The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.

Orientador : Fumio Yokoya / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-20T00:41:04Z (GMT). No. of bitstreams: 1 Leite_ClariceQueicoFujimura_D.pdf: 3804903 bytes, checksum: d606b7f70d7b2f4627cf89586684ed12 (MD5) Previous issue date: 1984 / Resumo: Dentro da linha de pesquisa de identificação e enumeração rápida de microrganismos, o presente trabaiho foi realizado no sentido de verificar a viabilidade do emprego da membrana filtrante na recuperação de Escherichia cali e Salmanella trphimurium, inoculadas experimentalmente em ovo de galinha (clara, gema e ovo liquido). Após ó tratamento com a protease bacteriana "Alcalase", viabilizou-se a filtragem de 5,0 g de clara, 2,5 g de ovo liquido e l,O g de gema, através da membrana HAWG 47 mm da marca Millipore. A recuperaçao de E..ca}i em clara foi realizada através do emprego siImultâneo da técnica de plaqueamento em profundidade e de um método por nós desenvolvido, utilizando a membrana filtrante. Os resultados obtidos com a técnica do plaqueamento foram próximos aos conseguidos com a técnica proposta. Colocando os dois resultados em gráfico, foi obtida uma reta de regressão com coeficiente de correlação de 0;95 e coeficiente angular de 0,99. Os resultados da experiência de inoculação de ovo liquido com E. cali, obtidos com o emprego das duas técnicas, forneceram uma reta com coeficiente de correlação de 0,98. O coeficiente angular"foi baixo, 0,39 .havendo necessidade da introdução de um fator de correção multiplicativo de 2,6, para compensar a menor recuperação obtida com o uso da técnica da membrana filtrante. O isolamento de E. coli da gema foi insatisfatório, com o emprego da técnica da membrana filtrante. A S. typhimurium, previamente .inoculada em clara e em ovo liquido, foi recuperada como emprego da técnica clássica de enriquecimento e isolamento em meios seletivos e com o método por nós desenvolvido. Os resultados demonstraram que mesmo em concentrações tão baixas como 0,3 microrganismo por grama de clara, é possivel recuperar a s. typhimurium através da técnica da membrana filtrante. A recuperação deste microrganismo do ovo liquido foi insatisfatória / Abstract: In the line of research for the identification and rapid enumeration of micro-organisms, the present work was carried out with the object of verifying the viabili ty of the use of a membrane filter in the recuperation of Escherichia coli and Salmonella typhimurium, inoculated experimentally into the hen'g egg (white, yolk, and whole liquid egg). After treatment wi th bacterian protease "Alcala se", 5.0 g of egg white, 2.5 g of whole liquid egg, and 1,0 g of egg yolk were filtered through the 47 mm HAWG filter (Millipore). The recuperation of E. coli from the egg white was done by means of the simultaneous use of pour plate method and a method developed by us, using a membrane filter. The results obtained using the pour plate technique were very similar to those developed by the authors of this research. When both results were plotted on a graph, a line of regression with a correlation coefficient of 0,95 and an angular coefficient of 0,99 was obtained. The results of the experiment involving the inoculationof the whole liquid egg with E. coli, using both techniques, showed a line with a correlation coeficient of 0.98. The angular coefficient of 0.39 was low, making the introduction of a manipulative correction factor of 2,6 necessary in order to compensate for a lesser recuperation obtained using the membrane filter technique. The isolation of E. coli from the yolk was not satisfactory using the membrane filter technique. The S.typhimurium, previously inoculated into the white and whole liquid egg, was recuperated us1ng the classic technique of enrichment and isolation in selective media and als,oby methods developed by USe The results show that even in concentrations as low as 0.3 micro-organisms per gram of egg white, it is possible to recuperate the S. typhimurium by use of the membrane filter technique. The recuperation of this microorganism from the who1e 1iquid egg was insatisfactory / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Determina la capacidad virulenta de una cepa aislada de la vesícula biliar de un cuy, con signos compatibles con salmonelosis, para identificar sus genes asociados a virulencia y resistencia a antibióticos. En este estudio, el genoma completo de S. Typhimurium cepa VET1 se sometió a un análisis in silico, que comenzó con el secuenciamiento mediante la plataforma Illumina HiSeq para generar lecturas de 2 × 101 pb. La calidad de los datos se verificó con FASTQC y los adaptadores fueron recortados con Trimmomatic. Las lecturas fueron ensamblados usando Velvet v.1.2.10 y se generaron 149 contigs con una cobertura de 111.0x, que dio como resultado un tamaño total del genoma de 4 905041 pb, con un contenido de G + C del 52.14%. La anotación génica mostró 4 885 genes, de los cuales 4 630 fueron CDS y 255 ARN, incluyendo 2 de ARNr, 56 ARNr y 197 ARNnc. Usando PHASTER, se identificaron tres regiones de profagos, incluyendo Gifsy-2, 118970_sal3 y RE-2010. La cepa VET1 fue asignada a ST19 utilizando el tipo de secuencia multilocus (MLST). Se identificó un total de 244 factores de virulencia. Entre ellos, 78 pertenecían a genes codificadores del sistema de secreción tipo 3 (T3SS), 68 a genes codificadores de la adherencia fimbrial y 51 a genes que codifican flagelos. Se consultó la base de datos CARD para identificar genotipos relacionados con la resistencia en el genoma de S. Typhimurium VET1. Se identificaron un total de 16 proteínas relacionadas a resistencia antimicrobiana, que pertenecen a 6 familias diferentes de genes de resistencia a antibióticos. Este estudio mostró que la cepa VET1 alberga genes que codifican adhesinas, proteínas flagelares, T3SS, sistemas de adquisición de hierro y genes de resistencia a antibióticos que pueden explicar la patogenicidad, capacidad de colonización y persistencia en el cuy. La existencia de elementos genéticos móviles sugiere que esta cepa podría adquirir y transferir material genético. El análisis genómico comparativo entre VET1 y otras cepas de Salmonella proporcionaría información fructífera para comprender la especificidad del huésped y desarrollar medidas de control contra la infección por S. Typhimurium. / Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú) / Tesis

abstract: The discovery that mechanical forces regulate microbial virulence, stress responses and gene expression was made using log phase cultures of Salmonella Typhimurium (S. Typhimurium) grown under low fluid shear (LFS) conditions relevant to those encountered in the intestine. However, there has been limited characterization of LFS on other growth phases. To advance the growth-phase dependent understanding of the effect of LFS on S. Typhimurium pathogenicity, this dissertation characterized the effect of LFS on the transcriptomic and phenotypic responses in both stationary and lag phase cultures. In response to LFS, stationary phase cultures exhibited alterations in gene expression associated with metabolism, transport, secretion and stress responses (acid, bile salts, oxidative, and thermal stressors), motility, and colonization of intestinal epithelium (adherence, invasion and intracellular survival). Many of these characteristics are known to be regulated by the stationary phase general stress response regulator, RNA polymerase sigma factor S (RpoS), when S. Typhimurium is grown under conventional conditions. Surprisingly, the stationary phase phenotypic LFS stress response to acid and bile salts, colonization of human intestinal epithelial cells, and swimming motility was not dependent on RpoS. Lag phase cultures exhibited intriguing differences in their LFS regulated transcriptomic and phenotypic profiles as compared to stationary phase cultures, including LFS-dependent regulation of gene expression, adherence to intestinal epithelial cells, and high thermal stress. Furthermore, the addition of cell-free conditioned supernatants derived from either stationary phase LFS or Control cultures modulated the gene expression of lag phase cultures in a manner that differed from either growth phase, however, these supernatants did not modulate the phenotypic responses of lag phase cultures. Collectively, these results demonstrated that S. Typhimurium can sense and respond to LFS as early as lag phase, albeit in a limited fashion, and that the lag phase transcriptomic and phenotypic responses differ from those in stationary phase, which hold important implications for the lifecycle of this pathogen during the infection process. / Dissertation/Thesis / Transcriptomic Data / Doctoral Dissertation Microbiology 2020

Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

This study was undertaken to compare the MIC (minimum inhibitory concentration) and MPC (mutant prevention concentration) values for oxytetracycline and florfenicol against strains of Pasteurella multocida isolated from cattle and pigs, and for enrofloxacin against strains of Salmonella Typhimurium isolated from horses. Isolates of P. multocida from cattle and pigs, and S. Typhimurium from horses were obtained from specimens or isolates from contributing laboratories. All the equine isolates and 50% of the cattle and pig isolates were from clinically sick animals. All isolates were tested in duplicate with both the MIC and the MPC methods. The MIC method used was the standardized microdilution method performed in microtitre plates. The MPC method used was according to the method described by Blondeau. This method was modified, to make use of smaller plates and lower volumes of antimicrobials, but retaining a final bacterial concentration of 109 colony-forming units per ml. The antimicrobials were dissolved as described in the certificates of analyses. Enrofloxacin and oxytetracycline were dissolved in water, and florfenicol was dissolved in alcohol. For the MPC method, an additional control was added to one quadrant of a four-quadrant 90mm plate/petri dish. The antimicrobials were tested as individual antimicrobials and not as combinations. Both the MIC and MPC methods included ATCC (American Type Culture Collection) strains as control organisms and were evaluated according to the guidelines of the CLSI (Clinical and Laboratory Standards Institute). The MIC50 values for enrofloxacin against Salmonella Typhimurium isolates from horses was 0.25 ìg/ml and the MPC50 values 0.5 ìg/ml. A comparative reference range was not available as enrofloxacin is not registered in South Africa for use in horses, and is used extra-labelly. The results for florfenicol against P. multocida yielded an MIC50 value of 0.5 ìg/ml and an MPC50 value of <2 ìg/ml. The close relationship of these two concentrations is an indication of the effectiveness of florfenicol when used against P. multocida. The PD/PK data with a value of 141.78 for AUC/MIC provided additional support for the efficacy of florfenicol against P. multocida. The PD/PK value of >125, is an effective parameter for treatment of Gram-negative bacteria. The corresponding results for oxytetracycline were above the MIC value but fell within the mutant selection window. The results point to the fact that the use of oxytetracycline against P. multocida may not be effective in preventing the appearance of first step mutant strains when used at current recommended dosages. The PK/PD data, using AUC/MIC, yielded a value of 56. Some of the isolates (55.17%) had an MPC value of 16 ìg/ml. Whereas the MIC method is used routinely in diagnostic laboratories, the MPC method can be employed to generate data that can be applied where antimicrobial treatment of certain bacteria is problematic and standard treatment may lead to the development of resistance. Data obtained from such studies will enable manufacturers of antimicrobial drugs to adapt antimicrobial therapy where practical and feasible to prevent the development of first step mutants. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

The Salmonella typhimurium/mammalian microsome test has provided a simple and sensitive short‐term assay for the detection of environmental mutagens. Metabolic activation of precarcinogens is usually achieved by incubating the compound to be tested, the bacterial strain and mammalian liver homogenates with NADPH. The results presented here utilize Salmonella typhimurium strain TA100, the precarcinogen 2‐aminofluorene and microsomal enzyme preparations prepared from liver homogenate of carp (Cyprinus carpio) taken from aquatic environments of northeastern Tennessee. Those environments range from virtually unpolluted to extremely polluted. The results show that the precarcinogen 2‐aminofluorene is activated either partially or totally in the presence of liver homogenates of carp taken from polluted aquatic environments (e.g., microsomal enzyme preparations made from rat liver with 2‐aminofluorene produced 808 revertants; whereas the liver preparations made from carp, taken from the Pigeon River, with 2‐aminofluorene produced 2,786 revertants). Revertant colony results correlated well with the degree of pollution within those waters. An increase (data were statistically different at the 0.05 level of significance) of TA100 revertant colonies was observed as aquatic contamination worsened. All data pairs of collecting sites in their order of increasing contamination, as well as those between collecting sites, were statistically different at the 0.05 level of significance.

Determina el efecto de la Salmonella Typhimurium sobre los parámetros productivos, composición química y calidad microbiológica de la carne de cuy (Cavia porcellus). El trabajo se realizó en la unidad de experimentación de cuyes del laboratorio de Bioquímica, Nutrición y Alimentación Animal de la Facultad de Medicina Veterinaria, UNMSM. Se utilizaron 40 cuyes machos de engorde que fueron distribuidos en 4 tratamientos con diez (10) repeticiones cada uno; T1: cuyes alimentados con dieta base + solución salina (control), T2: cuyes alimentados con dieta base + APC + solución salina, T3: cuyes alimentados con dieta base y desafiados experimentalmente con Salmonella Typhimurium, T4: cuyes alimentados con dieta base + APC y desafiados experimentalmente con Salmonella Typhimurium. En el día 11, los animales del T1 y del T2 fueron dosificados vía oral con solución salina, mientras que los T3 y T4 fueron desafiados con una dosis infectiva (2 x 106 UFC) de Salmonella Typhimurium, por única vez. Se evaluaron los parámetros productivos (ganancia de peso, consumo de alimento, conversión alimenticia), la composición química y la calidad microbiológica de la carne de cuy. Los cuyes de los tratamientos T3 yT4 presentaron, significativamente (p<0.05), menor ganancia de peso vivo total (T3: 534g; T4: 577 g) y mayor índice de conversión alimenticia (T3: 6.29; T4: 5.92) comparados con el grupo de animales no desafiados (T1: 761g, 4.04; T2: 828 g, 3.66). No se observó diferencia estadística significativa en el rendimiento de la canal de los cuyes en los cuatro tratamientos. El número de casos con mayor presencia de Salmonella sp. se observó en ganglios linfáticos, hígado, bazo, vesícula biliar y pulmón de las muestras de órganos de los grupos T3 y T4. Se concluye que el desafío oral a Salmonella Typhimurium causa, significativamente (p<0.05), una menor ganancia de peso vivo, menor porcentaje de proteína en la canal, mayor índice de conversión alimenticia y menor retribución económica en animales desafiados comparados con el grupo de animales no desafiados. / Tesis

Understanding the composition of camel milk coupled with studying the effects of thermal and non-thermal treatments on its components and bacterial inactivation were the general objectives of this dissertation. In the first study (Chapter 2), the gross composition of camel milk including milk protein, fat, casein, total solids, lactose, ash, and mineral content were analyzed. In addition, fatty acid profile, amino acid profile, protein fractions, and volatile compounds were evaluated as well. Our results revealed that camel milk has its unique nutrients profile. These findings make it easier for the researchers and consumers to understand some of the nutritional attributes of camel milk.The impact of non-thermal ultrasound treatment (900 W, 20 kHz, 100% power level) on some milk-borne microorganisms and the components of camel milk was studied in Chapter3. We reported that continuous ultrasound processing was efficient in inactivating Escherichia coli (E.coli) O157: H7 and Salmonella Typhimurium (S. Typhimurium) in camel milk without detrimental effects on milk fatty acids profile, lipid peroxides, and protein fractions except for some changes in milk volatile compounds (VC). In Chapter 4, another non-thermal technique, ultraviolet-C (UV-C) light, was applied to camel milk to study the effects of different UV-C light doses on the viability of E. coli O157:H7 and S. Typhimurium and the chemical changes to milk components. The main findings of this study were: (i) UV-C treatment at a dose of 12.45 mJ/cm2 resulted in only 3.9-log10 for both bacterial strains which did not meet the Food and Drug Administration (FDA) requirements for the 5-log pathogen reduction; (ii) the UV-C treatment at the above dose, had limited effects on camel milk components. Thermal pasteurization of milk was first introduced to prevent milk-borne infectious diseases, however, its effects on camel milk components and quality are still unknown. Therefore, in Chapter 5, we investigated the efficacy of three previously reported thermal methods: PAST-1 (65ºC/30 min), PAST-2 (72ºC/5 min), and PAST-3 (80ºC/5 min) on bacterial inactivation and some camel milk components such as the fatty acid profile, lipid peroxidation, VC, and milk protein fractions. Complete elimination (6 log10 CFU/ml reduction) of E. coli O157: H7 was achieved using all pasteurization methods, however, only 3.4 log10 CFU/ml reduction of the total viable counts was reported using PAST-1 and PAST-3 methods. We also reported that the PAST-1 and PAST-3 methods did not affect the chemical composition of camel milk. In conclusion, we assessed the main components of camel milk along with the amino fatty acid profile, acid profile, volatile compounds, and protein fractions. Thermal methods were more effective than the non-thermal methods in terms of microbial inactivation and most camel milk components were not significantly influenced by thermal and non-thermal methods.

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.