Doctor of Philosophy / Department of Biology / Lynn Hancock / Enterococci are the third leading cause of nosocomial infections including urinary tract infections (UTI), surgical site infections (SSI) and blood stream infections. Enterococci are also found in the gastrointestinal tracts of humans, and other mammals. We elucidated the influence of the transcriptional regulator RpoN on enterococcal biofilm formation, virulence potential and cell wall architecture and proposed a potential involvement for carbohydrate metabolism in these processes. Biofilms are held together by matrix (BM) components such as extracellular DNA (eDNA) released by cell death from a sub-population of cells. The rpoN mutant (ΔrpoN) was resistant to autolysis as well as fratricide-mediated cell death and eDNA was not detected in planktonic as well as biofilm cultures. Unlike the parental strain V583, the ΔrpoN mutant formed proteinase K sensitive biofilms, suggesting that protein as well as eDNA serves as an important matrix component. The rabbit model of endocarditis was used to assess the effect of rpoN deletion on enterococcal virulence. Rabbits infected with ΔrpoN had reduced bacterial burden in heart, blood, liver, kidney and vegetation in comparison to the parental strain. The growth defect of ΔrpoN in physiologically relevant glucose levels (5 mM) partially explains the reduced bacterial burdens observed in the virulence study. Microarray analysis of ΔrpoN showed that 10% of the genome is differentially regulated by RpoN. Deletion of rpoN also protects Enterococcus faecalis from lysis in the absence of known modulators of cellular lytic events such as O-acetylation and D-alanylation. Of the four identified enhancer binding proteins in E. faecalis, MptR regulates the RpoN-dependent mannose/glucose uptake system (MptABCD) and the ΔmptR mutant phenocopied the ΔrpoN mutant in the eDNA release and growth assays. Because MptC and MptD have been shown to be the cellular receptors for class IIa and IIc bacteriocins, we are presently testing the hypothesis that these receptors may serve as a global receptor for bacteriocins. In conclusion, our data demonstrates that alterations in the metabolic state of the bacterium, as observed in the ΔrpoN mutant could be responsible for the switch in biofilm matrix composition, and this switch in turn likely influences the virulence potential of the bacterium.

Cystic Fibrosis (CF) is the most common hereditary genetic disorder among Caucasians. Pseudomonas aeruginosa is a major cause of morbidity in cystic fibrosis patients. Chronic infection with P. aeruginosa eventually occurs and is associated with a switch to biofilm formation of the bacteria. The symptoms and pathology of acute and chronic P. aeruginosa infections differ greatly. The first line of defense within the lung is the physical barrier of the lung epithelia. The examination of established biofilm interactions with lung epithelia is difficult. Here, I use the Calgary Biofilm Device co-culture system to conduct the concurrent analysis of established biofilms and planktonic bacteria with A549 lung cells. Comparison of P. aeruginosa biofilm and planktonic bacteria’s effects on A549 lung cells showed that planktonic bacteria caused more A549 cell rounding and death, while biofilm stimulated more IL-8 release by epithelial cells. Biofilm was shown to secrete significantly more Pseudomonal Elastase than planktonic, causing A549 morphological changes and loss of tight junctions. The antimicrobial peptide LL-37 was shown to differentially affect biofilm and planktonic bacteria. LL-37 caused a decrease in twitching of planktonic bacteria and exposure to LL-37 for 48 hours resulted in a decrease in elastase secretion likely due to down-regulated type 2 secretion. When established biofilms were compared with newly adherent biofilms, young biofilms were shown to have characteristics similar to both planktonic bacteria and mature biofilms. From this data we can follow the pattern of bacterial virulence as P. aeruginosa transitions from the planktonic mode of growth to the eventual mature biofilm that is associated with chronic infection. In conclusion, this study provides the foundation for a co-culture system that can be used to study the host-pathogen interactions of mammalian epithelia with established P. aeruginosa biofilms. The future adaptations of this model will better represent the in vivo characteristics of chronic lung infection to delineate ongoing virulence mechanisms of the bacteria causing host cell stimulation and damage. / May 2016

O biofilme é um dos fatores primários para o desenvolvimento da gengivite, periodontite e outras alterações na saúde gengival do paciente. O tratamento ortodôntico é um fator predisponente para adesão de microrganismos deixando o paciente susceptível ao maior acúmulo de biofilme e, consequentemente, às doenças periodontais. Assim, o objetivo do presente estudo foi avaliar as alterações clínicas, microbiológicas e imunológicas no paciente em tratamento ortodôntico corretivo e testar as hipóteses nulas: 1 - não há diferença nos parâmetros clínicos, microbiológicos e imunológicos antes e durante o tratamento ortodôntico, 2 não há correlação entre os índices clínicos e imunológicos. Em 28 pacientes com necessidade de tratamento ortodôntico corretivo foram avaliados parâmetros clínicos como índice de Placa (IP), índice de Sangramento (IS), largura de gengiva queratinizada (LGQ); parâmetros microbiológicos por meio da contagem de 40 espécies subgengivais em amostras de biofilme (Checkerboard DNA-DNA Hybridization) e avaliação imunológica por meio da expressão dos níveis de Interleucina-1 β (IL-1β ), metaloproteínase da matriz-8 (MMP-8) e Fator de Necrose Tumoral (TNF-α) no Fluído Crevicular Gengival (FCG) (Luminex). Foram realizadas coletas em 3 momentos: T0 = antes da colocação do aparelho; T1 = 6 meses após e T2= 12 meses após o início do tratamento. Para as análises clínicas e microbiológicas foram aplicados teste de Friedman e Wilcoxon com correção de Bonferroni e para a análise imunológica, Análise de variância para medidas repetidas. Não foi observada diferença estatisticamente significante para a LGQ. IP apresentou aumento, sendo estatisticamente significante em T1 (70,58±28,56) e T2 (83,23±12,30) quando comparados ao T0 (24,44±11,56). O IS apresentou aumento estatisticamente significante em T1 (7,97±5,04) e diminuição em T2 (6,20±4,09), demonstrando valores sem diferença estatisticamente significante a T0 (4,54±4,98). Na análise microbiológica, dentre os seis complexos analisados, apenas o complexo vermelho apresentou frequência significativamente maior (p=0,001) em T2. Não houve diferença estatisticamente significante nos valores dos níveis das citocinas avaliadas, entre todos os tempos (p>0,05), porém em T2 houve correlação positiva moderada entre IS e IL-1β (r=0,49 p=0,01) e TNF-α (r=0,39 e p=0,05). As hipóteses nulas foram rejeitadas. O tratamento ortodôntico corretivo causou alterações periodontais clínicas com relação ao acúmulo de biofilme e sangramento gengival, alterações de bactérias periodontopatogênicas, além de gengivite após 6 meses de tratamento / The biofilm is one of the primary factors for the gingivitis and periodontitis development and changes in periodontal health. Orthodontic treatment is a predisposing factor for microorganisms adhesion leaving the patient susceptible to greater biofilm accumulation and, consequently, to periodontal diseases. Therefore, the study aimed to analyze clinical, microbiological and immunological parameters in patients in orthodontic treatment. The null hypothesis tested were: 1 - there is no difference in clinical, microbiologica and immunological parameters before and during orthodontic treatment; 2 there is no correlation between clinical and immunological parameters. In twenty-eight patients with corrective orthodontic treatment were evaluated clinical parameters, such as Plaque index (PI), Bleeding on Probing (BOP) and width of keratinized gingiva; microbiological parameters with counting of 40 subgingival species with Checkerboard DNA-DNA hybridization and immunological evaluation of cytokines levels IL-1β , MMP-8 and Tumor Necrosis Factor (TNF-α) in Gingival Crevicular Fluid (GCF). Samples were collected in three times: T0 = before orthodontic treatment; T1 = 6 months after and T2 = 12 months after starting orthodontic treatment. Data from clinical and microbiological evaluation were statistically analyzed with Friedman and Wilcoxon tests with Bonferroni correction and for the immunological analysis, Analysis of variance for repeated measures were applied. No significant difference was found for the width of keratinized gingiva. PI presented an increase, being statistically significant at T1 (70.58±28.56) and T2 (83.23±12.30) when compared with T0 (24.44±11.56). The BOP showed a statistically significant increase at T1 (7.97±5.04), however, at T2 (6.20±4.09) the values decrease with no statistically significant difference with T0 (4.54±4.98). In the microbiological analysis, the red complex showed significantly greater frequency (p=0.01) at T2. There was no statistically significant difference in the cytokine levels between the times, but there was a positive moderate correlation between BOP and IL-1β (r=0.49 p=0.01) and TNF-α (r=0.39 e p=0.05). The null hypothesis were rejected. Corrective orthodontic treatment caused clinical periodontal changes regarding biofilm accumulation and gingival bleeding, alterations of periodontopathogens, besides gingivitis after 6 months of treatment

No description available.

O objetivo deste trabalho é estudar o impacto dos processos erosivo, abrasivo e erosivo/abrasivo sobre a camada de glaze aplicada em materiais restauradores indiretos produzidos com tecnologia CAD/CAM quanto à rugosidade superficial (Ra), à perda de superfície (PS), à topografia superficial (T) e à deposição de biofilme (DB). Este estudo teve como fatores de variação: material restaurador [LuxaCam Zircon HT - Plus (LC) e IPS e.max CAD (IPS)] e tratamento de superfície [Sinterização (S), Glaze (G), Erosão (E), Abrasão (A), Erosão/Abrasão (EA)]. Foram produzidos espécimes de 6mm x 7 mm x 1,3mm, divididos em 10 grupos para cada resposta. Para as respostas Ra e PS (n=10) foi utilizada perfilometria óptica, já para a resposta T foi utilizado MEV e para DB, ensaio microbiológico (n=3 e n=5, respectivamente). O protocolo de erosão consistiu na imersão de espécimes em 5ml de HCl 0,06M, pH 1,2, por 30 horas a 37ºC. O protocolo abrasivo foi realizado com máquina de escovação, escovas de cerdas macias e slurry pasta dentifrícia/água destilada 1:2. Foram realizados 400.000 ciclos com carga de 200 gramas. O protocolo erosivo/abrasivo consistiu na combinação dos dois protocolos anteriores. Para DB foi acrescentada a variável tempo de leitura (5 e 24 horas). Para este ensaio foi utilizada a cepa de Streptococcus mutans UA159, cultivada em TSB suplementado com 1,7% de sacarose, usando o método indireto de coloração por safranina e leitura de absorbância. Os dados foram submetidos à análise de variância (ANOVA) e teste de Tukey (p<0,05). Quanto ao Ra, LC apresentou maior Ra do que IPS (p<0,05) para todos os grupos testados. O desafio E diminuiu o Ra do glaze (p<0,05), enquanto A e EA o aumentaram (p<0,05). Quanto à PS, LC apresentou PS maior que IPS (P=0,03). Os desafios A e EA geraram maior PS do que E (p=0,00). Para DB, não houve diferença para nenhum fator após 5 horas (p>0,05). Para 24 horas, LC apresentou maior DB que IPS (P=0,00) e os desafios A e EA geraram maior DB (p=0,01) que E. A DB após 5 horas foi maior do que após 24 horas (p=0,02). Todas as propriedades da camada de glaze testadas foram alteradas pelos desafios de superfície, porém a camada de glaze aplicada sobre zircônia foi mais suscetível a estas. A maior rugosidade da camada de glaze levou à maior deposição de biofilme. / The aim of this study is to evaluate the impact of erosive, abrasive and erosive / abrasive challenges on the glaze layer applied on indirect restorative materials produced with CAD/CAM technology for surface roughness (Ra), surface loss (SL), surface topography (ST) and biofilm deposition (BD). This study had as factors of variation: restoration material [LuxaCam Zircon HT - Plus (LC) and IPS e.max CAD (IPS)] and surface treatment [Sintering (S), Glaze (G), Erosion (E), Abrasion (A), Erosion / Abrasion (EA)]. Specimens of 6mm x 7mm x 1.3mm were produced and divided into 10 groups for each response. For Ra and SL responses (n = 10) was used optical profilometry, for ST was used SEM (n=3) and for BD was used microbiological assay (n = 5). The erosion protocol consisted of immersing specimens in 5ml of 0.06M HCl, pH 1.2, for 30 hours at 37ºC. The abrasive protocol was performed with brushing machine, soft bristle brushes and slurry toothpaste /distilled water 1: 2. 400.000 cycles were performed with a load of 200 grams. The erosive / abrasive protocol consisted of a combination of the two previous protocols. For BD the variable reading time was added (after 5 and 24 hours). Strains of Streptococcus mutans UA159, cultivated in TSB supplemented with 1.7% of sucrose, were used by the indirect method of staining with safranin and reading of absorbance. Data were submitted to analysis of variance (ANOVA) and Tukey test (p <0.05). Regarding Ra, LC presented higher Ra than IPS (p <0.05) for all groups tested. E decreased glaze Ra (p <0.05), while A and AE increased it (p <0.05). Regarding SL, LC presented SL higher than IPS (P = 0.03). A and EA challenges generated higher SL than E (p = 0.00). For BD, there was no difference for any factor after 5 hours (p> 0.05). For 24 hours, LC presented higher BD than IPS (P = 0.00) and A and EA challenges generated higher BD (p = 0.01) than E. BD after 5 hours was higher than after 24 hours (p = 0.02). All the glaze layer properties tested were altered by the surface challenges, however the glaze layer applied on zirconia was more susceptible to these. Greater roughness of the glaze layer led to greater biofilm deposition.

Staphylococcus aureus und Staphylococcus epidermidis gehören zu den häufigsten Erregern nosokomialer Infektionen bei immunsupprimierten Patienten. Gleichzeitig bilden diese Bakterien einen wesentlichen Teil der gesunden Hautflora des Menschen. Bisher ist wenig darüber bekannt, ob es Unterschiede in der genetischen Ausstattung zwischen klinischen und kommensalen Isolaten gibt und welche Faktoren zur Etablierung von Staphylokokken im Hospitalmilieu beitragen. Die Ergebnisse der vorliegenden Arbeit zeigen, daß die Fähigkeit zur Biofilmbildung offensichtlich ein wesentliches Merkmal pathogener Staphylokokken ist. Die Expression dieses Virulenzfaktors ist dabei hochvariabel und hängt von der genetischen Ausstattung der Stämme mit dem für die Biofilmbildung verantwortlichen ica-Operon, bestimmten Umweltfaktoren und dem Einfluß von Insertionssequenzen ab. In einer epidemiologische Untersuchung wurde gezeigt, daß in S. epidermidis das ica-Operon häufiger in klinischen als in kommensalen Stämmen vorkommt. Der überwiegende Teil dieser ica-positiven Stämme bildete phänotypisch einen Biofilm aus. Im Unterschied dazu enthielten alle untersuchten S. aureus-Stämme, unabhängig von ihrer Herkunft, das vollständige ica-Gencluster, wobei jedoch keiner dieser Stämme unter Laborbedingungen einen Biofilm bildete. Durch subinhibitorischen Konzentrationen bestimmter Antibiotika bzw. durch Osmostress ließ sich die Biofilmbildung in 30 Prozent der S. aureus-Stämme induzieren. Ebenso konnte in ica-positiven S. epidermidis-Stämmen die Biofilmbildung dirch diese Umweltfaktoren stimuliert werden. Die Studie ergab auch, daß es einen Zusammenhang zwischen der Biofilmbildung, der Antibiotikaresistenz und dem Vorkommen der Insertionssequenz IS256 gibt. So war IS256 signifikant häufig in klinischen S. epidermidis und S. aureus-Stämmen nachweisbar, während es keinen Unterschied im Auftreten von IS257 zwischen klinischen und saprophytären Isolaten gab. Die IS256-positiven S. epidermidis-Stämme wiesen überdurchschnittlich oft das ica-Operon auf und waren gegen mindestens zwei Antibiotika gleichzeitig resistent. Weiterhin konnte gezeigt werden, daß IS256 an der Phasenvariation der Biofilmbildung in vivo beteiligt ist. Bei einem klinischen S. epidermidis-Stamm, der von einem Patienten mit einer Katheter-assoziierten Harnwegsinfektion isoliert wurde, wurde die Insertion des Elementes im icaC-Gen nachgewiesen, was in einem Biofilm-negativen Phänotyp resultierte. Subkultivierung der Insertionsmutante führte nach wenigen Passagen zur Ausbildung eines Biofilms. Die Nukleotidsequenzierung ergab die vollständige Exzision von IS256 aus dem icaC-Gen einschließlich der duplizierten Zielsequenz von sieben Basenpaaren. Diese Daten stimmen vollständig mit den zuvor in einer in-vitro-Studie erhaltenenen Ergebnissen überein und sie zeigen, daß IS256 die Expression des ica-Operons offensichtlich auch in vivo während einer Infektion beeinflußt. Bei S. aureus konnte in dieser Arbeit ebenfalls eine Phasenvariation der Biofilmexpression nachgewiesen werden. Durch Mehrfachpassagen wurden aus ehemals Biofilm-negativen Einzelkolonien mehrere Biofilmproduzenten gewonnen, die auch wieder zum Biofilm-negativen Phänotyp revertieren konnten. Die DNA-Analyse mittels Pulsfeldgelelektrophorese zeigte, daß es in den varianten Stämmen zu größeren DNA-Rearrangements gekommen war, die neben der variablen Biofilmbildung auch mit Unterschieden in der Expression des alternativen Transkriptionsfaktors SigmaB einhergingen. Die Nukleotidsequenzierung des sigB-Systems ergab in den Varianten mehrere Punktmutationen in den SigB-Regulatorgenen rsbU und rsbW. Dies legt nahe, daß der SigB-Genlokus einer starken genetischen Variabilität unterliegt, die wiederum pleiotrope Effekte auf die Genexpression in S. aureus ausübt. Durch Northern-Blot-Analysen konnte allerdings gezeigt werden, daß die Biofilmbildung in den S. aureus-Varianten nicht mit der veränderten SigB-Expression in Zusammenhang steht. / Staphylococcus aureus and Staphylococcus epidermidis belong to the most frequent causes of nosocomial infections in immunocompromised patients. These bacteria form an essential part of the healthy skin flora of human beings. Little is known, whether there are differences in the genetic equipment between clinical and commensal isolates and which factors contribute to the setup of staphylococci in the hospital environment. The results of the presented work show that the ability to form biofilms is an essential feature of pathogenic staphylococci. The expression of this virulence factor is highly variable and depends on the presence of the ica operon which is responsible for biofilm formation, specific environmental factors and the influence of insertion sequences. In an epidemiological investigation, it was shown that the ica operon in S. epidermidis is more often present in clinical strains than in commensal ones. The predominant part of these ica-positive strains formed phenotypically a biofilm. In contrast, all examined S. aureus contained, independent of their origin, the complete ica gene clusters, while, however, none of these strains formed a biofilm under laboratory conditions. Biofilm formation could be induced by subinhibitory concentrations of specific antibiotics or osmotic stress in 30 percent of the S. aureus strains. Also, biofilm formation could be stimulated in ica-positive S. epidermidis strains through these environmental factors. The study also revealed that there is an association between biofilm formation, antibiotic resistance and the occurrence of the insertion sequence IS256. Thus, IS256 was significantly more often detected in clinical S. epidermidis and S. aureus strains, while there was no difference in the occurrence of IS257 between clinical and saprophytic isolates. Most of the IS256-positive S. epidermidis strains carried the ica operon and were simultaneously resistant against at least two antibiotics. Furthermore, it was shown that IS256 is involved in phase variation of biofilm formation in vivo. In case of a clinical S. epidermidis strain that was isolated from a patient with a catheter-associated urinary tract infection, the insertion of the element in the icaC gene was detected resulting in a biofilm-negative phenotype. Subcultivation of the insertion mutant resulted in biofilm-forming variants after a few passages. Nucleotide sequencing indicated the complete excision of IS256 from the icaC gene including the duplicated target site sequence of seven base pairs. These data are in agreement with the results received in a recent in vitro study and show that IS256 has an influence on the ica-expression during an infection. In this study, phase variation of biofilm formation was also shown in S. aureus. After serial passages, several biofilm producers were derived from formerly biofilm-negative single colonies which could also revert to the biofilm-negative phenotype again. DNA analysis by pulsed-field gel electrophoresis showed that in the variants large DNA-rearrangements took place. In addition to the variable biofilm production, differences in the expression of the alternative transcription factor SigmaB were observed in the variants. Nucleotide sequencing of the sigB system indicated several point mutations in the SigB regulatory genes rsbU and rsbW of the variants. This implies that the SigB gene locus is subject to a strong genetic variability that results, in turn, in pleiotropic effects on gene expression in S. aureus. However, Northern blot analysis revealed that the biofilm formation in the S. aureus variants are not associated with the varying SigB expression.

L'objectif de cette thèse est d'évaluer le concept d'un dispositif intégré comprenant un bioréacteur à biofilm fluidisé bio-augmenté, couplé à une membrane de nanofiltration de type polyelectrolyte multicouche, destiné à éliminer les micropolluants en traitement tertiaire des eaux usées domestiques, traitées conventionnellement. Les résultats montrent que, pour des micropolluants ciblés, chacun des procédés est efficace comme traitement tertiaire. Les mécanismes biologiques et de rétention membranaires sont explicités. Cependant des challenges restent à relever en particulier pour l'étape de bio augmentation (survie et implantation de la souche apportée) pour une exploitation de cette étape. D'autre part, des investigations plus poussées sont nécessaires à l'élaboration d'une membrane fiable et robuste. Un tel procédé couplé MBBR-NF pourra alors être entièrement justifié dans le contexte d'une élimination performante de micropolluants ciblés. Il aura toute sa place dans le panel des technologies vertes pour la préservation de l'environnement. / This thesis aims at answering whether the concept of an integrated layout comprised of a coupled "bioaugmented moving bed biofilm reactors (bMBBRs) - polyelectrolyte multilayer (PEM)-based nanofiltration (NF) membrane" can be considered as a promising technology to eliminate target MPs from conventionally-treated municipal wastewater. Results presented herein indicate that each given component of the layout is efficient in the tertiary removal of MPs. Still, several challenges ahead of the process bioaugmentation (such as the survival and maintenance of inoculated strains) must be in-depth studied to find convenient operating solutions. On the other hand, further investigations are definitely needed to achieve a robust PEM-based membrane as a long-lasting technology. Even though a coupled bMBBR-NF system for enhanced MPs removal can be experimentally justified is, however, practically questionable. "The tale of bMBBR-NF" deserves much more scientific endeavors as plenty of environmental considerations are placed in, whereby achieving a future Green technology will not be far from our expectation.

Neste trabalho foram preparados materiais híbridos do tipo silicatos organicamente modificados (ormosils) contendo fosfotungstato, [PW12O40]-3 e dopados com nanopartículas de TiO2. O objetivo é obter uma ação sinérgica destes dois fotocatalisadores na prevenção de formação de biofilmes e/ou sua fotodegradação. O fotocatalisador principal no sistema é o fosfotungstato, sendo o co-adjuvante o TiO2. Sendo assim, procurou-se manter a concentração deste no menor nível possível. Os materiais foram caracterizados por espectroscopias vibracionais, espectroscopia de fotoelétrons de raios X (XPS), Fluorescência de Raios X, Microscopia de força atômica e Microscopia eletrônica de varredura (MEV). Os filmes não mostraram eficácia na fotodegradação de biomoléculas como fosfolipídios encontrados na membrana celular. Os ensaios de inibição de crescimento de biofilmes de Escherichia coli sobre os ormosils mostraram que a maior inibição de bactérias é do filme contendo maior teor de nanopartículas de titânia portanto, são bons candidatos para filmes e revestimentos bactericidas/bacteriostáticos a serem usados em máscaras respiratórias, revestimentos de superfícies em salas de cirurgia e em filtros de ar em sistemas fechados (sistemas de ar condicionado e ventilação em geral). / This thesis deals with hybrid materials named organically modified silicates (Ormosils) with phosphotungstate, [PW12O40]-3 , and doped with TiO2 nanoparticles. The aim was to achieve a synergic action between both photocatalysts resulting on a more efficient coating for inhibition of the biofilm growing and/or its photodegradation. The photocatalyst in main system is the phosphotungstate, being the co-adjuvant the TiO2. Therefore, we tried to maintain the concentration of this at the lowest level possible The materials were characterized by vibrational spectroscopies, X-Ray Photoelectron Spectroscopy, X- ray Fluorescence Spectroscopy, Atomic Force Microscopy and Scanning Electron Microscopy. The films were unable to photodegradate biomolecules films such as phospholipids as well as they display interesting inhibition capacity against formation of biofilm of E.Coli bacteria. The tests of inhibition of growth of Escherichia coli biofilms on the ormosils showed that a greater inhibition of bacterias exists in the film containing higher content of nanoparticles of titania. Therefore, they are good candidates for bactericidal films and coatings to be used in respirators, surface coatings in surgery rooms and air filters in closed systems (systems of air conditioning and ventilation in general).

O objetivo do presente estudo foi avaliar a capacidade de isolados de Listeria monocytogenes em formar biofilmes em superfícies inertes, bem como sua resistência a agentes sanitizantes. Foram utilizados 37 isolados provenientes de ambiente de laticínios, amostras de queijos e salmoura, pertencentes à coleção do Laboratório de Microbiologia e Micotoxicologia de Alimentos (LMMA) do Departamento de Engenharia de Alimentos da FZEA/USP. Dos 37 isolados avaliados, 67,6% eram pertencentes ao sorotipo 4b. Três isolados de L. monocytogenes foram obtidos de salmoura, 5 foram obtidos de caixas plásticas, 1 de queijo Prato, 1 da mão de manipulador de embalagens, e 27 foram isolados de superfícies sem contato com alimentos (piso da sala de pasteurização, piso da câmara fria, ralo da câmara fria ou estrado da câmara fria). Os isolados de L. monocytogenes apresentaram maior capacidade de produzir biofilme nos ensaios com cupons de aço inox (43,2% dos isolados), seguido dos ensaios em microplaca de poliestireno (16,2%), cupons de borracha (13,5%) e discos de silicone (2,7%). As células de L. monocytogenes aderidas nas superfícies do aço inox foram visualizadas sob microscopia eletrônica de varredura após 48 horas de incubação a 37ºC. Dezenove isolados de L.monocytogenes, os quais produziram biofilmes nos ensaios com aço inox, borracha ou silicone, foram testados para determinação da eficiência de sanitizantes pelo método de concentração inibitória mínima (CIM), utilizando-se ácido peracético (2%), cloreto de banzalcônio (1%), digluconato de clorexidina (2%), hipoclorito de sódio (2%) e tintura de iodo (2%). Os isolados de L. monocytogenes analisados apresentaram resistência a ácido peracético, hipoclorito de sódio e tintura de iodo, cujos valores de CIM foram 3,12%, 6,25% e 6,25%, respectivamente. Nenhum isolado apresentou resistência a cloreto de benzalcônio e digluconato de clorexidina, os quais foram eficientes para inibição de isolados de L. monocytogenes provenientes de amostras de queijos e ambientes de laticínios. A L. monocytogenes apresenta capacidade de persistir em ambiente de laticínios sob a forma de biofilme em várias superfícies inertes como aço inox, borracha e silicone, o que pode representar uma fonte permanente de contaminação para produtos e processos de obtenção de leite e derivados. / The objective of the present study was to evaluate the ability of isolates of Listeria monocytogenes to form biofilms and their resistance to sanitizers. Thirty seven strains belonging to the collection of the Laboratory of Microbiology and Food Mycotoxicology (LMMA), Department of Food Engineering of FZEA / USP, were used. Of the 37 isolates, 67.6% belonged to serotype 4b. Three isolates of L. monocytogenes were obtained from brine, 5 were obtained from plastic boxes, one of Prato cheese, one from the packaging handler\'s hand, and 27 were isolated from non-food contact surfaces (pasteurization room floor, the floor of the cold room, the drain cold or pallet from the cold chamber). The isolates of L. monocytogenes showed greater ability to produce biofilm in the assays with stainless steel coupons (43.2% of isolates), followed by polystyrene micro plate (16.2%), rubber coupons (13.5%) and silicone disks (2.7%) assays. Cells of L. monocytogenes attached to stainless steel surfaces were viewed under scanning electron microscopy after 48 hours incubation at 37°C. Nineteen strains of L. monocytogenes, which were considered biofilms producers in the assays with stainless steel, rubber or silicone, have been tested to evaluate the efficiency of the sanitizing method by means of the minimum inhibitory concentration (MIC), using peracetic acid (2%), sodium chloride benzalkonium (1%), chlorhexidine digluconate (2%), sodium hypochlorite (2%) and iodine solution (2%). The isolates of L. monocytogenes analyzed showed resistance to peracetic acid, sodium hypochlorite and iodine tincture, with MIC values of 3.12%, 6.25% and 6.25%, respectively. No isolate showed resistance to benzalkonium chloride and chlorhexidine digluconate, which were effective for inhibiting the isolates of L. monocytogenes from samples of cheeses and dairy environments. In conclusion, L. monocytogenes has the ability to persist in the environment of dairy products by forming biofilms in several inert surfaces such as stainless steel, rubber and silicone, which may represent a continuing source of contamination to manufacture processes of dairy products.

The study aimed: 1) to compare the effect of two different nutrients supply models (static and semi-dynamic) on the microcosm biofilm viability and dentin carious lesions formation; 2) to compare micro-CT versus TMR to measure dentin demineralization; and 3) to evaluate the effect of 4% TiF4 varnish on the viability and metabolism of a microcosm biofilm and on development of dentin carious lesions. Microcosm biofilm was produced using pooled human saliva mixed with McBain saliva for the first 8 h; thereafter, only McBain saliva with 0.2% sucrose was applied daily (37°C, 5% CO2), for a total time of 5 days. In the study 1, the static model consisted of 24-wells microplate, where bovine root dentin samples were submitted to biofilm formation. The semi-dynamic model, consisted of artificial mouth with continuous flow of McBain saliva with 0.2% sucrose (0.15 ml/min, 37°C) during 10 h a day (for the other 14 h, no flow was applied). Biofilm viability was measured by fluorescence and dentin demineralization by TMR. For the studies 2 and 3, bovine root dentin samples were treated for 6 h: A) 4% TiF4 (pH 1.0, 2.45% F); B) 5.42% NaF (pH 5.0, 2.45% F); C) 2% CHX gel positive control D) placebo or E) untreated negative control. Treated samples were submitted to biofilm formation under static model as described above. Demineralization was measured using micro-CT (study 2) and TMR (studies 2 and 3). In the study 3, biofilm was analyzed with respect to viability by fluorescence and CFU counting for total microorganisms, total streptococci, mutans streptococci and Lactobacillus sp., and lactic acid and EPS production. In study 1, biofilm viability was lower for the static model (0.420±0.138) compared to semi-dynamic one (0.944±0.599). Both models were able to provoke dentin demineralization; however, the static model produced a higher number of typical subsurface lesions (83%) compared to the semi-dynamic (45%). In study 2, both fluorides were able to reduce dentin demineralization. Data obtained from micro- CT and TMR presented a significant and positive correlation (Z: r=0.78 p<0.0001 and LD: r=0.57 p<0.0001) In study 3, all treatments reduced the biofilm viability, but not the CFU counting, except NaF that significantly reduced the number of Lactobacillus sp. compared to control. No treatment was able to decrease the lactic acid production neither EPS production, except CHX that reduced the amount of insoluble EPS. Fluorides were able to reduce dentin demineralization compared to control, but TiF4 had the best effect in reducing mineral loss and lesion depth (reduction of Z: 70% and LD: 45%). In conclusion, 1) the nutrient supply model may have influence on the biofilm viability and the profile of dentin carious lesions; 2) micro-CT may be a suitable non-destructive method to measure dentin demineralization; and 3) despite TiF4 varnish has no relevant antimicrobial effect, it is the best option to reduce the development of dentin carious lesions under this model. / O estudo objetivou: 1) comparar o efeito de dois modelos diferentes de disponibilidade de nutrientes (estático e semi-dinâmico) sobre a viabilidade do biofilme microcosmo e formação de lesões de cárie em dentina; 2) comparar micro- CT versus TMR para mensurar a desmineralização da dentina; e 3) avaliar o efeito do verniz TiF4 4% na viabilidade e metabolismo do biofilme microcosmo e no desenvolvimento de lesões de cárie em dentina. O biofilme microcosmo foi produzido utilizando saliva humana misturada com saliva de McBain durante as primeiras 8 h; depois, apenas saliva de McBain com sacarose 0,2% foi aplicada diariamente (37°C, 5% de CO2), totalizando 5 dias. No estudo 1, o modelo estático consistiu de placa de 24 poços, onde amostras de dentina radicular bovina foram submetidas à formação do biofilme. O modelo semi-dinâmico, consistiu de boca artificial com fluxo contínuo de saliva de McBain com sacarose 0,2% (0,15 ml/min, 37°C) durante 10 h por dia (nas demais 14 h, não foi aplicado fluxo). A viabilidade do biofilme foi mensurada por fluorescência e a desmineralização da dentina por TMR. Para os estudos 2 e 3, as amostras de dentina radicular bovina foram tratadas por 6 h: A) TiF4 4% (pH 1,0, 2,45% F); B) NaF 5,42% (pH 5,0, 2,45% F); C) gel CHX 2% - controle positivo D) placebo ou E) não tratado - controle negativo. As amostras tratadas foram submetidas à formação do biofilme sob modelo estático conforme descrito acima. A desmineralização foi mensurada utilizando micro-CT (estudo 2) e TMR (estudos 2 e 3). No estudo 3, o biofilme foi analisado quanto à viabilidade por fluorescência e contagem das UFC para microrganismos totais, Streptococcus totais, Streptococcus mutans e Lactobacillus, e quanto à produção de ácido lático e PEC. No estudo 1, a viabilidade do biofilme foi menor para o modelo estático (0,420±0,138) comparado ao semi-dinâmico (0,944±0,599). Ambos os modelos provocaram desmineralização da dentina; entretanto, o modelo estático produziu maior número de lesões de subsuperfície (83%) comparado ao semi-dinâmico (45%). No estudo 2, ambos os fluoretos reduziram a desmineralização da dentina. Dados obtidos por micro-CT e TMR apresentaram uma correlação significativa e positiva (Z: r=0,78 p<0,0001 e LD: r=0,57 p<0,0001). No estudo 3, todos os tratamentos reduziram a viabilidade do biofilme, mas não a contagem de UFC, exceto o NaF que reduziu o número de Lactobacillus comparado ao controle. Nenhum tratamento diminuiu a produção de ácido lático e PEC, exceto a CHX que reduziu PEC insolúvel. Os fluoretos reduziram a desmineralização da dentina comparado ao controle, mas o TiF4 apresentou o melhor efeito em reduzir perda mineral e profundidade da lesão (redução de Z: 70% e LD: 45%). Em conclusão, 1) o modelo de disponibilidade de nutrientes pode influenciar a viabilidade do biofilme e o perfil das lesões de cárie em dentina; 2) micro-CT pode ser um método não destrutivo adequado para mensurar desmineralização da dentina; e 3) apesar do verniz TiF4 não apresentar efeito antimicrobiano relevante, é a melhor opção para reduzir o desenvolvimento de lesões de cárie em dentina neste modelo.