Orientadores: Wander José da Silva, Altair Antoninha Del Bel Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T12:52:17Z (GMT). No. of bitstreams: 1 Cavalcanti_YuriWanderley_D.pdf: 36598802 bytes, checksum: f05687dd3da4ac323b62e27ca3d8816c (MD5) Previous issue date: 2015 / Resumo: A prevalência das infecções por Candida é elevada; logo, melhor compreensão dos mecanismos de desenvolvimento do biofilme é necessária para a redução da virulência e apropriado manejo clínico. Objetivou-se analisar a virulência de biofilmes de Candida albicans desenvolvidos sob diferentes condições. O papel das superfícies de biomateriais, da película salivar, e do estágio de desenvolvimento dos biofilmes foi avaliado no Capítulo 1. A influência da presença de outros microrganismos na virulência de C. albicans e na interação com o epitélio foi avaliada no Capítulo 2. O papel da atmosfera e da população bacteriana dos biofilmes foi investigado no Capítulo 3. No Capítulo 1, biofilmes de C. albicans foram desenvolvidos sobre discos de resina acrílica e titânio recobertos com película de saliva, ou de saliva com plasma. A superfície dos materiais foi analisada quanto a rugosidade e energia livre de superfície (ELS). Avaliou-se o número de microrganismos viáveis, a concentração de DNA, a atividade metabólica, a expressão de fatores de virulência e a estrutura dos biofilmes. Não houve diferenças quanto a rugosidade das superfícies. A película minimizou as diferenças entre a ELS dos materiais, sendo maior ELS observada para películas de saliva com plasma. O número de microrganismos viáveis, a concentração de DNA e a atividade metabólica aumentaram ao longo da maturação do biofilme. Maior atividade metabólica, maior expressão de ALS1, ALS3 e HWP1, e maior número de hifas foram verificados em biofilmes desenvolvidos na presença de película de saliva com plasma. Não houve diferenças entre os materiais. Concluiu-se que a presença de plasma na película salivar aumenta a virulência de C. albicans. No capítulo 2, biofilmes maduros (72 h) de C. albicans, e de C. albicans com bactérias, foram desenvolvidos sobre a superfície de resina acrílica. Esses biofilmes foram então analisados ou utilizados para infectar um modelo de epitélio oral humano reconstituído (RHOE). Avaliou-se o número de hifas e a expressão de fatores de virulência. A resposta epitelial foi avaliada por meio da expressão de IL-18 e Dectin1, da atividade da enzima lactato-desidrogenase (LDH), e a invasão epitelial. Maior número de hifas e maior expressão de HWP1, SAP4 e SAP6 foram verificados em biofilmes multi-espécie desenvolvidos sobre o acrílico. Biofilmes multi-espécies que infectaram o RHOE também apresentaram maior número de hifas e maior expressão de ALS3, EPA1, SAP6 e HWP1. Consequentemente, esses epitélios apresentaram maior invasão tecidual, maior atividade de LDH e maior expressão de IL-18. Concluiu-se que a presença de bactérias aumenta a virulência e patogenicidade de C. albicans. No capítulo 3, biofilmes de C. albicans e de C. albicans com estreptococos foram desenvolvidos sobre titânio em atmosfera de aerobiose e anaerobiose. Biofilmes de C. albicans (com ou sem estreptococos) também foram desenvolvidos na presença de Porphyromonas gingivalis, em anaerobiose. Avaliou-se a formação de hifas e a expressão de fatores de virulência. A atmosfera de anaerobiose e o co-cultivo com estreptococos geraram maior número de hifas e maior expressão de fatores de virulência. Embora P. gingivalis tenha inibido a virulência de C. albicans em biofilmes duo-espécies, esse efeito foi revertido na presença de estreptococos. Concluiu-se que a atmosfera de anaerobiose e a presença de estreptococos estimulam a virulência de C. albicans / Abstract: Candida infections have high prevalence; therefore, better understanding of the development mechanisms of biofilms is required for the reduction of virulence and for more appropriated clinical management. The aim of this study was to analyse the virulence of Candida albicans biofilms developed under different conditions. The role of biomaterial surfaces, salivary pellicle, and stage of biofilm development has been reported in Chapter 1. The influence of the presence of other microorganisms in virulence of C. albicans and on the interaction with the epithelium was evaluated in Chapter 2. The role of the atmosphere and of the biofilm¿s bacterial population was investigated in Chapter 3. In Chapter 1, C. albicans biofilms were developed on acrylic resin and titanium discs coated with saliva pellicle, or saliva with blood plasma pellicle. The materials¿ surfaces were analysed with regards to surface roughness and surface free energy (SFE). We evaluated the number of viable organisms, the DNA concentration, the metabolic activity, the expression of virulence factors and the architecture of the biofilms. There were no differences in the surface roughness of materials. The pellicles eliminated the differences between SFE of materials. Higher SFE was observed for discs coated with pellicles of saliva with plasma. The number of viable organisms, the DNA concentration and the metabolic activity increased throughout the biofilm maturation. Higher metabolic activity, increased expression of ALS1, ALS3 and HWP1, and more hyphae were observed in biofilms grown in the presence of saliva with plasma pellicle. There were no differences between the materials. In conclusion, the presence of saliva with plasma pellicle increases the C. albicans virulence. In chapter 2, mature biofilms (72 h) of C. albicans, or C. albicans with bacteria, were developed on the surface of acrylic resin discs. These biofilms were then analysed or used to infect a reconstituted human oral epithelium model (RHOE). Biofilms were evaluated according to the number of hyphae and the expression of virulence factors. The epithelial response was evaluated by the expression of IL-18 and Dectin1, by the activity of lactate dehydrogenase enzyme (LDH), and with regards the epithelial invasion. Increased number of hyphae and increased expression of HWP1, SAP4 and SAP6 were observed in multi-species biofilms developed on acrylic. Multi-species biofilms that have infected RHOE also had a greater number of hyphae and increased expression of ALS3, EPA1, SAP6 and HWP1. Consequently, these epithelia showed greater tissue invasion, higher LDH activity and higher IL-18 expression. We concluded, therefore, that the presence of bacteria promotes the virulence and pathogenicity of C. albicans. In Chapter 3, biofilms of C. albicans and C. albicans with streptococci were developed on titanium, aerobically and anaerobically. C. albicans biofilms (mixed or not-mixed with streptococci) were also grown in the presence Porphyromonas gingivalis, anaerobically. Biofilms were evaluated according to the formation of hyphae and expression of virulence factors. Anaerobic conditions and co-cultivation of streptococci produced higher number of hyphae and increased expression of virulence factors. Although P. gingivalis has inhibited the virulence of C. albicans, this effect was reversed in the presence of streptococci. We concluded that the anaerobic atmosphere and the presence of streptococci stimulate the C. albicans virulence / Doutorado / Protese Dental / Doutor em Clínica Odontológica

De acordo com a Food and Agriculture Organization of the United Nations (FAO, 2013), o consumo mundial de carne de frango tem aumentado de maneira significativa nas últimas décadas. Por outro lado, a preocupação dos produtores de alimentos, com a inocuidade de seus produtos também tem aumentado na mesma proporção. Durante as etapas da operação de abate de maneira geral, as contaminações cruzadas são as principais causas de disseminação de microrganismos patogênicos nos produtos obtidos e causadores de gastroenterites no consumidor. Espécies da enterobactéria Salmonella se enquadram como um dos maiores riscos desse tipo de doença devido a sua associação com os inúmeros surtos ocorridos a nível mundial, após o consumo desses produtos. Algumas enterobactérias possuem um gene blaKPC, que codifica a enzima carbapenemase, que confere resistência a antibióticos carbapenêmicos, agravando mais a situação. Alguns fatores de virulência encontrados nesse gênero de bactéria podem ainda estar associados a capacidade de adesão e formação de biofilmes em superfícies inertes, dificultando operações de higienização nas linhas de processamento. Assim sendo, a presente pesquisa objetivou a verificação da capacidade de estirpes de Salmonella spp isoladas de aviário e linha de abate de frangos de um frigorífico no estado do Rio Grande do Sul produzirem biofilmes e apresentarem resistência a antibióticos carbapenêmicos. Na avaliação da produção de biofilme, foi empregada a técnica de microplacas de polietileno e produção de cápsula segundo Stepanovic et al. (2004) e Rodrigues et al. (2006), respectivamente.Foram pesquisados os fatores de virulência de salmonelas, representados pelos genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH,e spvC, utilizando o método de Reação de cadeia de Polimerase (PCR), descrita por Borges et al. (2013). As estirpes foram submetidas ao teste de resistência a antibióticos carbapenêmicos pelo método de disco difusão com carbapenêmicos segundo Clinical and Laboratory Standards Institute-CLSI (2010) e pesquisa do gene de resistência a carbapenêmicos blaKCP, pela técnica de PCR, segundo NAAS et al. (2007). Obteve-se quatro perfis genéticos das estirpes de Salmonella spp.: perfil 1: genes ifpA, agfA, invA e avrA; perfil2: genes ifpA, agfA, sefA, invA e avrA; perfil 3: genes invA e avrA; perfil4: genes ifpA,agfA e invA. Observou-se a resistência das estirpes somente ao antibiótico imipenen. Entre as 36 estirpes de Salmonella spp. isoladas, todas foram consideradas produtoras de biofilme in vitro, sendo que 69% destas, apresentaram-se como fortes produtoras, 25% como moderadas, e apenas 6% como fracas produtoras. O método Agar Vermelho Congo não se mostrou eficiente para teste presuntivo de produção de biofilme para estirpes de Salmonella spp. Não foi evidenciado o gene blaKPC nas estirpes de Salmonella spp. isoladas na presente pesquisa. / According to Food and Agriculture Organization of the United Nations (FAO, 2013), the world consumption of meat of chicken has been higher significantly in the last decades. On the other hand, the concern of the food producers with the safety of their products also has increased in the same proportion. During the steps of the slaughter operation in general, cross contamination are the main causes of pathogenic microorganisms dissemination in the obtained products and the causes of gastroenteritis in the consumer. Species of the Enterobacter Salmonella fall as the highest risks of this kind of disease, due to their association with countless outbreaks which occurred worldwide, after the consumption of these products. Some enterobacter have a blaKPC gene, which codes for the carbapenemase enzyme that confers resistance to carbapenems antibiotics, aggravating the situation. Some virulence factors found in this bacteria genes can also be associated to the ability of adherence and the formation of biofilms in inert surfaces, making it difficult the operations of sanitation in the processing lines. Thus, the present research aimed to verify the capacity of Salmonella spp. strains isolated from aviary and slaughter line of chicken in a fridge of Rio Grande do Sul state to produce biofilms and be resistant to carbapenems antibiotics. In the evaluation of biofilm production, it was used the polyethylene microplates and capsule production technique according to Stepanovic et al. (2004) and Rodrigues et al. (2006), respectively. The virulence factors of salmonella were researched, represented by the genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, e spvC, using the Polymerase Chain Reaction (PCR) method described by Borges et al. (2013). The lineages were submitted to the carbapenems antibiotic resistence test by the disk diffusion method with carbapenems according to the Clinical and Laboratory Standards Institute-CLSI (2010), and the research of the resistance to carbapenems gene blaKCP, by the strains Salmonella spp.: profile 1: genes ifpA, agfA, invA and avrA.; profile 2: genes ifpA, agfA, sefA, invA and avrA; profile 3: genes invA and avrA; profile 4: genes ifpA, agfA and invA. It was observed the resistance of the strains only to the imipenen antibiotic. Among the other 36 cultures of Salmonella spp. isolated, all were considered to produce biofilm in vitro, of which 69% were strong producers, 25% moderate, and only 6% were low producers. The method Congo Red Agar was not efficient to the presumptive test of biofilm production for the Salmonella spp. strains. It was not evidenced the gene blaKPC in Salmonella spp. strains isolates in this research.

Sessile bacterial communities which form on the solid surface or solid-liquid interface are known as biofilms. Both single species and multispecies biofilms are characterized by an extracellular matrix of polymeric substances which gives them several hundred times more antibiotic resistances than a planktonic bacterial culture. Though bacteria are the most common causative agent of various diseases, because of the high antibiotic resistance, biofilms cause complications of various diseases like cystic fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated urinary tract infections and several other diseases. From past studies, quorum sensing has been established as a novel target mechanism against biofilms; in this study, the two-component signal transduction systems (2CSTSs) have been focused. Once better understood, 2CSTSs can serve as a novel drug target and prevention mechanism for biofilm associated diseases. According to prior high-throughput experiments and phenotype microarray experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the global regulator FlhD/FlhC were hypothesized to have an important effect on various developmental stages of biofilm formation. From that past study, we postulated that acetate metabolism may be an important aspect for biofilm formation. In this study, we tested and confirmed this hypothesis. We observed biofilms formed by several mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of the acetate mutants when compared to their isogenic parental Escherichia coli strain. An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms whereas an additional mutation in dcuR results in the formation of less biofilms. So the structural and the quantitative differences of acetate mutant biofilms depend on additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The future aim of this study is to study the temporal as well as the spatial gene expression of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter plate assay was performed to study the temporal expression from the promoters by quantifying the fluorescence intensity in the planktonic culture. According to this experiment, the highest expression of flhD was after 20 hours whereas, the expression of ompR increases up to 7 days, which indicates that the flhD expresses earlier than ompR. The decreasing phase of flhD expression was paralleled by the sharpest increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD expression. / National Institutes of Health (NIH grant 1R15AI089403) / United States. Animal and Plant Health Inspection Service

The propensity of Salmonella to persist in water environments under unfavourable conditions is of concern as these water environments serve as contamination reservoirs. The role of contaminated water in the transmission of Salmonella in developing countries is largely unknown. The fate and persistence of non-typhoidal Salmonella in water environments and the specific influence of the indigenous microbiota on the survival and growth of Salmonella is poorly understood. A tagged Salmonella strain distinguishable in vivo from a mixed bacterial community would greatly facilitate the study of Salmonella in water environments. The clinically relevant S. enterica subsp. enterica ser. Typhimurium isolate was chromosomally tagged using the pUT mini–Tn5 Km transposon with the green fluorescent protein gene gfpmut3b*. Southern Blot hybridisation confirmed that the gfp gene had integrated into the chromosome. The gfp gene was stably maintained and the gfp-labelled recombinants were not growth rate impaired under low nutrient conditions. No significant changes were observed between the wild-type and the tagged strain. The survival fitness studies indicated the incorporation of the gfp gene did not have any noted detrimental effects on the survival and behaviour of the tagged strains. These tagged strains could therefore be used to study the fate and survival of Salmonella in biofilms of drinking water distribution systems. Genetic tagging of the target organism with the gfp gene, encoding the green fluorescent protein, allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella as a monospecies or in a complex mixed community. The fate and persistence of non-typhoidal Salmonella in drinking water biofilms was investigated. The ability of Salmonella to form biofilms independently and the fate and persistence of Salmonella in an aquatic biofilm was examined. </p.> In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, growing to form micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the flow, and was seen to re-colonize elsewhere. Results showed that Salmonella can enter into, survive and grow within, and be released from a drinking water biofilm. Once Salmonella has entered into a distribution system, it will be able to colonize an existing biofilm, grow in it and be released into the flow for re-colonization elsewhere, and possible subsequent infection of consumers. / Dissertation (MSc (Microbiology ))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

<div><div><div><p>Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, and biofilms are known to be the frequent source for infection and outbreaks of foodborne illness. Therefore, it is essential to understand the pathogenicity of bacteria in biofilms and methods to inactivate biofilm-forming microbes from food processing environments, including school cafeteria or other community-based food production facilities, and to prevent foodborne outbreaks. Pathogen transmissions occur primarily through raw or under cooked foods and by cross contamination during unsanitary food preparation practices. Then, pathogens can form biofilms on the surface and become persistent in food production facilities and can be a source for recurrent contamination and foodborne outbreaks. In this study, our first aim was to use L. monocytogenes as a model pathogen to study how an enteric infectious pathogen isolated from biofilm modifies its pathogenesis compared to its planktonic counterpart. Both clinical and food isolates with different serotypes and biofilm-forming abilities were selected and tested using cell culture and mouse models. L. monocytogenes sessile cells isolated from biofilms express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains revealed that at 12 and 24 h post-infection (hpi), L. monocytogenes burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile L. monocytogenes from intestinal content are about 6.0- and 280-fold higher than the sessile inoculum, respectively, suggesting sessile L. monocytogenes can still upregulate virulence genes shortly after ingestion (12 h).</p><p>After learning biofilm isolated L. monocytogenes cells have similar virulence potential as the planktonic counterparts, our next goal was to effectively prevent or inactivate biofilms using food-grade natural microbials. Since L. monocytogenes cells are usually found in multi-pathogen biofilm in nature, I combined two food-grade broad-spectrum natural antimicrobials, chitosan nanoparticles (ChNP) and ε-poly-L-lysine (PL), as ChNP-PL nanoconjugates and tested its function on single or mixed culture biofilms of L. monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa. ChNP- PL not only was able to significantly (P<0.05) prevent the biofilm formation but also inactivate pre-formed biofilms when analyzed by crystal violet staining and plate counting. In vitro cytotoxicity analysis (LDH and WST-based assays) using an intestinal cell line, indicated ChNP- PL to be non-toxic. In conclusion, our results showed ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens in food processing environment. Application of ChNP-PL could inhibit the colonization of foodborne pathogens, minimize cross-contamination during food production, and eventually reduce foodborne outbreaks.</p></div></div></div>

This study aims to rationalize the consumption of thermal energy in residential buildings by recovering heat from wastewater inside the building before entering the central sewage network outside the building, by conducting an analytical study for a residential tower in Syria to find out the coverage percentage of the heat energy recovered from wastewater for the heating and domestic hot water loads needed for the tower, and calculating the percentage of reduction in carbon dioxide (CO2) gases. It is a simple technology as the thermal recovery system consists of three main components, which are in order: a wastewater tank, heat exchangers, and a heat pump. The research begins with an introduction that consists of the importance of wastewater and the waste heat energy it carries. After that, there are some case studies, research problem, its importance, the aim of the research, and finally the research methodology. In the first chapter, we talked about the concept of heat recovery from wastewater in general, methods of heat recovery, and the most important advantages and disadvantages of this process. It also includes an identification of the main parts used in this technology and how it works, especially the exchangers and the heat pump. This chapter also addresses the problem of forming a layer of biofilms on the surface of heat exchangers from the wastewater side and the most important methods used to treat it. We move on to the second chapter, in which we review the most important facilities for heat recovery from wastewater that have been viewed. Then comes the third chapter in which the heat recovery process was conducted for a nine storey residential tower in Syria, each floor has four apartments, where we first calculated the rate of wastewater flow for the entire tower, and we proposed a heat recovery system (physical model) inside the tower. Then the mathematical equations for heat recovery and the solution of these equations were developed based on some necessary assumptions needed in the solution process to know the most important results desired in this field. It also included the calculation of the coverage ratio of the heat energy recovered from the wastewater for the domestic hot water and heating loads, as well as the calculation of the mass and percentage of the reduction of carbon emitted to the atmosphere. Then simple economic feasibility was also conducted in this chapter to know the daily financial savings as a result of using this technology. The research ends with the most important conclusions and future research that have been reached and the conclusion of the research. The most important results show that the average coverage percentage of heat energy recovered from wastewater for heating load in residential buildings ranges between [30-56%]. It was also found that the average coverage percentage of heat energy recovered from wastewater for domestic hot water load ranges between [65-100%].

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.

Indiana University-Purdue University Indianapolis (IUPUI) / Tooth decay is a complex dieto-bacterial disease with an association of social, behavioral and biological factors. Streptococcus mutans plays a major role in tooth decay. This endogenous oral microorganism adheres to tooth surfaces and grows and develops into micro-communities that mature and form dental biofilm. Development of cariogenic biofilm is one of the major factors associated with the tooth decay process. The use of tobacco is considered a great risk factor for oral diseases. Several studies demonstrated the association of tooth decay and the use of tobacco as effects of first-hand or second- hand smoking. Nicotine has been reported to increase the biofilm growth and metabolism of S. mutans in a dose-dependent manner up to 16 mg/ml of nicotine. However, its effects on biofilm formation of S. mutans strains isolated from smokers are not known and should be investigated. Therefore, we proposed the use of an in-vitro model to better understand the effects of nicotine on biofilm formation of strains of S. mutans isolates from smokers versus non-smoking subjects. Objectives: To investigate the effects of nicotine on biofilm formation of S. mutans isolates from oral washes of smoker and non-smoker human subjects. Materials and Methods: This study was conducted using three S. mutans isolates collected from oral washes of 10 smoking subjects and 10 non-smoking subjects. The oral wash samples were stored at -80oC before S. mutans isolation. S. mutans isolates were obtained by plating on Mitis Salivarius Sucrose Bacitracin plates and species identity confirmed by carbohydrate fermentation assays. Nicotine from Sigma-Aldrich (St. Louis, MO, USA) was used. Biofilm was formed by overnight culturing of each S. mutans strain (10 μl) in 190 μl of tryptic soy broth (TSB) supplemented with 1.0-percent sucrose (TSBS) containing 0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 4.0 mg/ml, 8.0 mg/ml, 16.0 mg/ml, and 32.0 mg/ml of nicotine for 24 hours in 5.0-percent CO2 at 37oC in sterile (8 x 12) 96-well microtiter plates (Fisher Scientific, Newark, DE, USA). The absorbance values of biofilm were measured at 490 nm in a microplate spectrophotometer (SpectraMax 190; Molecular Devices, SunnyVale, CA, USA) after crystal violet staining. Null Hypotheses: 1) Nicotine will not increase biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations will not increase biofilm formation in both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) Nicotine will not produce significant differences in biofilm formation between smoker and non-smoker S. mutans isolates. Alternative Hypotheses: 1) Nicotine increases the growth of biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations increase biofilm formation of both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) However, nicotine increases biofilm formation of smoker S. mutans strains more than non-smoker S. mutans isolates. The rationale for this hypothesis is that our preliminary data indicated that S. mutans can become resistant to increased nicotine concentrations and that this resistance appears to be stable and may allow the smoker isolates to be able to respond more vigorously to higher nicotine concentrations than the non-smoker isolates. Results: There was a significant effect (p < 0.05) of both nicotine concentrations and smoking on the growth of biofilm, planktonic cells, and total absorbance, for all strains of S. mutans (p < 0.0001). Isolates from smokers had significantly more biofilm at 0 mg/ml to 16 mg/ml of nicotine compared with those from non-smokers (p-value < 0.0001). Conclusion: S. mutans smoker isolates are more affected by high nicotine concentrations than non-smoker isolates.

No description available.

The present work examines the antimicrobial efficacy of the essential oils of thyme, oregano and carvacrol against L. monocytogenes biofilms produced on stainless steel coupons and for control of L. monocytogenes growth on raw catfish fillets stored at 4°C for 10 days. At 0.5%, all three essential oils were highly effective in completely eliminating L. monocytogenes cells from stainless coupons within 24 h as compared to the untreated control yielding ~7 log CFU/cm2 L. monocytogenes. When catfish inoculated with L. monocytogenes were dipped for 30 min at 4°C in essential oil solutions of thyme and oregano at 1%, 2% and 5%, there were no significant reductions in L. monocytogenes counts on the fresh catfish fillets as compared to untreated control. For the same conditions, treatment with 2% carvacrol resulted in a complete reduction of 4 log CFU/g of L. monocytogenes counts from fresh catfish fillets.