Individual cells employ mechanisms of cell-autonomous immunity to defend their cytosol against bacterial invasion. One such mechanism involves indirect detection of the pathogen through recognition of pathogen-induced disturbances causing the appearance of specific host molecules in an abnormal location. For example, glycans, which are located on the extracellular leaflet of the plasma membrane under homeostatic conditions, become hidden inside bacteria-containing vacuoles (BCVs) during bacterial entry into the cell. Upon BCV rupture, glycans become exposed to the cytosol where they act as a danger signal and are detected by the cytosolic danger receptor, Galectin 8. My research reveals that sphingomyelin, a host lipid predominantly located on the outer leaflet of the plasma membrane, is exposed to the cytosol on damaged BCVs. I visualised the appearance of intracellular sphingomyelin by utilising Lysenin - a sphingomyelin-specific toxin from earthworms – as a cytosolic sphingomyelin reporter. Lysenin is recruited to BCVs in a sphingomyelin-dependent manner upon cytosolic entry of both Gram-negative and Gram-positive bacteria. Lysenin co-localises with Galectin 8 on a proportion of BCVs, indicating that sphingomyelin exposure occurs upon membrane damage. Moreover, I elucidated that sphingomyelin exposure occurs before glycan exposure on damaged BCVs indicating that BCV rupture may proceed through two stages: ‘minor’ and ‘major’ damage. My investigations into possible causes of vacuole rupture are on going. To identify endogenous cellular receptors for cytosol-exposed sphingomyelin, I established and executed an assay to compare enrichment of mammalian cell lysate proteins on liposomes containing or lacking sphingomyelin. Following mass spectrometry analysis, 49 candidate proteins were tested for recruitment to Salmonella. Twelve candidates were recruited to BCVs upon infection. Of these twelve, I pursued five candidates in greater detail due to their recruitment to Salmonella being either entirely unknown, or known, but via a non-sphingomyelin mechanism. Further analysis of one candidate in particular, TECPR1, elucidated that TECPR1 is recruited to Salmonella in a sphingomyelin-dependent manner and possesses sphingomyelin-specific binding properties in vitro. Therefore, my thesis research identifies TECPR1 as an endogenous sphingomyelin-binding protein.

Orientador: José Paes de Almeida Nogueira Pinto / Banca: Vera Lúcia Mores Rall / Banca: Luciano dos Santos Bersot / Resumo: Apesar dos recentes avanços tecnológicos na indústria de alimentos, ainda se observa a ocorrência de inúmeras enfermidades de origem alimentar, devido à ingestão de alimentos contaminados por micro-organismos patogênicos, dentre eles os do gênero Salmonella. Dentre os produtos envolvidos em processos de contaminação por este agente, as carnes são as mais importantes, sendo a de aves o veículo em numerosos casos de infecções humanas. Além dos problemas em saúde pública, o micro-organismo causa grandes problemas devido à formação de biofilmes na indústria, sendo que esta ocorre em virtude da adesão dos micro-organismos em uma superfície de contato, a qual se fixam, constituem uma matriz de exopolissacarídeos e iniciam seu crescimento. Estes representam uma preocupação à indústria de alimentos por seu efeito como fonte de contaminação crônica, que aumenta as chances de veiculação do patógeno ao consumidor. Inúmeros são os fatores que condicionam o desenvolvimento do biofilme, entres eles o tipo de superfície e suas propriedades físico-químicas, já que as mesmas exercem influência direta sobre a adesão dos micro-organismos. Diversas medidas de controle para resolver o problema são empregadas na indústria, como o uso de sanitizantes, em destaque os à base de ácido peracético, que embora tenham o seu efeito sob células sésseis comprovado, a sua ação sob biofilmes ainda necessita de maior comprovação científica. Nesse sentido, os dados obtidos por este trabalho ratificaram a importância de Salmonella spp. como formadora de biofilme em diferentes superfícies, o que pode induzir a uma maior permanência destes micro-organismos no ambiente de processamento na indústria e, consequentemente, a uma maior chance de contaminação dos alimentos, com riscos ao consumidor. Nossos resultados também confirmaram que as propriedades físico-químicas da superfície dos diferentes materiais influenciaram ... / Abstract: Despite recent technological advances in the food industry, still observed the occurrence of numerous food borne illnesses due to ingestion of contaminated by pathogenic microorganisms, including members of the genus Salmonella spp. Among the products involved in processes of contamination by this agent, the steaks are the most important, being the vehicle of birds in numerous cases of human infections. In addition to problems in public health, the microorganism causes big problems due to the formation of biofilms in the industry, and this occurs because of the accession of microorganisms on a surface of contact, which are fixed, are an array of exopolysaccharides and begin their growth. These are a concern to the food industry for its effect as a source of chronic infection, which increases the chances of placement of the pathogen to the consumer. There are many factors that affect biofilm development, entres they the type of surface and its physicochemical properties, since they will directly influence the adhesion of microorganisms. Several control measures to solve the problem are employed in the industry, such as the use of sanitizers, featured in the peracetic acid, though they have proven their effect on sessile cells in biofilms its action still needs more scientific evidence. In this sense, the data obtained from this study confirm the importance of Salmonella spp. as forming biofilm on different surfaces, which can induce a greater permanence of these microorganisms in the processing environment in the industry and hence a greater chance of food contamination, with risks to the consumer. Our results also confirmed that the physicochemical surface properties of different materials directly influence the adhesion of microorganisms and, consequently, the development of the biofilm. In simple terms, the greater the hydrophobicity of the surface, the greater the ability to promote the development of the biofilm, in order that the ... / Mestre

Para avaliar a diversidade de Salmonella enterica, foram analisados os sorovares presentes em amostras de Salmonela isoladas de aves e seu ambiente do Sul do Brasil (n=155), Colômbia (n=141) e Mississippi (EUA, n=50). No total, foram examinados 346 isolados de Salmonela pela técnica de ribotipificação de sequências Intergênicas (ISR) usando PCR convencional (para bactérias vivas isoladas no Estado de Mississippi) ou nested PCR (para o DNA de Salmonelas do Brasil e da Colômbia, em cartões FTA). O sequenciamento da região intergênica do gene dkgB avalia polimorfismos de nucleotídeos simples que ocorrem em torno do gene ribossomal 5S. A concordância geral entre o esquema Kauffman-White-LeMinor (KWL) e a ISR foi de 85,2% em isolados do Brasil, e de 89,58% em isolados do Mississippi. É possível que a divergência seja devida à capacidade da ISR de detectar as misturas de sorovares numa cultura. Os sorovares de Salmonella enterica identificados nos isolados brasileiros foram: Heidelberg, Enteritidis, Hadar, Typhimurium, Gallinarum, Agona, Cerro, Livingstone, Infantis, Isangi, Mbandaka, Montevideo e Senftenberg. Três sorovares ISR únicos foram detectados (UN0041, UN0042, UN0043). Nos isolados colombianos, foram identificados os sorovares Enteritidis, Gallinarum, Isangi, Heidelberg, Paratyphi B var. Java, Tennessee, Saintpaul, Agona, Isangi, Mbandaka, Urbana, Albany, Javiana, Fresno, Miami, Muenster, Rissen, Braenderup, Yoruba e um sorovar ISR Único (UN0048). Já no tocante aos isolados de Mississippi, foram identificados os sorovares Enteritidis, Typhimurium, Kentucky, Bredeney, Mbandaka, Saintpaul, Montevideo, Cubana, Lille, Senftenberg, Johannesburg e um ISR Único (UN0094). Em geral, a ISR forneceu mais informações do que KWL sobre a ecologia de Salmonella enterica das aves comerciais. Em 73 isolados da Colômbia e 50 isolados de Mississippi, foram estabelecidas as resistências para 15 e 17 agentes antimicrobianos, respectivamente. Não foram identificados isolados pansusceptíveis. Todas as amostras analisadas foram classificadas como multirresistentes. Os resultados deste estudo mostram uma grande diversidade entre os isolados analisados, não só com respeito aos sorovares identificados em cada país, mas também sobre a sua resistência aos antimicrobianos. Os resultados desta pesquisa irão beneficiar a indústria avícola do sul do Brasil, da Colômbia e de Mississippi, porque a caracterização de cepas isoladas de aves comerciais poderá ser usada no futuro para o desenvolvimento e adaptação de programas de controle. / To assess diversity of Salmonella enterica serotypes present in poultry and their environment from Southern Brazil (n=155), Colombia (n=141) and Mississippi (EUA, n=50); 346 isolates were examined with conventional PCR (live bacteria from Mississippi) or nested PCR (Brazilian and Colombian Salmonella DNA in FTA cards) and sequencing of the dkgB-linked intergenic sequence ribotyping (ISR) region that assesses single nucleotide polymorphisms occurring around a 5S ribosomal gene. Overall agreement between Kauffman-White-LeMinor (KWL) scheme and ISR was 85.2% in Brazilian Salmonella isolates, and 89.58% in Mississippi´s isolates. It is possible that the disagreement was due to the ability of ISR to detect mixtures of serotypes in culture. Salmonella enterica serotypes identified among Brazilian isolates were Heidelberg, Enteritidis, Hadar, Typhimurium, Gallinarum, Agona, Cerro, Livingstone, Infantis, Isangi, Mbandaka, Montevideo, and Senftenberg. Three unique ISRs were detected (UN0041, UN0042, UN0043). Regarding the Colombian isolates, serotypes Enteritidis, Gallinarum, Isangi, Heidelberg, Paratyphi B var. Java, Tennessee, Saintpaul, Agona, Isangi, Mbandaka, Urbana, Albany, Javiana, Fresno, Miami, Muenster, Rissen Braenderup, Yoruba, and One Unique ISR (UN0048) were identified and among Mississippian isolates, serotypes Enteritidis, Typhimurium, Kentucky, Bredeney, Mbandaka, Saintpaul, Montevideo, Cubana, Lille, Senftenberg, Johannesburg, and one Unique ISR (UN0094). Overall, ISR provided more information than KWL about the ecology of on-farm Salmonella enterica. In 73 isolates from Colombia and 50 isolates from Mississippi, there were established the antimicrobial resistance against 15 and 17 antimicrobial agents, respectively. No pansusceptible isolate was identified. All analyzed isolates were classified as Multidrug resistant. The results of this study show a great diversity among analyzed isolates, not only in respect to the serotypes, but also about their antimicrobial resistance. The results of this research will benefit the poultry industries of Southern Brazil, Colombia and Mississippi, because the characterization of strains isolated from poultry can be used in the future to the development and adaptation of Salmonella control programs.

Resumo: Foi desenvolvido um ensaio imunoenzimático do tipo ELISA indireto para a detecção de resposta sorológica de aves para Salmonella sorotipos Gallinarum, Pullorum, Enteritidis e Typhimurium. Utilizou-se antígeno solúvel obtido por meio de sonicação de cultura de Salmonella Gallinarum (AgSG), Salmonella Enteritidis cepa aflagelar (AgSE) e Salmonella Typhimuirum cepa aflagelar (AgTM), os conjugados peroxidase e fosfatase alcalina e amostras de soros positivos e negativos de vários sorotipos de salmonelas. Os resultados demonstraram que o AgSG pode ser utilizado diluído a 1:25.000 (peroxidase e fosfatase alcalina). Observou-se que o ELISA contendo S. Gallinarum como antígeno e fosfatase alcalina como enzima, propicia a separação de reações positivas para Gallinarum e Pullorum de Enteritidis. O AgSE pode ser utilizado diluído a 1:10.000 (peroxidase) ou 1:5.000 (fosfatase alcalina). Nestas condições, o ELISA/AgSE detectou resposta sorológica para os sorotipos Enteritidis, Gallinarum e Pullorum. O ELISA com o AgTM demonstrou que o antígeno pode ser diluído a 1:20.000 para ambos os conjugados. O ELISA/AgTM demonstrou reatividade entre salmonelas dos grupos B e D. Todas as amostras de soros testes devem ser analisadas diluídas a 1:1.000. Concluindo, o ELISA mostrou-se um teste útil para identificar aves com reação sorológica contra S. Gallinarum, S. Pullorum, S. Enteritidis e S. Typhimurium, podendo ainda identificar aves com sorologia positiva para S. Gallinarum, S. Pullorum sem que haja reação cruzada com amostras de soro de aves vacinadas ou infectada por S. Enteritidis. / Abstract: This study was done to assess the enzyme-linked immunosorbent assays (ELISA) for detection chicken serologic response against Salmonella enterica sorotypes Gallinarum, Pullorum, Enteritidis and Typhimurium. The test was performed using soluble proteins from Salmonella Gallinarum strain 9 (AgSG), from non-flagellate Salmonella Enteritidis strain (AgSE) and from not flagellate Salmonella Typhimurium (AgTM) strain as detecting antigen and peroxidase and alkaline phosphatase enzymes, as conjugate. According to the results, the antigen has to be diluted at 1:25.000 (AgSG, peroxidase and alkaline phosphatase). In addition, using alkaline phosphatase enzyme, the assay was helpful to separate positive serological reaction to serotypes Gallinarum and Pullorum from Enteritidis. To the ELISA/AgSE, the antigen has to be diluted at 1:10.000 for peroxidase assay and at 1:5.000 for alkaline phosphatase assay. In this condition, the ELISA/AgSE can detect serological reaction to S. Enteritidis, S. Gallinarum and S. Pullorum. To the ELISA/AgTM the antigen has to be diluted at 1:20.000 to both enzymes. In this condition the ELISA/AgTM showed sensibility but was no possible to separate positive serological reaction to serotype concerning at the group B and group D. In all test, the sample of serum has to be diluted at 1:1.000. Therefore, the ELISA was able to identity reactors birds to Salmonella antigens and also to detect serological response to S. Gallinarum, S. Pullorum antigen with no cross-reaction with serum samples taken from birds either challenged or vaccinated against S. Enteritidis. / Orientador: Angelo Berchieri Júnior / Coorientador: Hélio José Montassier / Banca: Raul José Silva Girio / Banca: Fernando Antonio de Ávila / Banca: Paulo Lourenço da Silva / Banca: Ana Maria Iba Kanashiro / Doutor

Metals are used as cofactors for enzymes, but are toxic in excess. In order to avoid the deleterious effects posed by metals, the cell must employ strict metal homeostasis systems. One such system is the Cue copper-resistance system in Salmonella enterica serovar Typhimurium (S. Typhimurium) which includes the periplasmic copper binding protein CueP. Previous studies have shown CueP to be a major periplasmic copper-sequestering protein that has a role in supplying copper to, and thus activating, the periplasmic Cu,Zn-superoxide dismutase enzyme SodCII (Osman et al., 2013). SodCII protects the cell from reactive oxygen species (ROS), due for example to the actions of the respiratory burst oxidase in host macrophages. However, despite its ability to sequester copper and activate SodCII, the precise physiological role of CueP in S. Typhimurium has remained unresolved since cueP mutants of S. Typhimurium strain SL1344 (the wild-type stain used in this study) do not exhibit a phenotype with respect to tolerance to copper or reactive oxygen species. In addition, the copper-binding mechanism of CueP and its interactions with other copper-binding proteins, including SodCII, have not been examined. An aim of this study was to establish a phenotype for a cueP mutant of S. Typhimurium with respect to copper and/or ROS tolerance. It was hypothesised that the possession of KatG (catalase) and multiple superoxide dismutases (SodCI, SodA and SodB), in addition to SodCII, by S. Typhimurium may confer functional redundancy with respect to copper and ROS tolerance. Hence mutants lacking katG (ΔkatG) or the various superoxide dismutase encoding genes (ΔsodA/ΔsodB/ΔsodCI/ΔsodCII) with and without functional cueP were generated. The ΔkatG mutants exhibited reduced catalase activity and reduced tolerance to hydrogen peroxide, consistent with the loss of KatG, however the additional loss of cueP did not reduce tolerance to hydrogen peroxide further. Similarly, tolerance to copper and extracellular superoxide was also unaltered in the ΔkatG/ΔcueP mutant. The tolerance of the various superoxide dismutase mutants to copper and various ROS was also unaffected by the presence or absence of CueP. To examine the role of CueP in SodCII activation in vivo, SodCII was over-expressed in S. Typhimurium (in a ΔsodA/ΔsodB/ΔsodCI/ΔsodCII background) with and without functional cueP and superoxide dismutase activity measured in both whole cells and periplasmic extracts. SodCII-dependent superoxide dismutase activity was successfully identified within the periplasmic extracts. However, surprisingly, the level of activity was unaffected by the presence 16 or absence of CueP and/or the addition of copper. It is possible that SodCII is thus able to scavenge sufficient copper for activity from the reagents used in these assays. Similarly, in an alternative approach to examine the role of CueP in vitro, both SodCII and CueP (WT and potential metal-binding residue mutant forms) were successfully over-expressed in E. coli and methods for their purification optimised (without the use of affinity tags). ICP-MS analysis indicated that a CuePC104S mutant contains > 18-fold less copper than the CueP WT protein. Furthermore, superoxide dismutase activity assays using purified proteins, indicated that the CuePC104S mutant was less able to activate SodCII than the WT CueP. Taken together, these results are consistent with a role for the Cys104 residue in copper-binding by CueP. Bioinformatics results suggest the presence of CueP or homologous genes in the presence of other bacteria, including pathogens such as Klebsiella, Yersinia and Shigella spp. Further understanding of the role of CueP and the systems used by S. Typhimurium to avoid both copper and ROS stress may inform the development of novel treatment strategies for bacterial diseases.

O interesse da Medicina Veterinária nas espécies silvestres tem aumentado gradativamente, principalmente no estudo dos contextos ecológicos de saúde. Dentro desse contexto, autores realizaram estudos com o objetivo de conhecer a importância de Salmonella sp. na saúde das aves silvestres e seu potencial de transmissão para humanos e outros animais. Informações sobre a prevalência e distribuição dos sorovares de salmonelas na população de animais silvestres e domésticos são essenciais para relacionar os possíveis reservatórios que possam ser responsáveis pela transmissão dessa zoonose. Este trabalho teve como objetivo a detecção de Salmonella sp. em psitacídeos clinicamente sadios por Reação em Cadeia da Polimerase (PCR). Foram coletados suabes cloacais de 280 psitacídeos mantidos em cativeiro no Estado do Rio Grande do Sul, pertencentes a treze espécies, provenientes de um zoológico, um criadouro conservacionista e um criadouro comercial. O DNA das amostras foi extraído pelo método de fenol-clorofórmio e examinados pela PCR com a utilização de um par de iniciadores que amplifica um fragmento de 284 pb do gene invA pertencente ao gênero Salmonella, resultando em 37 amostras positivas. Não houve diferença na prevalência de salmonela entre os três plantéis nem entre as 13 espécies analizadas. Não foi possível a detecção desse patógeno pela PCR com iniciadores para a identificação de S. Typhimurium, S. Enteritidis, S. Pullorum e S. Gallinarum, nem através da Técnica Microbiológica Convencional nas amostras detectadas pela PCR genérica, provavelmente devido a maior sensibilidade e especificidade da PCR genérica. De acordo com a revisão bibliográfica realizada, este foi o primeiro trabalho de detecção direta de Salmonella em psitacídeos utilizando a PCR. Os resultados indicaram que aproximadamente 13,2% dos psitacídeos mantidos em cativeiro eram portadores assintomáticos ou eram transientemente infectados pelo gênero Salmonella.

Este trabalho foi conduzido com o objetivo de gerar dados de resistência a agentes antimicrobianos de Salmonella Enteritidis (SE) isoladas de amostras clínicas e do ambiente criatório de aves, nos anos de 1999, 2000 e 2001, de cortes de frango, no ano de 1996, ambos na região Sul, bem como de carcaças de frango, nos anos de 2004 e 2005, na região Nordeste, detectar mutação no gene gyrA da região determinante de resistência a quinolonas das cepas que apresentaram resistência ao ácido nalidíxico e fagotipá-las. Realizou-se também a ribotipagem de 28 cepas de SE isoladas de carcaças resfriadas de frango no ano de 2004, na região Sudeste. Cento e dezesete cepas de SE foram submeitdos foram submetidas a testes de sensibilidade a agentes antimicrobianos e os resultados indicaram que 84,6% (99/117) das cepas de Salmonella Enteritidis apresentaram resistência a um ou mais agentes antimicrobianos, sendo que o maior percentual está entre as cepas isoladas de carcaças resfriadas de frango, 100% (17/17), seguidas pelas isoladas de cortes de frango, 85,7% (18/21) e das de amostras clínicas e de ambiente criatório de aves, 81% (64/79). Cepas com diferentes níveis de resistência foram encontradas para ampicilina (0,8%), canamicina (1,7%), ciprofloxacina (1,7%), enrofloxacina (10,2%), gentamicina (14,5%), estreptomicina (16,2%), ácido nalidíxico (35,9%), nitrofurantoína (47%) e tetracicilna (59%). Nenhuma das 117 cepas de Salmonella Enteritidis foi resistente ao cloranfenicol, norfloxacina e polimixina B. Dentre as 99 amostras de SE que apresentaram resistência, 66,6% (66), foram resistentes a dois ou mais agentes antimicrobianos. Trinta e três cepas (33,3%), foram resistentes somente a um agente antimicrobiano, 16 a tetraciclina, 12 a nitrofurantoína, três ao ácido nalidíxico, uma a estreptomicina e uma a gentamicina. Para detectar mutação no gene gyrA da região determinante de resistência a quinolonas, 42 cepas de SE que apresentaram resistência ao ácido nalidíxico foram submetidas a reação em cadeia da polimerase e sequenciamento. Das 42 cepas, 30 (71,4%) apresentaram algum tipo de mutação no gene gyrA da região determinante de resistência a quinolonas. As alterações gênicas ocorreram nos aminoácidos dos codons Gly-81 (3,5%), Asp-82 (3,5%), Ser-83 (31%) ou Asp-87 (62%). As mutações continham alterações de Gly para Asp (n: 1) no codon 81, Asp para Asn (n: 1) no codon 82, Ser para Phe (n: 9) no codon 83 e Asp para Tyr (n: 9) ou Asn (n: 9) no codon 87. Uma amostra apresentou uma inclusão do aminoácido prolina entre os codons 56 e 57. A fagotipificação de 116 cepas de SE apresentou que 68,9% (80/116) pertencem ao fagotipo (FT) 4, 15,5% (18/116) ao FT 4a, 12,2% (14/116) ao FT 1, 0,9% (1/116) ao FT6, 0,9% (1/116) ao FT 6a, 0,9% (1/116) ao FT 7 e 0,9% (1/116) ao FT 7a. Quando leva-se em consideração a origem dos isolados, observamos que nas SE isoladas de amostras clínicas e ambientais criatório de aves, 56,4% pertencem ao FT 4, 21,8% ao FT 4a, 17,9% ao FT 1, 1,3% ao FT 6, 1,3% ao FT 6a e 1,3% ao FT 7a. Nas amostras isoladas de carne cortes de frango, o FT 4 predomina com 95,2% (20/21) e 4,8% (1/21), pertencem ao FT 7. Nos isolados de carcaças resfriadas de frango 94,1% são do FT 4 e 5,9% (1/17) do FT 4a. A caracterização por ribotipagem foi realizada utilizando-se o RiboPrinter® system (DuPont), e apresentou quatro diferentes ribotipos, sendo que o mais comum foi o 25-S-1 (82,1%), seguido pelo 29-S-5 (10,7%) e 28-S-5 e 38-S-3 com 3,5% cada um. Baseados nos dados do presente estudo, conclui-se que: houve uma elevada percentagem de cepas de Salmonella Enteritidis resistente a um ou mais agentes antimicrobianos, indicando que levantamentos contínuos são necessários na indústria avícola e que existe a necessidade de um uso responsável dos agentes antimicrobianos, baseado na compreensão da ecologia da resistência, da transmissão da bactéria resistente e de genes de resistência, da relação entre o uso do antibiótico e aumento da resistência e de um conhecimento de intervenções efetivas; Que assim como em outros trabalhos amostras de S. Enteritidis resistentes ao ácido nalidíxico, isoladas no Brasil, também apresentam mutação no gene gyrA, da região determinante de resistência a quinolonas; Que o FT 4 foi o predominante e que entre as cepas de S. Enteritidis isoladas de aves e de seu ambiente criatório existe uma maior diversidade de fagotipos quando comparada as isoladas de carcaças de frango; E que ao avaliarmos os dados gerados pela ribotipagem observamos um baixo grau de diversidade gênica entre as cepas de Salmonella Enteritidis utilizadas no presente estudo. / This work aimed to evaluate the antimicrobial resistance of Salmonella Enteritidis (SE) isolated from clinical and environmental poultry samples, during the years of 1999, 2000 and 2001, broiler chicken parts, in 1996, both in Southern Brazil, broiler chicken carcasses, during the years 2004 and 2005, in Northeastern Brazil, detect mutations in the gyrA gene from nalidixic acid resistant and identify their phage type. Also, 28 SE strains isolated during the year 2004 in Southeastern Brazil were characterized by ribotyping. The antimicrobial resistance test was performed using the disk diffusion method on Mueller-Hinton Agar. The results indicated that 84.6% (99/117) of SE strains were resistant to at least one of the antimicrobial agents tested. Resistance at different levels was found to ampicillin (0.8%), kanamycin (1.7%), cyprofloxacin (1.7%), enrofloxacin (10.2%), gentamycin (14.5%), streptomycin (16.2%), nalidixic acid (35.9%), nitrofurantoin (47%), and tetracycline (59%). None of the SE strains were resistant to chloramphenicol, norfloxacin and polimyxin B. Among the 99 SE strains showing resistance, 66.6% (66) presented multiple resistance, to two or more antimicrobial agents. Thirty-three strains (33.3%) were resistant to only one of the antimicrobial agents, 16 to tetracycline, 12 to nitrofurantoin, 3 to nalidixic acid, 1 to gentamycin, and 1 to streptomycin. Fourty-two nalidixic acid resistant strains were submited to PCR and sequencing to detect gyrA mutation genes. Thirty SE strains (71.4%) showed at least one mutation in gyrA genes of quinolone resistance determining region (QRDR), in the codons corrosponding to Gly-81 (3,5%), Asp-82 (3,5%), Ser-83 (31%) or Asp-87 (62%). These mutants contained a change from Gly to Asp (n: 1) at codon 81, Asp to Asn (n: 1) at codon 82, Ser to Phe (n: 9) at codon 83 and Asp to Tyr (n: 9) or Asn (n: 9) at codon 87. In one sample there was a Pro inclusion between the 56 and 57 codons. The phage typing of 116 SE isolates showed that 68.9% (80/116) belonged to the phage type (PT) 4, 15.5% (18/116) to the PT 4a, 12.2% (14/116) to the PT 1, 0.9% (1/116) to the PT 6, 0.9% (1/116) to the PT 6a, 0.9% (1/116) to the PT 7, and 0.9% (1/116) to the PT 7a. The ribotyping characterization was done using RiboPrinter® system (DuPont), and showed four different ribotypes. The most common ribotype was 25-S-1 (82.1%). The other ribotypes were 29-S-5 (10.7%), 38-S-3 (3.6%) and 28-S-5 (3.6%). In conclusion, the antimicrobial resistance levels presented here suggest a high occurrence of Salmonella Enteritidis strains resistant to at least one antimicrobial agent, indicating the need for continuous surveillance in the poultry industry, and the need for responsible use of antimicrobial agents in food animals, based on a understanding of the ecology of resistance, the transmission of both bacteria and resistance genes, the relationship between antimicrobial agents use and resistance amplification, and the knowledge of effective interventions. S. Enteritidis nalidixic acid resistant strains isolated in Southern and Northeastern Brazil showed mutation in the gyrA gene of QRDR. Phage type 4 was the most common isolated and among S. Enteritidis strains isolated from clinical and environmental poultry samples there were a higher phagetype diversity when compared with broiler chicken carcasses. The S. Enteritidis strains that were ribotyping showed a lower degree of genetic diversity.

Salmonella enterica sv. typhimurium is a major food-borne pathogen, in part because of its ability to persist and multiply at low temperatures. Adaptation to refrigerated temperatures involves induction of a multigenic cold shock response (CSR); where gene expression is co-ordinately modified, to express cold shock proteins (CSPs). Characterisation of CspA, the major cold shock protein, instigated the identification of other CspA paralogues; which are highly conserved and widespread across species. Six CspA paralogues have previously been identified in S. typhimurium and a csp null strain, lacking all CspA paralogues made. This strain is unable to grow following cold shock, demonstrating that the CspA paralogues play an essential role during low temperature adaptation. The individual CspA paralogues exhibit distinct expression profiles; including expression of CspC and CspE at optimal temperature and CspA and CspB following cold shock. This work investigates the transcriptional changes of S. typhimurium during cold shock and the role of the CspA paralogues under both optimal and cold shock conditions. Using a bacteriophage Mu transposon library (Francis and Gallagher, 1993) this study identifies 7 novel cold induced targets and analyses their native expression levels in SL1344 and the csp null strain during cold shock. This revealed that the regulation of 5 discrete loci including tRNApro2, cpxP and 3 uncharacterised ORFS are mediated by CspA paralogues. In addition, the transcriptional profiles of a highly conserved and essential set of genes encoding known cold shock proteins, NusA, IF2, RbfA, PNPase and CsdA have been characterised. Comparative Northern analysis of SL1344 and the csp null strain has identified a role for CspA paralogues in mediating low temperature induction of three of these genes, through transcription anti-termination. Taken together these results demonstrate that during adaptation to low temperature CspA paralogues regulate expression of genes involved in the translational machinery and metabolic biosynthetic pathways: possibly through a number of transcriptional and post transcriptional processing events. Furthermore this study provides in vivo evidence of the RNA binding activity of the S. typhimurium CspA paralogues. Using fusion proteins, the RNA targets of CspE at 37°C and CspA at 10°C were isolated and analysed. This work identifies 17 direct binding targets for CspE and these indicate that CspE performs a role at optimal growth temperature in regulating components of metabolic (coaA and plsX), translational (EF-Tu, EF-G and IF3) and virulence associated (hha) pathways. Functional redundancy between CspE and CspA was suggested as both paralogues bound 16s rRNA. In light of these findings, the functions of CspA & CspE at optimal and low temperature are discussed. Overall this study has revealed novel information about low temperature adaptation of S. typhimurium, expanding our knowledge of the complexity and importance of the CSR in bacterial pathogens. In addition this work enhances our comprehension of the roles of the CspA paralogues at both optimal and low temperature.

In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures. However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA paralogues), it has been shown that CspA paralogues function during other environmental stresses, outwith temperature reduction, and play an essential role in colony formation of an SL1344 rpoS mutant at 37°C. The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of stresses including oxidative stress, temperature changes, low pH and stationary phase. Under such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS degradation, and a specific recognition factor that targets RpoS to this protease, MviA. The present study has shown that the CspA paralogues of S. Typhimurium are involved in the expression of RpoS and aims to elucidate the role of these proteins in RpoS production. Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain, were observed. CspA paralogues and Hfq are functionally distinct, not only in their involvement in RpoS expression, but also in RpoS-independent processes, such as cold acclimation, motility and to some extent, growth at 37°C. The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the molecular level. A combination of qRT-PCR analysis, transcriptional fusions and immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C or cold shock in exponential phase. Immunoblotting and translational fusions have identified different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock in exponential phase. Hfq is involved during the former condition only, whilst CspA paralogues are involved in both. Protein stability experiments have shown that the CspA paralogues do not play a major role in stabilising RpoS protein against degradation. Together, these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the efficient translation of rpoS mRNA. An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a phenomenon found when introducing combinations of mutations to SL1344 for phenotypic assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony forming ability to the rpoS mutant. Further complementation experiments pointed to faulty cell division, due to abnormal RNase E activity, as the cause.

Chronic infections are often attributed to bacterial biofilms. These biofilms are extremely tolerant to antimicrobial treatment due to the presence of dormant persister cells. Whilst a number of persister genes and pathways have been identified, it is likely that others remain. Investigating persistence of S. Typhimurium was therefore undertaken. A csp null mutant of Salmonella enterica sv. Typhimurium, lacking all six cold shock protein (CspA) paralogues was previously constructed (Hutchinson 2005). At 10°C, this strain is unable to divide, but remains viable for several weeks. However it remains capable of growth at 37°C and thus is conditionally dormant. Using this strain, the link between dormancy and persistence was investigated. Treatment of stationary phase planktonic cultures with fluoroquinolones revealed persister cells in SL1344. In contrast the csp null mutant was completely eliminated by treatment at 37°C; this could be prevented by cspC or cspE expression, implicating a role for cspA paralogues in persistence. Cold shock (10°C) substantially increased persister levels, although csp null cultures remained hypersensitive. Chloramphenicol pre-treatment also reduced elimination of the csp null mutant, linking translation with the persister phenotype. Mutations in 5 genes affecting chromosomal structure and function were investigated, 3 of which (hns, hfq, rpoS) were found to reduce persister levels, suggesting a possible role for DNA supercoiling in persistence. Plasmid topologies in the csp null mutant were highly supercoiled compared to SL1344, a phenotype prevented by cspC or cspE expression. Altered supercoiling is therefore proposed as a mechanism for fluoroquinolone sensitivity in the csp null mutant. Persister levels were also characterised in biofilms of SL1344 and the csp null mutant. In contrast to stationary phase planktonic cultures, the CspA paralogues did not appear to play a role in biofilm persistence under the experimental conditions tested. However, the study revealed a novel role for CspA paralogues in pellicle formation at the air-liquid interface. A plasmid library was used to identify chromosomal regions capable of rescuing the planktonic persister phenotype of the csp null mutant. One region which delayed fluoroquinolone elimination of the csp null mutant, contained components of the hpa gene cluster, replicated in 11 isolates. This locus is involved in hydroxyphenylacetate (HPA) catabolism, indicating a possible role of cellular metabolism in the persistence. Overall this study has revealed novel information about antibiotic persistence in S. Typhimurium and the involvement of the CspA paralogues. These results provide an important foundation for further investigations and contribute towards knowledge of the complex processes of dormancy, persistence and biofilm formation in bacteria.