Microorganisms, particularly bacteria, can associate together to form complex communities called biofilms. These communities are embedded in extracellular polymeric substances and can form on numerous surfaces such as implanted devices (catheters, central lines, joint replacement) in patients. These biofilms cause bloodstream and systemic infections that are difficult to treat and increase the chances of sepsis. Previously, our laboratory has identified a protein secreted by Klebsiella that has inhibitory effects on other members of the Enterobacteriacea bacterial family, namely Citrobacter and Enterobacter. Our current interest lies in the ability of the protein to potentially inhibit this bacterial family from establishing biofilms. In the present study, we wanted to explore: (1) if it is possible to form Citrobacter and Enterobacter biofilms in 6-well plates and on microscope coverslips; (2) if treating these biofilms with the secreted protein shows inhibition similar to previous planktonic cultures; (3) if these biofilms and inhibition could be visualized by a variety of staining techniques. To determine if it is possible to create Citrobacter and Enterobacter biofilms, bacteria were inoculated into 6-well plates, grown under static conditions in a 37°C incubator for 24 hours, and stained with crystal violet. Images showed that robust biofilms grew in the 6-well control plates while wells treated with the Klebsiella protein displayed reduced biofilms. To determine if it was possible to see Citrobacter and Enterobacter biofilms at a microscopic level, microscope slides were placed into 6-well plates, treated as above, and the slides were Gram stained. Images show thick biofilms consisting of Gram negative rods on control slides, while slides treated with the Klebsiella molecule become sparse and poorly grown. To determine if Citrobacter and Enterobacter biofilms could be fluorescently labeled for visualization, the same process was employed as above, but stained with a LIVE/DEAD cell viability kit where live cells fluoresce green and dead cells fluoresce red. Control slides showed bright thick green fluorescing biofilms while slides treated with the Klebsiella molecule had fewer green fluorescing cells and some red cells. From these observations, it is our conclusion that: (1) it is possible to grow Citrobacter and Enterobacter biofilms on both 6-well plates and microscope slides; (2) Citrobacter and Enterobacter biofilms can be visualized both by simple staining and fluorescent staining; (3) Citrobacter and Enterobacter biofilms are inhibited by Klebsiella secreted proteins. Currently, the identity of this protein is unknown. However, it is possible that this unknown protein could be of future use in the treatment of bacterial biofilms one identified.

No description available.

Enterobacter sakazakii has been associated with powdered infant formula outbreaks which caused high mortality rate illnesses in infants in recent years. Current research was mainly focused on searching for natural antimicrobial agents which may be incorporated into baby foods to control this emerging pathogen. Yogurt and muscadine products were used in this study. The antimicrobial effects of yogurt were evaluated on agar plates and in a simulated gastrointestinal model. In the agar spot tests, diluted yogurt sample containing lactic acid bacteria at 106 CFU/mL was antagonistic toward E. sakazakii. However, the antimicrobial effect of yogurt on E. sakazakii in the simulated GI model was not noted. Certain numbers of tested E. sakazakii and lactic acid bacteria in yogurt were able to survive the acidic gastric condition and recovered in the intestinal model. By measuring the viable E. sakazakii cells in liquid cultures, the strong antimicrobial activities of muscadine juices and muscadine seed extracts were demonstrated. Within two hours, all inoculated E. sakazakii at 106 CFU/mL were decreased to non-detectable level. Juice and seed extract from dark-skinned muscadine demonstrated stronger antimicrobial activities than those form white-skinned muscadine. The characteristics of muscadine juices and seed extracts were also analyzed. The high phenolics and organic acid contents, such as ellagic, gallic, tannic, and tartaric acids in muscadine were correlated to the inhibitory effect observed.

A interação da bactéria diazotrófica Enterobacter sp.ICB481 com plântulas de cana-de-açúcar foi avaliada em culturas com adubação orgânica ou convencional. As plântulas foram separadas em grupos: orgânico, convencional e sem adubação (inoculadas e não inoculadas com a bactéria). Após 60 dias, os fertilizantes orgânico e convencional atuaram de forma semelhante nos parâmetros de crescimento: comprimentos foliar, volume radicular, massas fresca e seca em folhas, concentrações de nitrato, amônio, aminoácidos, proteínas, açúcares e teor de clorofila. O parâmetro de massa fresca radicular foi maior no grupo orgânico. A inoculação de Enterobacter promoveu maior volume e massa fresca radicular nos grupos orgânico e convencional. No grupo sem adubação, a inoculação aumentou todos os parâmetros de crescimento, clorofila total, incremento protéico e conteúdo de nitrogênio. / The interaction of diazotrophic bacterium Enterobacter sp. ICB481 in sugarcane plantlets was evaluated in cultures with organic or conventional fertilization. Plantlets were separated into groups: organic, conventional and no fertilization (inoculated or not with the bacterium). After 60 days, the organic and conventional fertilizers presented similar growth parameters for: leaf length, root volume, fresh and dried matter of leaves, concentrations of nitrate, ammonium, amino acids, proteins, sugars and chlorophyll content. The parameter of fresh matter was higher in the organic group. The inoculation of Enterobacter promoted greater root volume and fresh matter in organic and conventional groups. In the group without fertilizer, inoculation increased all growth parameters, chlorophyll, protein and nitrogen content.

Made available in DSpace on 2015-07-22T20:49:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-26. Added 1 bitstream(s) on 2015-08-11T03:25:45Z : No. of bitstreams: 1 Publico-10833.pdf: 1360435 bytes, checksum: a345766b88aa33fb633cad88c29a3a08 (MD5) / Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq) / Introducao: A hiper-producao de AmpC associada a impermeabilidade de membrana externa em Enterobacter spp. pode contribuir no fenotipo de resistencia a cefalosporinas de quarta geracao e aos carbapenemicos. O objetivo do estudo foi caracterizar os mecanismos de resistencia a cefepima e a ertapenem em 11 amostras de E. cloacae subespecie cloacae isoladas de hemoculturas no Hospital sao Paulo. Materiais e Metodos: O perfil de sensibilidade foi avaliado por disco difusao e pela tecnica de diluicao em agar, de acordo com o CLSI. A analise das proteinas de membrana externa (OMPs) foi realizada pela tecnica de SDS-PAGE. A similaridade genetica foi avaliada pela tecnica de PFGE e o coeficiente similaridade entre os padroes de PFGE obtidos foi calculado pelo programa Bionumerics. Os isolados foram tambem testados contra cefepima, ertapenem, imipenem e meropenem na presenca e na ausencia dos inibidores de bomba de efluxo PAƒÀN (25 ƒÊg/ml) e reserpina (20 ƒÊg/ml), pela tecnica de diluicao em agar. Foram realizados testes de hidrolise, disco-aproximacao para a deteccao fenotipica de ESƒÀL e testes de inducao para a deteccao de AmpC induzida. A presenca de genes codificadores de ESƒÀL, carbapenemases e AmpC plasmidiais foram avaliados pela tecnica de PCR e confirmadas por sequenciamento, bem como a presenca de integrons. Os niveis de expressao de ampC, ompC e ompF foram medidos pela PCR em tempo real. Resultados: Todos os isolados de E. cloacae subespecie cloacae foram resistentes as cefalosporinas, aztreonam, associacoes com inibidores de ƒÀ-lactamases, tetraciclina, ciprofloxacina e amicacina por disco difusao. A tecnica de diluicao em agar demonstrou altas CIMs para cefotaxima (CIM50, > 256 ƒÊg/mL), ceftazidima (CIM50, 256 ƒÊg/mL) e cefepima (CIM50, 256 ƒÊg/mL). Todos os isolados foram sensiveis a imipenem (CIM50, 0.25 ƒÊg/mL) e meropenem (CIM50, 0.25 ƒÊg/mL). Entretanto, 7 isolados apresentaram CIMs de 4 ƒÊg/ml para ertapenem, 3 isolados foram resistentes (CIMs de 8 ƒÊg/ml) e um isolado foi sensivel (CIM de 2 ƒÊg/ml). Quatro padroes de PFGE foram encontrados entre os isolados de E. cloacae subespecie cloacae multirresistentes. Pelo programa Bionumerics, 3 clusters foram observados com coeficiente de similaridade acima de 80%. Todas as amostras foram positivas para ESƒÀL no teste de hidrolise e no disco aproximacao. Os resultados da PCR apresentaram amplificacao para blaTEM e blaCTX-M. Hiperexpressao do gene ampC foi detectado em todos os isolados pela qRT-PCR. Somente uma amostra apresentou uma pequena diminuicao da expressao de OmpC. Atraves do gel de SDS-PAGE foi observado um perda de uma proteina de 43 kDa quando comparado ao controle sensivel. Nenhuma reducao significativa nas CIMs para ƒÀ-lactamicos foi observado na presenca dos inibidores de bomba de efluxo. Conclusoes: Os resultados mostraram que a hiper-producao de AmpC associada a producao de ESBL sao os responsaveis pelos altos niveis de resistencia a cefepima e a perda de uma proteina de 43 kDa associada a hiper-producao de AmpC provavelmente foram os responsaveis pela diminuicao da sensibilidade a ertapenem verificada entre os isolados de E. cloacae subespecie cloacae. / Background: The overproduction of AmpC in Enterobacter spp. can contribute to resistance phenotype to fourth-generation cephalosporins and to carbapenens. The mechanisms of resistance to cefepime and ertapenem were studied among 11 E. cloacae subespecie cloacae clinical isolates from blood cultures isolated at Sao Paulo Hospital. Methods: Susceptibility testing by disk diffusion and agar dilution techniques was performed according to CLSI. Analysis of outer membrane protein (OMP) was performed by SDS-PAGE. The genetic relatedness was evaluated by PFGE and the dendogram was obtained by Bionumerics program. The isolates were also tested against cefepime, ertapenem, imipenem and meropenem with and without of PAƒÀN (25 ƒÊg/ml) and reserpine (20 ƒÊg/ml), an efflux pump inhibitors by agar dilution. Genes for ESƒÀLs, carbapenemases and plamidial AmpCs were screened by PCR and confirmed by sequencing. The expression level of ampC, ompC and ompF genes was measured by real time PCR (qRT-PCR). Results: All isolates were resistant to cephalosporins, aztreonam, ƒÀ-lactamases association inhibitors, tetraciclin, ciprofloxacin and amikacin, by disk diffusion. All isolates showed high resistant phenotype to cefotaxime (MIC50, > 256 ƒÊg/mL), ceftazidime (MIC50, 256 ƒÊg/mL), and cefepime (MIC50, 256 ƒÊg/mL) but they were susceptible to imipenem (MIC50, 0.25 ƒÊg/mL) and meropenem (MIC50, 0.25 ƒÊg/mL). Seven isolates showed ertapenem MICs of 4 ƒÊg/ml, while other three isolates showed MICs of 8 ƒÊg/ml and one isolate showed MIC of 2 ƒÊg/ml. Four clusters were founded by the Bionumerics program. The PCR results showed amplification for blaTEM and blaCTX-M. Hyperexpression of ampC was detected in all isolates by qRT-PCR. Only one sample showed a decreased in the OmpC expression. The loss of an OMP of 43 kDa was observed for all isolates in the SDS-PAGE technicque compared to the susceptible control. No significant reduction for â-lactam’s MICs was observed in the presence of PAâN. Conclusions: The results showed that the hyperproduction of AmpC coupled with ESBL production were responsible for the high resistant rates to cefepime. The hyperproduction of AmpC coupled with the loss of an OMP of 43 kDa is likely to be responsible for the reduced susceptibility to ertapenem in the E. cloacae subespecie cloacae isolates, which seems not to be associated with efflux systems. / TEDE / BV UNIFESP: Teses e dissertações

Orientadores: Arnaldo Yoshiteru Kuaye, Marcelo Palma Sircili / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T20:40:16Z (GMT). No. of bitstreams: 1 Esper_LucianaMariaRamires_D.pdf: 999342 bytes, checksum: 3f9fabfdcb18d73bafce5fd0ce00935e (MD5) Previous issue date: 2010 / Resumo: A contaminação de fórmulas infantis por Enterobacter sakazakii (Cronobacter spp.) e Bacillus cereus pode ter como origem o contato do alimento com biofilmes formados em ambientes, utensílios e equipamentos empregados na sua produção ou posterior reconstituição nos locais de distribuição. A grande preocupação em relação a estas bactérias é a presença das mesmas em fórmulas infantis, produtos estes utilizados como fonte de alimentação para lactentes de forma exclusiva ou em combinação com outros alimentos. A formação de biofilmes, assim como outros mecanismos celulares como por exemplo a produção de bacteriocinas e fatores de virulência, podem ser modulados pelo processo de comunicação célula-célula ou quorum sensing - mecanismo de sinalização célula-célula mediada pelo acúmulo de uma classe ou mais de moléculas sinalizadoras produzidas pela célula e excretadas para o meio externo. Por sua vez, a quebra deste sistema, pela degradação das moléculas sinalizadoras de comunicação, denomina-se quorum quenching. Neste trabalho objetivou-se, primeiramente, a avaliação da dinâmica de formação de biofilmes mono e multi-espécies de E. sakazakii (Cronobacter spp.) e B. cereus em superfície de aço inoxidável utilizando-se como meios de cultivos fórmula Infantil (FI) e caldo Luria Bertani (LB) e a eficácia de soluções de ácido peracético e de hipoclorito de sódio na inativação desses biofilmes. Outro objetivo principal foi pesquisar a ocorrência dos sistemas quorum sensing e quorum quenching em E. sakazakii (Cronobacter spp.) e B. cereus e a possível influência das moléculas sinalizadoras na sensibilidade destas bactérias aos antimicrobianos. A formação de biofilmes ocorreu de forma mais intensa ao utilizar-se a fórmula infantil, quando comparado com o meio de cultivo LB, fato este relevante por ser a fórmula infantil o mais conhecido e importante veículo de transmissão de E. sakazakii (Cronobacter spp.). Tanto em cultivo mono-espécie quanto em cultivo misto, o nível de contagem de E. sakazakii (Cronobacter spp.) superou o de B. cereus e para ambos o maior desenvolvimento ocorreu em cultivos mono-espécie. Em todos os biofilmes de E. sakazakii (Cronobacter spp.) e B. cereus isoladamente e em cultura mista, produzidos em caldo LB e independente do tempo de formação, as contagens foram reduzidas a níveis inferiores a 1 logUFC/cm2 quando em contato com soluções de ácido peracético ([500mg/L]) e hipoclorito de sódio ([100mg/L]) por 15 minutos. No entanto, para os biofilmes produzidos em fórmula infantil ocorreram algumas situações, em que as contagens de microrganismos após o contato com as soluções de sanitizantes, foram superiores a 1 logUFC/cm2, evidenciando assim a ineficácia do procedimento de sanitização para alguns biofilmes formados a 5 dias ou mais e a necessidade da higienização das superfícies ser realizada o mais próximo do término do processamento ou reconstituição destes alimentos. Evidenciou-se a existência dos sistemas quorum sensing e quorum quenching, através dos testes de atividades biológicas das culturas, extratos e suas frações. Os testes com os biossensores revelaram-se positivos para a produção de homoserinas lactonas em cepa de E. sakazakii (Cronobacter spp.) e negativos para a espécie B. cereus. Em cultivo misto de E. sakazakii (Cronobacter spp.) e B. cereus observou-se a redução e/ou a não detecção das homoserinas lactonas fato este possivelmente associado ao fenômeno quorum quenching. A possível presença de moléculas sinalizadoras AI- 2 e AI-3 foi evidenciada, sendo confirmada a presença de moléculas sinalizadoras AI-1. A caracterização do AI-1 realizada por cromatografia gasosa acoplada à espectrometria de massa (CG-EM) revelou a capacidade da cepa de E. sakazakii (Cronobacter spp.) em produzir três compostos, N-heptanoil-HSL, N-dodecanoil- HSL e N-tetradecanoil-HSL, substâncias estas ainda não reportadas na literatura para o microrganismo em estudo. Os autoindutores sintéticos C7-HSL, C12-HSL e C14-HSL adicionados a cultura de E. sakazakii (Cronobacter spp.) e B. cereus não exerceram efeito sobre a sensibilidade aos antimicrobianos, sugerindo que estas moléculas não estariam envolvidas em mecanismos de resistência a estes antimicrobianos. Em resumo este trabalho representa um importante passo no estudo da formação de biofilmes e dos sistemas quorum sensing e quenching para as bactérias em questão, cujos conhecimentos são de grande interesse na segurança dos alimentos / Abstract: The contamination of infant formulas by Enterobacter sakazakii (Cronobacter spp.) and Bacillus cereus can occur due to contact with biofilms formed in the environment and on equipment used in their production and reconstitution. There is concern about these bacteria due to their presence in foods used as a source of nutrition for infants. Biofilm formation as a wide spectrum of important processes is reported to be regulated by quorum sensing, including, for example, antibiotic production and virulence. Cell-to-cell communication or bacterial quorum sensing is a signaling mechanism that refers to the ability of bacteria to respond to chemical molecules called autoinducers, in response to cell density. Degradation of the signaling molecule prevents it from accumulating in sufficient amounts, leading to disruption of the communication system, known as quorum quenching. The aim of this study was first to evaluate the formation of mono and multi-species biofilms of Enterobacter sakazakii (Cronobacter spp.) and B. cereus on stainless steel surfaces using infant formula (FI) and Luria Bertani (LB) broth as the culture media, and the efficacy of sodium hypochlorite and peracetic acid in inactivating these biofilms. The other objective was to investigate the involvement of Enterobacter sakazakii (Cronobacter spp.) and Bacillus cereus in quorum sensing and quorum quenching systems and the possible influence of the signaling molecules on the sensibility of these bacteria to antimicrobials. The formation of biofilms was greater when using the infant formula than the Luria Bertani broth, this fact being important since the infant formula is considered to be an important vehicle in the transmission of Enterobacter sakazakii (Cronobacter spp.) to infants. In both cases, the growth of the mono species was greater than in the multi species culture. In all the biofilms formed in the Luria Bertani broth, independent of the time of formation, the counts were reduced to less than 1 log CFU/cm2 when in contact with sodium hypochlorite ([100mg/L]) or peracetic acid ([500mg/L]). However for the biofilms produced in the infant formula, many situations occurred, with counts more than1 log CFU/cm2 in some situations after the contact with sanitizers highlighting the need for na efficient sanitization of the surfaces as soon as the processing or reconstitution of these foods has finished. The experiments with biosensors provided evidence of the occurrence of quorum sensing and quorum quenching systems using bioassays with the cultures and extracts of the microorganisms under examination. These bioassays were positive for the production of homoserine lactone by E. sakazakii (Cronobacter spp.) but negative for B.cereus. In the mixed culture a reduction and/or non-detection of the homoserine lactone was observed, a fact that could be associated with quorum quenching. A possible presence of the signaling molecules AI-2 and AI-3 was evident and the presence of AI-1 was confirmed. Gas chromatography-mass spectrometry (GC-MS) analyses allowed for a chemical identification of the production of N-heptanoyl-HSL, N-dodecanoyl- HSL and N-tetradecanoyl-HSLby the E. sakazakii (Cronobacter spp.), a fact not previously reported in the literature. The addition of synthetic signaling molecules (N-heptanoyl-HSL, N-dodecanoyl-HSL and N-tetradecanoyl-HSL) had no significant effect on the sensibility of the E. sakazakii (Cronobacter spp.) and B. cereus strains to antimicrobials. In summary this study represents an important step in biofilm's formation, quorum sensing and quorum quenching in these bacteria that are of great interest in food safety / Doutorado / Doutor em Tecnologia de Alimentos

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-08-21T18:43:09Z No. of bitstreams: 2 Thaís Luft da Silva Neitzke.pdf: 946176 bytes, checksum: 7f73bad9b6143ec1effc7bfee257ab65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-21T18:43:09Z (GMT). No. of bitstreams: 2 Thaís Luft da Silva Neitzke.pdf: 946176 bytes, checksum: 7f73bad9b6143ec1effc7bfee257ab65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-20 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná (FA) / In the search for agronomic alternatives that are less aggressive to the environment, the substitution of mineral fertilizers by biofertilizers based on plant-beneficial micro-organisms was initiated, among them plant growth promoting bacteria. The objective of this work was to characterize rhizobacteria isolated from soils with different crop managements, grouping the samples based on their genetic characteristics (16S rDNA sequencing, amplification of the ITS 16–23S rDNA region, feoB, entC and entF genes) and biochemical (indole-acetic acid (IAA) production, acetoin production and phosphate solubilization), and verify their diversity within groups. Based on the results, the presence of the genes feoB, entF and entC was verified in 81.5%, 48.1% and 81.5% of the samples, respectively. Most of the strains (96.3%) had the capacity to produce IAA, 59.2% were able to produce acetoin and 81.5% of them to solubilize phosphate. By the analysis of congruence and grouping, there was similarity of genetic or biochemical patterns between the groups; however, there was great diversity among the isolates within each group. In general, it was observed that the conservationist managements presented the best performances. The study revealed that strains 130, 219, 302 and 326 presented biotechnological potential for at least two of the characteristics evaluated. / Na busca por alternativas agronômicas que sejam menos agressivas ao meio ambiente, iniciou-se a substituição do fertilizante mineral por biofertilizante a base de microrganismos benéficos às plantas, entre estes as bactérias promotoras do crescimento vegetal. O objetivo deste trabalho foi caracterizar rizobactérias isoladas de solos com diferentes manejos de cultivo, agrupando as amostras com base em suas características genéticas (sequenciamento 16S rDNA, amplificação da região ITS 16-23S rDNA, genes feoB, entC e entF) e bioquímicas (produção de ácido-indol-acético (AIA), produção de acetoína e solubilização de fosfato), e verificar a sua diversidade dentro dos grupos. Baseado nos resultados, foi verificada a presença do gene feoB, entC e entF em 81,5%,48,1% e 81,5% das amostras, respectivamente. A maioria (96,3%) das estirpes apresentou a capacidade de produzir AIA, 59,2% foi capaz de produzir acetoína e 81,5% delas à solubilizar fosfato. Pela análise de congruência e agrupamento, verificou-se semelhança de padrões genéticos ou bioquímicos entre os grupos, no entanto, apresentaram grande diversidade entre os isolados dentro de cada grupo. De forma geral, observou-se que os manejos conservacionistas apresentaram os melhores desempenhos. O estudo revelou que as estirpes 130, 219, 302 e 326 apresentaram potencial biotecnológico para pelo menos duas das características avaliadas.

Orientador: Jose Luiz Pereira, Valeria Chritina Amstalden Junqueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T20:30:44Z (GMT). No. of bitstreams: 1 Santos_RosanaFranciscoSiqueirados_M.pdf: 1367464 bytes, checksum: 1f8a0a9b0f9ec558cd3f583d15def385 (MD5) Previous issue date: 2006 / Mestrado / Mestre em Ciência de Alimentos

The fungal pathogen Candida albicans and the opportunistic bacterial pathogens Enterobacter aerogenes and Enterobacter cloacae are common sources of human disease. The colonization of proximal anatomical locations by these pathogens suggests that interspecies polymicrobial interactions between Candida albicans and Enterobacter species occur. In order to understand mechanisms of diseases caused by these pathogens and to further the study of disease prevention, analyzation of their combined activities was conducted in this study. Changes in fungal morphology, cellular viability, and colony density were investigated using fungal and bacterial co-cultures. The effects of the Candida secreted quorum sensing molecule farnesol on Enterobacter aerogenes and Enterobacter cloacae was studied to observe changes in Enterobacter viability and colony density. The effects of the presence of Enterobacter species on Candida albicans was studied by observing changes in Candida morphology and colony density. The mutant strain of Candida albicans AlS6-/- was also cultured with Enterobacter to determine if the presence of the ALS6 surface glycoprotein gene affected Candida viability and colony density in the presence of Enterobacter species. Statistically significant decreases were observed in all studied metrics between experimental and control groups. This indicated that the interactions observed between Candida albicans and Enterobacter species represent reciprocal inhibitions of cellular functionalities. As Candida albicans is the primary cause of human fungal infections and Enterobacter species are common causes of opportunistic infections, the study of polymicrobial interactions between Candida and Enterobacter species as conducted in this study is important to furthering efforts of human disease inhibition.

Enterobacter sakazakii é um bacilo Gram-negativo, pertencente à família Enterobacterieceae. Este microrganismo vem ganhando a atenção das autoridades de saúde pública ao redor do mundo, não tanto pela morbidade, que é baixa, mas pela elevada taxa de mortalidade que varia de 40-80%. O patógeno afeta principalmente recém-nascidos de baixo peso e bebês com até seis meses de idade. Em comum, estas crianças têm o fato de serem alimentadas com fórmula infantil desidratada, a base de leite. Em nosso país ainda não existem muitos estudos sobre a ocorrência deste patógeno em fórmulas infantis, nem no ambiente de preparo das mesmas. O objetivo deste trabalho foi avaliar as condições de produção de mamadeiras para recém-nascidos em maternidades da Grande São Paulo, além de determinar a população de E. sakazakii em fórmulas infantis desidratadas e reidratadas. A população de Enterobacteriaceae e a presença de E. sakazakii também foram avaliadas em amostras ambientais, de utensílios e mão de manipuladores. Avaliou se ainda o comportamento do patógeno em fórmula infantil reidratada simulando as condições de oferecimento aos bebês. Coletou-se amostras de três hospitais maternidades diferentes (A-escola/B-público/C-particular) e analisou-se a presença de E. sakazakii usando método ISO. Para fórmulas desidratadas e reidratadas usou-se a mesma metodologia e a técnica de número mais provável (NMP). A população de Enterobacteriaceae foi determinada usando-se PetrifilmTM 3M. E. sakazakii foi detectada em duas amostras do Hospital A (na sobra da mamadeira que voltou do berçário e de uma amostra da lata lacrada da fórmula infantil desidratada. A população nesta amostra foi de 0,03 NMP/100g.) No Hospital B, foi detectada em apenas uma amostra (na esponja de lavagem das mamadeiras contaminadas). No Hospital C, E. sakazakii não foi detectada nas amostras analisadas. Quanto à população de Enterobacteriaceae nos lactários, observou-se uma variação, sendo que as amostras colhidas no hospital C foram as que apresentaram populações mais elevadas. As cepas de E. sakazakii isoladas apresentaram comportamento similar àquele da cepa padrão, ocorrendo um aumento de 2 log na população do patógeno quando simulou-se as condições de serviço das fórmulas, via naso-gástrica, aos bebês nos berçários. / Enterobacter sakazakii is a bacillus belonging to the Enterobacteriaceae family. It is considered an opportunistic pathogen that has been gaining attention from health authorities all over the world. While morbidity associated with this bacterium is low, mortality rates can range from 40-80%. The pathogen affects mainly low-birth-weight neonates (first 28 days), but babies less than 6 month old are also at risk. Powdered infant formula has been incriminated as the possible source of the microorganism to the infected babies. ln Brazil, as in several other countries, there is scarce information regarding the incidence of E. sakazakii in powdered infant formula, in reconstituted formula, and in milk kitchens areas in hospitals. The objective of this study was to evaluated the presence of E. sakazakii in the environment, utensils, handlers, powdered and rehydrated infant formula from milk kitchens from different maternity wards in Sao Paulo, Brazil. Moreover, it was evaluated the behavior of the pathogen in rehydrated infant formula. Samples were collected from 3 hospitals maternities (A-school/B-public/C-particular) and analyzed for E. sakazakii using the ISO method. For formula (powdered or rehydrated) the MPN technique was used. Enterobacteriaceae population was determined using PetrifilmTM 3M. E. sakazakii was found in one unopened formula can collected from Hospital A (0,03 MPN/100g), although the pathogen could not be detected in other cans from the same lot. E. sakazakii was also found in leftovers from one nursing bottle from the same hospital and from one cleaning sponge from Hospital B. E. sakazakii was not detected in none of the samples from Hospital C. A variation in Enterobacteriaceae population in milk kitchens was observed. Samples collected in Hospital C presented the highest population. Isolated strains of E. sakazakii presented similar behavior to standard strains, When spiked in rehidrated infant formula. A 2 log increase in the population of the pathogen was observed when simulating the conditions of formula administration to the babies by naso-gastric tubing.