’adhésion bactérienne de fils de nylon et de polyéthylène. Design expérimental—Etude in vitro. Matériel—Des brins non stérilisés, stérilisés au HPGP, à l’EO et ST; de fil nylon leader (FNL), de fil de nylon pêche (FNP) et de fil de polyéthylène (PE) ont été utilisés. Méthodes—Une analyse de surface au spectroscope photo-électronique à rayons X (XPS), une mesure de l’angle de contact, une analyse par microscopie à force atomique (AFM) et l’adhésion bactérienne de Staphylococcus intermedius et d’Escherichia Coli ont été testés sur les brins. Résultats—Une oxydation de la surface de tous les échantillons stérilisés a été observée quelque soit la méthode de stérilisation. La stérilisation a augmenté significativement l’angle de contact pour tous les types de fil quelque soit la méthode. La rugosité n’a pas été affectée significativement par la méthode de stérilisation pour le FNL et FNP. L’adhésion bactérienne a été affectée significativement par la méthode de stérilisation. Le PE a un angle de contact, une rugosité et une adhésion bactérienne significativement plus élevée que le FNL et FNP, peu importe la méthode de stérilisation. Conclusion—La stérilisation au HPGP constitue une alternative intéressante à la vapeur et l’EO. Le PE n’est peut être pas un matériel idéal par sa capacité d’adhésion bactérienne. De futures études sont nécessaires pour déterminer la signification clinique de ces trouvailles.

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV preinfection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (,1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon c. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.

Streptococcus mutans Biofilm Adhesion on Composite Resin Surfaces After Different Finishing and Polishing Techniques

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)