As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

Freilebende, sich von Bakterien ernährende Nematoden fressen, beherbergen und verbreiten menschliche Pathogene. In dieser Arbeit wurden die Wechselwirkungen zwischen Nematoden und Pathogenen sowohl in aquatischen als auch in terrestrischen Umgebungen untersucht, wobei der Schwerpunkt auf Legionella pneumophila (Süßwasser) und Salmonella enterica (Boden) lag. Das Potenzial von Nematoden, menschliche Pathogene zu beherbergen und/oder zu verbreiten, wird hauptsächlich durch ihre Anziehung zu den bakteriellen Pathogenen bestimmt. Daher konzentrierte sich diese Arbeit auf das Fressverhalten von Nematoden. Pharyngeale Pumptests zeigten, dass L. pneumophila die Fressaktivität der Nematodenarten Plectus similis und Plectus sp. im Vergleich zum Standardlaborfutter Escherichia coli OP50 beeinträchtigte. Darüber hinaus wurden natürliche Biofilme mit mCherry-markierten L. pneumophila allein oder in Kombination mit GFP-markierten E. coli inokuliert und das Beweiden der Bakterien durch die Nematoden wurde mithilfe von Laser-Scanning-Mikroskopie als Fluoreszenzmuster im Nematodendarm untersucht. Bemerkenswerterweise ernährten sich die getesteten Nematodenarten bevorzugt von L. pneumophila, wenn sie die Wahl zwischen E. coli und dem Pathogen hatten. Darüber hinaus wurde die Räuber-Beute-Beziehung zwischen Nematoden und S. enterica in einer Bodenumgebung untersucht. Binäre Auswahltests zeigten, dass sich der weit verbreitete Bodennematode Acrobeloides buetschlii sowie die beiden Modellorganismen Caenorhabditis elegans und Pristionchus pacificus sowohl von S. enterica als auch von gewöhnlichen Bodenbakterien ernährten, was auf eine Anpassung an mehrere bakterielle Ressourcen hindeutet. Insgesamt zeigte diese Arbeit, dass verschiedene freilebende Nematodenarten L. pneumophila und S. enterica als Ressource nutzen, was ihr Potenzial unterstreicht, als Umweltreservoir und/oder Vektor für menschliche Pathogene sowohl in Wassersystemen als auch in landwirtschaftlichen Böden zu dienen. / It is becoming increasingly clear that free-living bacterial-feeding nematodes not only consume, but also harbor and disseminate human pathogens. In this thesis, the nematode-pathogen interactions were investigated in both, aquatic and terrestrial environments, with the emphasis on Legionella pneumophila (freshwater) and Salmonella enterica (soil). The potential of harboring and/or dissemination of human pathogens by nematodes is mostly shaped by their attraction to the bacterial pathogen. Therefore, this thesis focused on the feeding behavior of nematodes. Pharyngeal pumping assays revealed that L. pneumophila impaired the feeding activity of the nematode species Plectus similis and Plectus sp. compared to the standard lab food Escherichia coli OP50. Further, natural biofilms were inoculated with mCherry-labeled L. pneumophila solely or in combination with GFP-labeled E. coli and nematode grazing was assessed as fluorescence pattern in the gut, using laser scanning microscopy. Remarkably, the tested nematode species preferentially fed on L. pneumophila when given a choice between E. coli and the human pathogen. Additional feeding experiments with L. pneumophila and common bacterial taxa as resources revealed that food preference was not necessarily associated with an upregulation of pumping activity. Besides, the predator-prey relationship between nematodes and S. enterica was investigated in a soil environment. Binary choice assays revealed that the widespread soil nematode Acrobeloides buetschlii as well as the two model organisms Caenorhabditis elegans and Pristionchus pacificus fed on both, S. enterica and common soil bacteria, indicating an adaption to multiple bacterial diets. Overall, this thesis showed that different free-living nematode species use L. pneumophila and S. enterica as a resource, highlighting their potential to serve as environmental reservoir and/or vector for human pathogens in both, water systems and agricultural soil.

Published Article / The development of tolerance to low pH levels and the existence of cross-resistance may promote survival of bacteria in acidic foodstuff and in acidic environments such as the human stomach, in so doing escalating the probability of food poisoning. Similar to antimicrobial resistance developing, there is growing concern that effectiveness of organic acids may decrease as a result of the emergence of acid-tolerant food-borne pathogens. The objectives of this study were to determine the development of acid tolerance in selected food-borne pathogenic bacteria and to explore the activity of organic acids against acid tolerant pathogens. Bacterial strains were screened for acid-tolerance and susceptible strains were induced through exposure to increasing concentrations of an inorganic acid, as well as acidic foodstuffs. Susceptibility to six organic acids at various pH levels was assessed in order to evaluate the possible relationship between altered antimicrobial activity and acid tolerance. Salmonella enterica sv. Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 were found to rapidly develop acid tolerance, while intrinsic acid tolerance was noted in Salmonella enterica sv. Typhimurium ATCC 14028. Pseudomonas aeruginosa ATCC 27853 demonstrated intermediate intrinsic acid tolerance. As expected, pH played a significant role in inhibitory activity of the organic acids as these compounds exhibit optimum antimicrobial activity at a lower pH (pH ≤5). It is, however, necessary to further elucidate the two-way role of pH in foodstuff concomitant to the addition of an organic acid.

Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-29 21:18:18.966

This study characterized Salmonella enterica serovar Heidelberg from human, abattoir poultry and retail poultry isolates to examine the molecular relationships of cefoxitin resistance between these groups. A total of 147 S. Heidelberg (70 cefoxitin-resistant and 77 cefoxitin-susceptible) isolates were studied. All cefoxitin-resistant isolates were also resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur and ceftriaxone, and all contained the CMY-2 gene. Pulsed-field gel electrophoresis typing illustrated that 93.9% isolates clustered together with ≥ 90% similarity. Core genome analysis using whole genome sequencing identified 12 clusters of isolates with zero to four single nucleotide variations. These clusters consisted of cefoxitin-resistant and susceptible human, abattoir poultry and retail poultry isolates. Analysis of CMY-2 plasmids from cefoxitin-resistant isolates revealed all belonged to incompatibility group I1. Analysis of plasmid sequences using WGS revealed high identity (95-99%) to a previously described plasmid (pCVM29188_101) found in Salmonella Kentucky. When compared to pCVM29188_101, all sequenced cefoxitin-resistant isolates were found to carry one of ten possible variant plasmids. The discovery of several clusters of isolates from different sources with zero to four SNVs suggests that transmission between human, abattoir poultry and retail poultry sources may be occurring. The classification of newly sequenced plasmids into one of ten sequence variant types suggests transmission of a common CMY-2 plasmid amongst S. Heidelberg with variable genetic backgrounds. / October 2016

Memoria para optar al Título Profesional de Médico Veterinario / Los serotipos de Salmonella tradicionalmente se han clasificado mediante métodos serológicos que determinan antígenos somáticos y flagelares específicos. Sin embargo, este método diagnóstico es caro y tarda mucho tiempo en dar un resultado ya que requiere implementar una batería de anticuerpos para detectar los más de 2.500 serotipos de Salmonella enterica que se han identificado. Por otra parte, la identificación molecular de los genes responsables de la expresión de antígenos flagelares son más rápidos y más sensibles que la identificación serológica. Es por esto que, en la presente memoria se implementaron pruebas de PCR para identificar S. enterica serotipos Typhimurium y Hadar, ambas incluidas en un plan nacional de control de Salmonella en establecimientos comerciales de aves. Se analizaron 135 cepas, 50 correspondientes a S. Typhimurium, 50 a S. Hadar y 35 enterobacterias como controles negativos. Estas cepas, previamente serotipificadas en el Instituto de Salud Pública (ISP), fueron sometidas a la prueba de PCR para determinar si existen diferencias de diagnóstico entre ambas técnicas. Del total de cepas analizadas, sólo 46 cepas de S. Typhimurium dieron positivas a la prueba de PCR y cuatro dieron negativas, mientras que las 50 cepas de S. Hadar dieron positivas a las dos pruebas de PCR. Las 35 enterobacterias dieron negativas a las 3 pruebas de PCR. De acuerdo a los resultados obtenidos en el estudio, la detección de antígenos mediante serotipificación y PCR para las cepas de S. Typhimurium fue menor al esperado (92%), a diferencia de lo que ocurrió con las cepas de S. Hadar en que hubo 100% de acuerdo entre ambas técnicas, sugiriendo que esta prueba de detección de Salmonella es posible reemplazarla por los métodos tradicionales de identificación y así poder acelerar el diagnóstico de esta bacteria de gran importancia para la salud pública

Una de las características de la célula bacteriana es la capacidad de adaptarse a los cambios ambientales del entorno en el que se desarrolla modificando el patrón de expresión génica. Uno de los ejemplos mejor caracterizados es la respuesta frente al cambio de osmolaridad del medio, pero la mayoría de trabajos se han centrado en el estudio de genes cuya expresión se induce a elevada osmolaridad. Mediante mutagénesis al azar con el transposón MudJ se diseñó una estrategia para identificar genes en Salmonella enterica serovar Typhimurium cuya expresión se viera reducida a elevada osmolaridad. Siguiendo esta estrategia se identificó el gen yfeR, que codifica para una proteína, YfeR, descrita en los bancos de datos como un hipotético regulador de la familia LysR de reguladores transcripcionales. Los miembros de esta familia son generalmente proteínas activadoras de la transcripción y se encuentran en muchos géneros procariotas, regulando genes u operones que codifican funciones extremadamente diversas. En base a la regulación por osmolaridad del gen yfeR, y a la posibilidad de pertenecer a la familia LysR, se planteó como objetivo de esta Tesis Doctoral estudiar el modelo de regulación de YfeR.El análisis de secuencia de la proteína YfeR reveló características distintivas de los reguladores transcripcionales LysR: un extremo N-terminal altamente conservado con un dominio "helix-turn-helix" de unión al ADN; una cadena aminoacídica de 308 residuos (proteínas LysR tienen 300 +/- 20 AAs); una relación Lys/Arg anómala; además de la homología tanto con reguladores de la familia descritos como tal como con hipotéticos reguladores LysR. Al proporcionar el producto del gen yfeR en trans su expresión se autorregula negativamente, capacidad que presentan la mayoría de reguladores LysR. En la región promotora del gen yfeR se localizó una secuencia de unión de las proteínas LysR, T-N11-A, con la T y la A formando parte de una región invertida. Mediante ensayos de retardo en gel se demostró la capacidad de unión de la proteína YfeR a esta región, y por lo tanto al ADN. Todos estos resultados definen a YfeR como un miembro de la familia LysR.Los resultados de la regulación de la expresión del gen yfeR muestran tanto de forma indirecta, mediante una fusión génica yfeR::lacZ (MudJ), como de forma directa mediante el ensayo de protección frente a la RNasa-ONE, que el genyfeRestá osmoregulado reprimiéndose a elevada osmolaridad. A 89 pb del inicio de traducción del gen yfeR se localizó una pauta abierta de lectura, denominada yfeH, que se transcribía de forma divergente, característica compartida entre muchos reguladores de la familia LysR y sus genes regulados. Así, se planteó como hipótesis de trabajo que éste era el gen regulado por YfeR. Sin embargo, el estudio de la regulación de la expresión del gen yfeH demuestra que ésta se induce en fase estacionaria, pero de forma independiente a la presencia o ausencia de su posible regulador YfeR y de su regulación por osmolaridad. En base a este resultado se planteó la posibilidad de que la proteína YfeR regulase la expresión de otros genes alternativos al adyacente. El análisis de extractos celulares de un mutante yfeR respecto a la cepa salvaje mediante geles 2D demostró la presencia de diferentes proteínas con expresión diferencial. Dos de ellas pudieron ser identificadas por MALDI-TOF: IbpA, implicada en la protección frente a un estrés por superóxidos, y Lrp, un regulador global implicado en la modulación de una gran variedad de funciones metabólicas, así como de genes que se inducen en fase estacionaria y en respuesta a cambios ambientales. Este resultado posiciona a YfeR como una proteína LysR implicada en una red de regulación global frente a estímulos ambientales. / Bacteria adapt to changes in their environment by modifying its pattern of gene expression. Adaptation to the medium osmolarity is one of the best characterized examples, however many of the well-known situations correspond to genes whose expression is induced when medium osmolarity increases. Previous work identified by random mutagenesis in Salmonella enterica serovar Typhimurium gene yfeR, a hypothetical LysR-like regulator repressed at high osmolarity. This work is focused on the study of the model of regulation of YfeR.The sequence analysis of YfeR protein showed most of the common features of LysR family: an N-terminal conserved domain, 308 amino acids long, an anomalous Lys/Arg ratio, and homology with members of the LysR family. As for the majority of LysR regulators, YfeR negatively autoregulated its own transcription. In the promoter region of yfeR was located a sequence having characteristics of a LysR-type target consensus motif. Gel retardation assays demonstrated that YfeR binds specifically to this region. All these results clearly related YfeR to the LysR family of transcriptional regulators. About the osmoregulation of yfeR gene expression we confirmed, by indirect and direct methods (transcriptional fusion yfer::lacZ and RNasa protection assays, respectively), that its expression is repressed at high osmolarity.An ORF, yfeH, was found oriented in the opposite direction to yfeR, a common property of genes regulated by members of the LysR family. However, expression of yfeH gene is induced in stationary phase independently of the presence of YfeR and osmolarity conditions. Then we decided to search for other possible regulated genes by YfeR but not in adjacent position. Protein extracts of an yfeRmutant and wild type strains were analyzed by 2D electrophoresis. Some differences were found and two of them were identified as IbpA and Lrp. These results suggest that YfeR could be implied in a global regulation network related to environmental stimuli.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Freiburg i. Br., Univ., Diss., 2007.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-132). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;