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The aim of this study was to observe biofilm formation by Enterobacter sakazakii (E. Sakazakii) from different clinical, dairy and environmental origins on three infant feeding tubes made of different materials. Infant formula milk was selected as the medium for E. sakazakii growth. Seventeen isolates from different origins were retrieved and tested for purity, using a plating method and biochemical tests to eliminate the non E. sakazakii strains from this study. A method to rapidly and accurately detect viable cells of E. sakazakii on infant feeding tube surfaces using of the BacTrac® 4000 microbiological growth analyser was developed. The sources of errors such as from cleaning, operation and handling procedures were assessed prior to experimental runs. The strength of biofilm formation by different isolates of E. sakazakii on plastic surfaces was scrutinised using a microtiter plate assay. The results from the microtitre plate assay were based on the absorbance at 550 nm of crystal violet stained films and showed that all the clinical isolates were able to attach and form strong biofilms on the plate. Some environmental isolates formed strong or weak biofilms and some did not produce biofilm at all. However, dairy isolates formed both strong and weak biofilms in the microtitre plate when incubated in 10% reconstituted infant formula milk. The further studies were to quantify biofilm formation by three isolates of different origin on three different materials of infant feeding tubes using a batch system. Tubing pieces were incubated with infant formula milk inoculated with E. sakazakii cells at approximately 8 log CFU mL-1 and the biofilm formation was assessed at three time intervals: 4, 12 and 24 hours. Biofilm formation on the tubing by clinical isolates was also observed using epifluorescence microscopy and the scanning electron microscope. E. sakazakii from clinical, dairy and environmental isolates were able to form biofilm on three different materials of infant feeding tubes. The results showed that the initial attachment at 4 h on silicone tubing was low compared with the other two tubes. The scanning electron micrographs showed the surface characteristics of each tubing and the biofilm formation by E. sakazakii clinical isolates after 4, 12 and 24 hours. Silicone tubing appeared to be the best choice for premature babies that need feeding using feeding tubes, as it was slow to become colonised compared with the PVC and polyurethane tubing.

Made available in DSpace on 2014-07-29T15:13:47Z (GMT). No. of bitstreams: 1 TESE Julia de Miranda Moraes.pdf: 2895250 bytes, checksum: e061f736f6922f781b2c671bf4f90f32 (MD5) Previous issue date: 2012-08-17 / The aim of this work was to evaluate the regeneration of equine ulnar nerves submitted to neurotomy, silicone tubing and cell therapy with bone marrow mononuclear cells fraction (MCF) or adipose derived mesenchymal stem cells (ADSC). Fifteen adult horses were divided into three groups with five animals each: control group (CG) with the use of saline solution, group with FCM deposition and group with ADSC deposition. The same surgical procedure was performed in all groups, using both nerves of each animal (right and left), establishing two moments of biopsy: on the 13th week on the right limb (CG1, MCF1 and ADSC1) and on the 26th week on the left limb (CG2, MCF2 and ADSC2). The MCF and ADSC were obtained respectively, from bone marrow and adipose tissue from each animal, both used as an authologous implant. After 13 and 26 weeks, biopsies were performed in all groups and immediately it was made some fragments slide imprints for viewing the nanocrystal fluorescent label. The fragments were fixed in 10% buffered formalin for histological analysis, with HE, luxol fast blue, Masson's trichrome, immunohistochemistry against the antibodies neurofilament (NF), S-100, FGF-2 and GDNF. Microscopically it was observed the presence of axonal growth, connective tissue, inflammatory infiltrate, Schwann cells, neural growth factors, myelin sheath and wallerian degeneration. For histologic analysis, it was established qualitative scores and for immunohistochemistry it was performed quantitative analysis with Image J program. It was observed wallerian degeneration reduction, better fascicular reorganization, new collagen increased, myelin sheath early formation, and NF antibody stronger staining in the experimental groups compared to CG. The ADSC group presented the best results than the other groups, showing ADSCs efficiency compared to MCF and CG for peripheral nerve regeneration. However, it was proved a longer time necessity to occur complete regeneration of peripheral nerve after injury. / Este trabalho teve por objetivo avaliar a regeneração do nervo ulnar de equinos, submetidos à neurotomia, tubulização com tubo de silicone e terapia celular com fração de células mononucleares da medula óssea (FCM) ou células-tronco mesenquimais do tecido adiposo (ADSC). Foram utilizados 15 equinos adultos alocados em três grupos, com cinco animais em cada: grupo controle (GC) com utilização de soro fisiológico; grupo com deposição de FCM; e grupo com deposição de ADSC. Foi realizado o mesmo procedimento cirúrgico em todos os grupos, utilizando-se os dois nervos de cada animal (direito e esquerdo), e estabelecido dois momentos de biopsia: na 13ª semana com biopsia no membro direiro (GC1, FCM1 e ADSC1) e na 26ª semana com biopsia no membro esquerdo (GC2, FCM2 e ADSC2). A FCM e a ADSC foram obtidas respectivamente, da medula óssea e tecido adiposo de cada aninal, ambos utilizados como implantes autólogos. Após 13 e 26 semanas, realizaram-se as biopsias de todos os grupos e imediatamente, faziam-se imprints dos fragmentos para visualização do marcador fluorescente nanocristal. Os fragmentos foram fixados em formol tamponado a 10%, para análise histológica, com as coloraçãoes de HE, luxol fast blue, tricrômio de Masson e imuno-histoquímica com os anticorpos neurofilamento (NF), S-100, FGF-2 e GDNF. Microscopicamente avaliou-se a presença de proliferação axonal, tecido conjuntivo, infiltrado inflamatório, células de Schwann, fatores de crescimento neurais, bainha de mielina, e degeneração walleriana. Para a análise histológica estabeleceram-se escores qualitativos e para a imuno-histoquímica realizou-se análise quantitativa com o programa Image J. Foi observada diminuição da degeneração walleriana, melhor reorganização fascicular, aumento do colágeno novo, início de formação de bainha de mielina, além de maior marcação do anticorpo NF nos grupos experimentais em relação ao GC. O grupo ADSC apresentou melhores resultados em relação aos demais, enfatizando a eficiência das ADSCs em relação à FCM e ao GC para a regeneração nervosa periférica. Porém, há a necessidade de um tempo maior para ocorrer completa regeneração do tecido nervoso periférico após lesão.