Dissertations / Theses on the topic 'Pathogenic microorganisms'

The organisms in this study were chosen due to their associations with foods and their potential as food borne pathogens. Food borne diseases are an import public health problem in most countries. Bacteria of the genera Campylobacter, Salmonella and Listeria can be transported by poultry and poultry products to humans. Gastroenteritis, typhoid fever, diarrhea, dysentery may originate from the infection. This study was undertaken to determine the incidence of pathogens in a poultry processing plant using polymerase chain reaction and conventional tests and to determine the formation and survival of biofilm cells of food pathogens in trisodium phosphate.

The ability to mount an efficient immune response should be an important life-history trait as parasitism can impact upon an individual's fecundity and survival prospects, and hence its fitness. However, immune function is likely to be costly as resources must be divided between many important traits. Whilst many studies have examined host resistance to particular parasite types, fewer have considered general immune responses. Studies that have considered general immune responses tend to do so in vertebrate models. However, the complexity of the vertebrate immune system makes the examination of evolutionary aspects of immune function difficult. Using larvae of the genus Spodoptera (Lepidoptera: Noctuidae) as a model system, this study examines' genetic and phenotypic aspects of innate immunity. The aims were to assess the levels of additive genetic variation maintained in immune traits, to consider possible costs that could maintain this variation, and to assess the role of phenotypic plasticity in ameliorating those costs. A key finding of this study was that high levels of additive genetic variation were maintained in all of the measured Immune traits. Analysis of the genetic correlations between traits revealed potential trade-offs within the immune system and between immune components and body condition. In addition, it was shown that larvae living at high densities invest more in immune function than those living in solitary conditions, suggesting that larvae can minimise the costs of immune function by employing them only when the risk of pathogenesis is high.

Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.

Thesis (M.S. in zoology)--Washington State University, May 2009.
Title from PDF title page (viewed on May 22, 2009). "School of Biological Sciences." Includes bibliographical references (p. 18-25).

The purpose of this study was to determine the removal efficiency of natural systems for the reduction of enteric protozoa (Giardia and Cryptosporidium), and enteric viruses in wastewater. The first part of the study used bench-scale soil columns to determine the potential effectiveness of Soil Aquifer Treatment (SAT) for the removal of Cryptosporidium oocysts and Giardia cyst from treated wastewater. Sand and sandy loam were used to pack 18 to 200-cm long columns. Results from this study showed that removal of oocysts increased as increasing length of the soil column. Although substantial removal of Cryptosporidium occurs (>99.99%) within 200 cm of soil, oocysts are likely to penetrate beyond this depth. Giardia was removed far below detectable levels, probably due to its larger size. The next phase of the project investigated the removal of pathogenic and indicator microorganisms from untreated wastewater by a surface flow wetland, the importance of plants in wetlands, as well as the potential for groundwater contamination passed by pathogens with the use of constructed wetlands. This small-scale study was conducted in a large tank divided into two cells. Both cells were filled with sand and one cell was planted with bulrushes and the other was unplanted. About 90 percent of all microorganisms were removed by either of the systems. Neither Giardia nor Cryptosporidium were found to penetrate through the 2-m of sand in either the planted or unplanted cells. Lower numbers of viruses and bacteria were transported through the sand in the planted wetland cell versus the unplanted cell. This could indicate that vegetated wetlands are more likely to prevent microbial transport to groundwater. The objective of the last part of this study was to determine the survival of Cryptosporidium oocysts in wastewater effluent applied to a constructed vegetated wetland, when exposed to and when protected from sunlight, and the effect of temperature during different seasons. Viability of Cryptosporidium oocysts was determined using the excystation technique. Results from this study indicated that sunlight and/or temperature play a significant role in the survival of Cryptosporidium. Thus, it was concluded that oocyst reduction in wastewater applied to wetlands can be enhanced by natural die-off due to the effects of temperature or UV irradiation in sunlight, and greater removal could be achieved if designing of wetland systems take into consideration such factors.

Estuarine environments are biologically productive ecosystems that are both economically and socially important. Consequently, a decline in the microbiological water quality can pose a risk to human health and have severe socioeconomic consequences, especially for areas that rely on tourism and shellfisheries for income. The enumeration of faecal indicator bacteria (FIB) in water samples has been the paradigm for estimating water quality in coastal zones, but there is an emerging view that sediments are a poorly studied and yet a significant reservoir of FIB. The aims of this thesis were: (I) to investigate the role of sediments as a reservoir for FIB and other potentially pathogenic bacteria; (II) to examine the spatial ecology of FIB in relation to sediment composition (grain size and organic matter content); (III) to investigate the influence of point and diffuse pollution sources on the abundance of bacteria in marine and estuarine sediments; (IV) to investigate the abundance of human pathogenic bacteria in the Conwy estuary, North Wales, UK, and (V) to investigate the influence of suspended particulate matter (SPM) on the survival of FIB in both fresh and brackish water. Culturable E. coli, total coliforms, enterococci (FIB), Salmonella, Campylobacter, Vibrio spp. and heterotrophic bacteria were enumerated in sediments and water from the Conwy estuary that is subject to various point and diffuse sources of pollution. FIB counts were three orders of magnitude greater in sediments compared with the overlying water column, demonstrating that estuarine sediments are a significant reservoir for FIB and other potential pathogens. In addition, sediment grain size analysis and organic matter content determinations revealed that finer sediments such as clay, silt and very fine sand contained significantly higher concentrations of all bacterial groups enumerated. The enumeration of FIB in marine sediments surrounding an offshore sewage outfall pipe revealed that spatial variations in FIB abundance reflected the course of the sewage effluent plume as predicted by a hydrodynamic model, demonstrating the impact of point sources of microbial pollution on the underlying sediments. To address the actual pathogen content of sediments in the Conwy estuary (rather than only indicator bacteria), PCR and qPCR were utilized to detect and quantify known pathogen virulence genes, revealing that estuarine sediments are a reservoir for pathogenic bacteria. Furthermore, qPCR suggested greater concentrations of FIB compared with culture counts from the same sample, indicating the possible presence of viable but non-culturable (VBNC) bacteria. Consequently, sediment-associated bacteria pose a risk to human health if they are resuspended into the water column under certain hydrodynamic processes, as tide-dominated estuaries usually contain large areas of fine sand that are easily mobilized. To investigate the influence of SPM concentration on FIB survival, fresh and brackish water containing low (~16 mg/l), high (~160 mg/l) and extreme (~1650 mg/l) SPM concentrations were inoculated with crude sewage and sheep faeces. FIB were enumerated every 24 hrs for 5 days, revealing that SPM concentrations influence FIB survival in brackish water but had minimal influence in freshwater over time. In general, FIB concentrations increased with a decrease in SPM concentration. These data add to a limited body of evidence on the role of sediments as a reservoir for pathogenic bacteria, with implications for routine monitoring protocols that assess the microbial pollution of environmental waters. In addition, these data suggest that catchment-based risk assessments of microbial pollution in aquatic systems should consider the source of FIB, the hydrodynamics of the environment, and the subsequent influence of SPM concentrations, all of which determine the survival of FIB in aquatic environments.

The work presented in this thesis is concerned with investigating the use of UV-rich light pulses for the inactivation of problematic microorganisms. UV radiation is an effective means of disinfecting surfaces and liquids and of reducing contamination in air. The germicidal effects are primarily due to the UV-C region of the electromagnetic spectrum, which interferes with the nuclear core of a microorganism resulting in a loss of ability to replicate and initiate infection. When UV radiation is delivered as pulses of light however, the results are even more appealing, with higher levels of microbial inactivation achieved in much shorter timescales. Drinking water and wastewater disinfection is normally provided by the use of chemicals such as chlorine. These are disadvantaged by the production of harmful chemical by-products and the resistance of certain types of microorganism to chemical treatment. The main aim of this investigation was therefore to look at the role of pulsed UV-rich light for inactivating a range of microorganisms suspended in liquid media and to determine how successful the treatment process would be as an alternative disinfection method. The results show that pulsed UV-rich light treatment is extremely effective against many types of bacteria, virus and Cryptosporidium. It was also demonstrated that the sensitivities of microorganisms to UV radiation can vary significantly depending on the cell-wall structure, growth phase, strains and nucroorgarusm specIes. Studies were also undertaken to identify electrical and biological parameters that may influence the inactivation success. It was found that high operating voltages and low pulse repetition frequencies give desirable levels of inactivation Other important factors investigated were sample depth, volume and reflection. Finally, the possible limitations to pulsed UV-rich light treatment were investigated. It was found that the success of the treatment is primarily determined by the transmittance of UV pulses through a sample. As expected, transmittance depends upon microorganism size and population. The major limitation of UV treatment is the ability of microorganisms to reactivate following exposure to visible light (photoreactivation). Studies showed photoreactivation to occur following pulsed UV-rich light treatment, only when the microorganisms do not receive sufficient UV treatment or if they are exposed to high intensities of visible light.

This project was designed to assess the potential for contamination of produce during irrigation with wastewater from animal operations. Dairy wastewater from the University of Arizona Campus Dairy Research Center was used to irrigate three different types of vegetable crops: lettuce, carrot, and bell pepper. This study was conducted over two consecutive years. The crops were planted in February and vegetables were harvested from May through July. Irrigation water and vegetable samples were examined for Escherichia coli, Clostridium perfringens, Listeria monocytogenes, and coliphage. In the dairy wastewater, E. coli concentrations averaged 5.7 x 10⁵ MPN/100 mL in the first year (2000), and 9.9 x 10⁷ MPN/100 mL in the second year (2001). C. perfringens concentrations were nearly the same in both years (1.7 x 10⁴ and 3.4 x 10⁴ CFU per 100 mL). Coliphage averaged 2.0 PFU/mL in 2000 and 1.3 x 10⁴ PFU/mL in 2001 in wastewater. E. coli was detected with greater frequency on carrots (100 and 96%) succeeded by lettuce (67 and 96%) and bell peppers (63 and 58%). The same was true for C. perfringens : carrots (100%), lettuce (86 and 88%), and bell peppers (100 and 50%). Coliphages were not detected on any of the vegetable crops except for average concentrations of 2 PFU/g on lettuce in the first year. L. monocytogenes was not detected on any of the vegetable samples. ANOVA test results indicates that E. coli and C. perfringens concentrations on three crops were statistically different (p < 0.0001) which suggest that the degree of contamination on the surface of the vegetables depends on where the edible portion of the crop is situated (above the soil or under the soil). The greatest contamination occurred on the carrots followed by lettuce and bell peppers. E. coli and C. perfringens were recovered from the carrots, bell peppers, and soil 55 days after wastewater irrigation of the plots had ceased. Positive correlations (p < 0.05) were found between E. coli and C. perfringens density and soil moisture content. The greatest risk of infection from pathogenic E. coli (O157:H7) occurs from consumption of lettuce and carrots. The annual risk of infection from consumption of all three vegetables was above the acceptable risk of 1:10,000 per year. The results of this study suggest that a more strict irrigation water quality standard for root and leafy vegetables might be appropriate to prevent the risk of infection in exposed population.

Thesis (M.S.) University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (June 27, 2007) Includes bibliographical references.

Streptococcus pneumoniae is a highly diverse and adaptable opportunistic pathogen that can infect and colonise different niches within the human host to cause a wide range of invasive disease (sepsis and meningitis) and noninvasive disease (pneumonia, otitis media and sinusitis). The molecular mechanisms that contribute to the different patterns of pneumococcal infection remain largely unknown. This thesis aims to determine the physiological and proteomic responses that allow the pneumococcus to survive and adapt to invasive and non-invasive sites. The comparative proteomic analyses of clinical S. pneumoniae isolates recovered from blood cultures (classified as invasive site isolates) and mucosal surfaces such as sputum, skin and ear swabs (classified as non-invasive site isolates) was initiated. The pneumococci were grown in vitro under standard conditions and the total cellular bacterial proteins extracted and analysed using both gel based and non-gel based proteomic approaches. Analysis of the pneumococcal isolates by two-dimensional polyacrylamide gel electrophoresis (2DGE) revealed that a high degree of heterogeneity existed between the pneumococcal isolates particularly among isolates in the invasive site isolates. Differential patterns of protein synthesis were observed that discriminated the pneumococcal isolates according to their sites of isolation. These were proposed to be associated with the bacterial adaptation to invasive and non-invasive sites of infection. Mass spectrometry was used to identify selected significant (ANOVA, p < 0.05) protein spots, which were further categorised into functional groups by Gene Ontology analysis. An extension of the 2DGE data using an integrated approach comprising bioinformatics, surfome analysis and a shotgun proteomic workflow provided a comprehensive qualitative and quantitative analyses of the pneumococcal intracellular and cell-surface proteomes. Proteins potentially involved in pneumococcal niche-specific adaptation and surface proteins with potential for further investigation and inclusion in the pipeline of vaccine candidates were identified. Quantitative regulation of proteins involved in energy metabolism, genetic competence, stress response, surface adhesion and virulence were considered important for pneumococcal adaptation to invasive and non-invasive sites. The anatomical sites colonised by the pneumococcus vary in their V availability for iron. The 2DGE method was also used on selected pneumococcal isolates from the two sites of infection to define the proteome variability linked to the effect of iron starvation that may contribute to the different disease outcomes associated with pneumococcal infections. The iron restricted condition was generated by cation depletion of the growth medium using Chelex-100. Quantitative differences in protein abundance were demonstrated that correlated with pneumococcal adaptation to iron restriction. The identification of selected significant spots by liquid chromatography-mass spectrometry and systems biology analysis of the identified proteins contributed to the elucidation of the molecular mechanisms underlying pneumococcal survival under iron limitation. The expression/repression of proteins functionally associated with metal ion binding, oxidative stress response, translation and virulence mainly constituted the pneumococcal adaptive responses to growth under conditions of limited iron availability. The data presented in this thesis extended our understanding of the molecular events underlying pneumococcal physiological adaptation and provide the basis of future work in this area.

Microorganisms are present in a variety of sources, including food, water, animals, environment as well as the human body. They can be harmless or harmful. The latter is also called pathogenic and their detection is extremely important due to health and safety reasons.

It is well known that food contaminated with bacteria can produce a number of foodborne diseases. As a consequence, thousands of euros are invested each year in medical treatments trying to keep the population healthy. There are more than 250 known foodborne diseases. For example, outbreaks of salmonellosis have increased in many countries in the last decades being Salmonella Infantis one of the most important etiological agents associated with this enteric disease. Moreover, due to the wide distribution of the microorganisms, they can also contaminate foods in the field as well as during the storage stage. In that sense, filamentous fungi are one of the etiological agents responsible for most post-harvest food spoilage producing quality losses and economic devaluation.

On the other hand, the invasive fungal infections due to yeast have risen considerably in recent years. Candidiasis is the so-called disease produced by Candida albicans. This is an opportunistic infection that affects immunocompromised patients requiring costly treatment with advanced medicine.

Several methods have been proposed so far to detect pathogenic microorganisms. Conventional culture is highly selective and sensitive but they also require several days to yield the results. To simplify and automate the identification of both bacteria and fungi rapid biochemical kits have been developed. Although the results obtained with these kits are comparable to the traditional biochemical tests they also need 1 or 2 days to obtain results. Enzyme-linked immunosorbent assays (ELISA) can be applied for the direct identification of pathogenic microorganisms in real samples. This immuno-based method has been widely used in both food and the medical sector with high sensitivity. Nevertheless, the main disadvantage of this method is that it can also be time-consuming because a pre-enrichment of the sample is often required in order to achieve low limits of detection. As a consequence, many researchers have addressed their efforts towards the development of alternative methods to allow the rapid detection of pathogens.

Molecular biology-based methods, specifically polymerase chain reaction (PCR) and real-time PCR are nowadays the most common tools used for pathogen detection. They are highly sensitive and allow the quantification of the target. In addition, microarray platforms of DNA have been developed in order to analyse hundreds of targets simultaneously. However, this technique is costly and reagent-consuming.

The introduction of biosensors has brought new alternatives in pathogenic detection. Biosensors are the most used tools in pathogenic detection after PCR, culture methods and ELISA. They provide rapid results after the sample has been taken. However, their real application lies in achieving selectivities and sensitivities comparable to the established methods and at low cost.

Since carbon nanotubes (CNTs) were discovered by Iijima, many papers have reported their unique electronic and optical properties which, together with their size, make these nanostructures interesting materials in the development of biosensing platforms. Their very high capacity for charge transfer between heterogeneous phases makes them suitable as components in electrochemical sensors. The electrical conductivity of the CNTs is highly sensitive to changes in their chemical environment and, as a result, they have been successfully applied in the study of molecular recognition processes.

An approach for the direct electrical detection of biomolecules integrates CNTs as transducer elements within a field-effect transistor (FET) configuration. The main advantages of this kind of configuration lies in that the conducting channel is usually located on the surface of the substrate and as a result, they are extremely sensitive to any change in the surrounding environment. Moreover, CNTFET devices can operate at room temperature and in ambient conditions.
At the beginning of this research (2006) electrochemical CNTFETs based on single walled carbon nanotubes had not been applied to detect bacteria or fungi. Only the interaction between CNTs and bacteria had been explored, but without sensing purposes. Therefore, this thesis reports the first CNTFET devices applied to the detection of pathogenic microorganisms. First, the background and the introduction containing the state of the art are presented covering relevant investigations made in the last years. Next, the main analytical methods are described. These descriptions involve detailed information of all procedures, analytical tools and materials used throughout this research work.

In the following chapters, the application of the CNTFETs for the determination of bacteria, yeast and moulds is presented throughout the scientific articles published along the development of the thesis. Briefly, the first device developed was applied to the detection of Salmonella Infantis in a simple matrix (0.85 % saline solution) and it was proven for first time, that this kind of sensor was able to detect, at least, 100 cfu/mL of the bacteria in just one hour with high selectivity. Subsequently, we enlarged the application field to other types of microorganisms: Candida albicans. In this study we improved not only the detection limit of the devices to 50 cfu/mL but also we proved the selectivity of the CNTFETs against possible interference that can be present in real samples like serum proteins. Finally, the devices were applied to the detection of the mould Aspegillus flavus in real samples. In this assay the response time was 30 minutes and a high sensitivity (10 µg of A. flavus / 25 g of rice) was obtained.

As the final chapters, general conclusions extracted from the overall work and annexes are reported. It can be stated that nanomaterials displaying extraordinary properties like carbon nanotubes can be combined with biological entities to obtain highly sensitive and selective biosensors able to detect bacteria, yeasts and moulds in a very short time. In future work, other performance parameters such as, long term stability, robustness and reusability must be studied further and contrasted with standard methods before thinking of the commercialization of the devices.
Los microorganismos están presentes en una gran variedad de orígenes, incluyendo alimentos, agua, animales, medio ambiente también como en el propio cuerpo humano. Estos pueden ser beneficiosos o perjudiciales. Los microorganismos perjudiciales reciben el nombre de patógenos y su detección es de gran importancia por razones de salud y seguridad.

Es bien conocido que los alimentos contaminados con bacterias pueden producir cierto número de enfermedades. Como consecuencia de esto, miles de euros se invierten cada año en tratamientos médicos para mantener la salud de la población. Existen más de 250 enfermedades transmitidas por alimentos. En las últimas décadas se ha incrementado por ejemplo, la incidencia de brotes de salmonelosis en muchos países, siendo Salmonella Infantis uno de los agentes etiológicos más importantes asociados con la producción de esta enfermedad entérica. Debido a la amplia distribución de los microorganismos, estos pueden llegar también a contaminar alimentos durante su cultivo como durante la fase de almacenamiento. En este sentido, los hongos filamentosos son en gran parte los agentes etiológicos responsables del deterioro de alimentos después de la cosecha produciendo pérdidas en la calidad y devaluación económica.

Por otra parte, las infecciones fúngicas invasivas producidas por levaduras han aumentado considerablemente en los últimos años. Candidiasis, es la enfermedad producida por Candida albicans. Esta es una de las infecciones más comunes que afectan pacientes inmunocomprometidos requiriendo tratamientos de elevado coste.

Se han propuesto varios métodos hasta la fecha para la detección de microorganismos patógenos. El cultivo es el método de referencia utilizado para la detección y cuantificación de bacterias. Tiene la ventaja de ser altamente selectivo y sensible pero tiene el inconveniente de requerir varios días para obtener un resultado. Para simplificar y automatizar la identificación de bacterias y hongos se han desarrollado kits bioquímicos rápidos. Aunque los resultados obtenidos usando esta clase de kits son comparables a las pruebas bioquímicas tradicionales, también 1 o 2 días son requeridos para la obtención de resultados. El enzimoinmunoensayo ("Enzyme Linked Immunosorbent Assay", ELISA) es un método immunológico de gran sensibilidad que se utiliza ampliamente para detectar y cuantificar microorganismos patógenos, tanto en el sector médico como en el alimentario. Sin embargo, su principal desventaja es que a veces el tiempo de análisis puede aumentar considerablemente, específicamente cuando se realizan etapas de pre-enriquecimiento de la muestra para disminuir el límite de detección. Como consecuencia, muchos investigadores han dirigido sus esfuerzos hacia el desarrollo de métodos más rápidos.

Los métodos basados en el uso de la biología molecular, específicamente la reacción en cadena de la polimerasa (PCR) y la PCR en tiempo real, son hoy en día las herramientas más comúnmente usadas para la detección de patógenos. Estas técnicas son altamente sensibles y permiten la cuantificación del patógeno. Adicionalmente, se han desarrollado chips con plataformas de DNA para analizar cientos de patógenos simultáneamente. Sin embargo, esta técnica es costosa y requiere el uso de muchos reactivos.

La introducción de los biosensores ha contribuído a generar nuevas alternativas para la detección de patógenos. Los biosensores son las herramientas más usadas en la detección de patógenos después de la PCR, los métodos convencionales y el ELISA. Tienen la ventaja de proporcionar respuestas rápidas entre la toma de muestra y la obtención de los resultados. No obstante, el reto para su aplicación en muestras reales radica en alcanzar selectividades y sensibilidades comparables a los métodos convencionales ya establecidos y a un costo económico reducido.

Desde que Iijima descubrió los nanotubos de carbono (CNTs) se han publicado numerosos trabajos sobre sus excelentes propiedades electrónicas y ópticas, las cuales, en conjunción con su tamaño, hacen de estas nanoestructuras materiales interesantes en el desarrollo de plataformas de biodetección. Los CNTs presentan una gran capacidad de transferencia de carga entre estructuras heterogéneas. Ello les confiere una gran utilidad en la elaboración de sensores de tipo electroquímico. Su conductividad eléctrica varía de forma muy acusada con cambios en su ambiente químico y, como resultado, se han aplicado con éxito en el estudio de procesos de reconocimiento molecular.

Una metodología para la detección directa de biomoléculas integra los CNTs como elementos transductores dentro de una configuración de transistor de efecto campo (FET). Las principales ventajas de esta clase de configuraciones radican en que el canal conductor se localiza sobre la superficie del substrato y, como resultado, es altamente sensible a cualquier cambio en el medio ambiente. Además, los CNTFETs pueden operar a temperatura y, humedad ambientales.

Al inicio de esta tesis (2006), todavía no se habían aplicado los CNTFETs basados en nanotubos de carbono monocapa a la detección de bacterias y hongos. Sólo se había estudiado la interacción entre los CNTs y bacterias, pero sin el objetivo de detección. Por tanto, esta tesis aporta los primeros CNTFETs aplicados a la detección de microorganismos patógenos. En primer lugar, se presentan los antecedentes y la introducción, donde se realiza una revisión crítica y actualizada de los métodos e investigaciones más relevantes para detectar microorganismos patógenos. Posteriormente, se incluye un capítulo con la información detallada de todos los procedimientos experimentales, herramientas analíticas y materiales utilizados a lo largo del trabajo de investigación.

En los siguientes capítulos, se presenta la aplicación de CNTFETs en la determinación de bacterias, mohos y levaduras mediante artículos científicos publicados a lo largo del desarrollo de la tesis. Brevemente, el primer dispositivo desarrollado se aplicó a la detección de Salmonella Infantis en una matriz simple (solución salina 0.85 %) y se comprobó por primera vez que esta clase de sensores eran capaces de detectar al menos 100 ufc/mL de la bacteria en tan solo una hora con alta selectividad. Seguidamente, se amplió el campo de aplicación a otro tipo de microorganismo, Candida albicans. En este estudio, se mejoró no sólo el límite de detección de los dispositivos a 50 ufc/mL sino que también se mejoró la selectividad de los CNTFETs frente a posibles interferentes que pueden estar presentes en muestras reales, tales como proteínas séricas. Finalmente, se aplicaron los dispositivos a la detección del moho Aspergillus flavus en muestras reales. En este ensayo, el tiempo de respuesta fue de 30 minutos y se obtuvo una buena sensiblidad (10 µg de A. flavus / 25 g de arroz).



Como parte final de la tesis, se presentan las conclusiones generales extraídas a lo largo del trabajo completo junto con los anexos. Puede concluirse que, gracias a las propiedades únicas de los nanotubos de carbono, dichos nanomateriales pueden combinarse con entidades biológicas (como los anticuerpos) para obtener biosensores altamente sensibles y selectivos capaces de detectar bacterias, levaduras y mohos en un tiempo de análisis muy reducido. Como trabajo futuro, se deberán estudiar otros parámetros de calidad de los dispositivos tales como la estabilidad a lo largo del tiempo, la robustez o su reutilización con el fin de contrastarlos con los métodos estándar antes de poder iniciar la comercialización de este tipo de sensores.

Aquesta tesi s'ha focalitzat en el desenvolupament de noves eines immunoquímiques per al diagnòstic de malalties infeccioses amb l'objectiu d'incrementar l'eficiència dels actuals mètodes de diagnòstic. Concretament, aquesta tesi s'ha centrat en el desenvolupament de tècniques immunoquímiques per a la detecció de Staphylococcus aureus i Pseudomones aeruginosa en mostres biològiques. L'estratègia ha consistit a seleccionar dianes específiques de cadascun dels microorganismes i, mitjançant el disseny racional d'haptens d'immunització, desenvolupar anticossos policlonals específics. Els anticossos han estat avaluats mitjançant assajos de tipus ELISA (enzyme-linked immunosorbent assay) i posteriorment s'han implementat en immunoassajos ELISA en microplaca o de micromatrius (microarrays) fluorescents per a l'anàlisi de mostres biològiques (bacteris en medi de cultiu) i mostres clíniques d'origen respiratori (esput, rentats broncoalveolars i broncoaspirats). Els immunoassajos obtinguts per ambdós bacteris són capaços de detectar les dianes seleccionades amb nivells de detecció útils per a la seva implementació en l'àmbit de diagnòstic, oferint una alternativa prometedora al diagnòstic de malalties causades per aquests bacteris. Com a resultat de la investigació realitzada, s'han generat dues patents (PCT/ES2014/070161 i P201530780), les quals es troben en fase de negociació amb empreses de l'àmbit del diagnòstic. A més s'ha publicat un article de revisió que descriu l'estat de la qüestió pel que fa al potencial de la nanobiotecnologia en el diagnòstic de microorganismes patogen, un article de recerca original, que ja ha estat publicat en la revista Analytica Chimica Acta i que descriu el treball realitzat pel que fa a la detecció immunoquímica de S. aureus, i un tercer article que es troba sota avaluació pels revisors i editors de la revista Analytical Chemistry i que reporta la recerca realitzada pel que fa al desenvolupament d'eines immunoquímiques per al diagnòstic de malalties causades per P. aeruginosa. Per aquest motiu, la tesi s'ha estructurat en format de compendi de publicacions. La tesi també inclou un annex fruit d'una estada predoctoral al grup del Prof. Kim Janda de The Scripps Research Institute (TSRI, CA, EEUU). L'objectiu de l'estada era l'avaluació com a eines terapèutiques dels anticossos produïts en aquesta tesi contra la P. aeruginosa. En aquest sentit, es pretenia investigar el potencial dels anticossos generats per neutralitzar els efectes citotòxics causats per factors de virulència d'aquest microorganisme sobre línies cel•ulars de macròfags. Tot i l'interès d'aquests estudis, aquest treball no es va poder concloure, i no es descarta reprendre'l més endavant.
This thesis has focused on the development of immunochemical new tools for the diagnosis of infectious diseases with the aim of increasing the efficiency of current diagnostic methods. Specifically, this work has focused on the development of immunochemical techniques for the detection of Staphylococcus aureus and Pseudomonas aeruginosa in biological samples. The strategy consisted in selecting specific targets for each of the microorganisms and, specific polyclonal antibodies were developed through rational design of immunization haptens. The antibodies were evaluated by ELISA tests (enzyme-linked immunosorbent assay) and later implemented in microplate ELISA immunoassays and fluorescent microarrays for the analysis of biological samples (bacteria in culture medium) and clinical samples of respiratory origin (sputum and broncoalveolars lavages and broncoaspirats). The immunoassays obtained for both bacteria are capable of detecting the selected targets with detection levels useful for its implementation in the diagnosis field, offering a promising alternative for diagnosing diseases caused by these bacteria. As a result of the research carried out two patents have been generated (PCT/ES2014/070161 and P201530780), which are under negotiation with companies in the field of diagnosis. It has published a review article that describes the state of the art regarding the potential of nanobiotechnology in the diagnosis of pathogenic microorganisms, an original research paper, which was published in the Analytica Chimica Acta journal describing the work regarding the immunochemistry detection of S. aureus, and a third article that is under evaluation by the reviewers and editors of the Analytical Chemistry journal and reporting research conducted regarding the development of immunochemistry tools to diagnose diseases caused by P. aeruginosa.

The thesis presents the development of functionalized electrospun nylon 6 nanofibers for the eradication of pathogenic microorganisms in drinking water. Imidazole derivatives were synthesized as the antimicrobial agents and were characterized by means of NMR spectroscopy, IR spectroscopy, elemental analysis and X-ray crystallography. The first set of compounds (2-substituted N-alkylimidazoles) consisted of imidazole derivatives substituted with different alkyl groups (methyl, ethyl, propyl, butyl, heptyl, octyl, decyl and benzyl) at the 1-position and various functional groups [carboxaldehyde (CHO), alcohol (CH2OH) and carboxylic acid (COOH)] at the 2-position. It was observed that the antimicrobial activity of the compounds increased with increasing alkyl chain length and decreasing pKa of the 2-substituent. It was also observed that the antimicrobial activity was predominantly against a Gram-positive bacterial strains [Staphylococcus aureus (MIC = 5-160 μg/mL) and Bacillus subtilis subsp. spizizenii (MIC = 5-20 μg/mL)], with the latter being the more susceptible. However, the compounds displayed poor antimicrobial activity against Gram-negative bacterial strain, E. coli (MIC = 150- >2500 μg/mL) and did not show any activity against the yeast, C. albicans. The second set of compounds consisted of the silver(I) complexes containing 2-hydroxymethyl-N-alkylimidazoles. The complexes displayed a broad spectrum antimicrobial activity towards the microorganisms that were tested and their activity [E. coli (MIC = 5-40 μg/mL), S. aureus (MIC = 20-80 μg/mL), Bacillus subtilis subsp. spizizenii (MIC = 5-40 μg/mL) and C. albicans (MIC = 40-80 μg/mL)] increased with the alkyl chain length of the 2-hydroxymethyl-N-alkylimidazole. The third set of compounds consisted of the vinylimidazoles containing the vinyl group either at the 1-position or at the 4- or 5- position. The imidazoles with the vinyl group at the 4- or 5-position contained the alkyl group (decyl) at the 1-position. For the fabrication of the antimicrobial nanofibers, the first two sets of imidazole derivatives (2-substituted N-alkylimidazoles and silver(I) complexes) were incorporated into electrospun nylon 6 nanofibers while the third set (2-substituted vinylimidazoles) was immobilized onto electrospun nylon 6 nanofibers employing the graft polymerization method. The antimicrobial nylon nanofibers were characterized by IR spectroscopy and SEM-EDAX (EDS). The electrospun nylon 6 nanofibers incorporated with 2-substituted N-alkylimidazoles displayed moderate to excellent levels of growth reduction against S. aureus (73.2-99.8 percent). For the electrospun nylon 6 nanofibers incorporated with silver(I) complexes, the levels of growth reduction were >99.99 percent, after the antimicrobial activity evaluation using the shake flask method. Furthermore, the grafted electrospun nylon 6 nanofibers showed excellent levels of growth reduction for E. coli (99.94-99.99 percent) and S. aureus (99.93-99.99 percent). The reusability results indicated that the grafted electrospun nylon 6 nanofibers maintained the antibacterial activity until the third cycle of useage. The cytotoxicity studies showed that grafted electrospun nylon 6 nanofibers possess lower cytotoxic effects on Chang liver cells with IC50 values in the range 23.48-26.81 μg/mL. The thesis demonstrated that the development of antimicrobial electrospun nanofibers, with potential for the eradication of pathogenic microoganisms in water, could be accomplished by incorporation as well as immobilization strategies.

In recent decades, most countries of the world have experienced a shortage of water and increase its rate of consumption. Today, every country in the world are interested in this problem by trying to find alternatives to address this shortage. One solution is reuse greywater (GW) for irrigation after treatment. GW is all water generated from Household except toilet water. The risks associated with the reuse of these waters are the presence of pathogens that can infect humans, animals and plants. In this thesis focused on studying treatment by slow sand filtration and the survival of representatives of pathogens, such as E. Coli, P. aeruginosa , E. Faecalis and Bacteriophage MS2 which could be found in the greywater. The study factors was a physico-chemicals factors such as; temperature (6±2,23±2,42±2°c), salinity (1.75 and 3.5% Nacl), oxygen (aerobic and anaerobic condition), nutrient ( rich media , 50%: 50% salt and poor media ), light with photocatalysis ( UV and Visible lights) and slow sand filter (Egyptian desert sand and swimming pool sand). A combination of high temperature, sunlight and photocatlysis are mainly responsible for the rapid decline of bacteria and MS2 coliphage. Slow sand filter have clearly less influence on the survival of bacteria in the greywater, but it effective to decline turbidity and COD for short times.

SHEFET, SARID M.

    More than 10% of the U.S. population experience at least one incident of foodborne disease annually (Todd, 1989). From 1983 to 1987, infections contribute to at least 1,000 deaths per year in the United States.

     Poultry products are considered to be the single most important food source of contamination rates for live chickens can vary from about 13% to 80% of the flock and are invariably higher after processing (Mead, 1976; Roberts, 1988; Budnik, 1990). In 1992, the U.S. was ranked first in the world in poultry consumption with 94.8 pounds per capita, followed by Israel with 83.7 pounds, and Hong Kong with 79.3 pounds (Brown, 1993). In 1993 over 27.6 billion pounds of ready-to-cook poultry products were produced in the U.S. Per capita consumption of poultry products has increased substantially over the last two decades relative to other meat products; therefore, exposure of the consumer to poultry product-associated microorganisms including pathogens has correspondingly increased and no doubt contributes to these foodborne disease statistics.

    Besides bacterial pathogens, poultry products are also contaminated with a variety of spoilage microorganisms which can contribute to the development of strong off odors and/or slime formation and shortened product shelf life. These organisms, however, are not generally associated with human illness. A reduction in the population of these microorganisms or suppression of their growth often results in increased product shelf life and greater consumer acceptability. Some reports have estimated that the presence of pathogenic and spoilage microorganisms on poultry may cost the American public over two billion dollars annually in foodborne disease-related expenditures and spoiled products (Roberts, 1988; Todd, 1989).

     The bacteriocin nisin was approved by the United States Food and Drug Administration in 1988 as a GRAS (general recognized as safe) substance for use in pasteurized cheese spreads to control outgrowth and toxin production by Clostridium botulinum. Blackburn when combined with chelating agents such as disodium ethylenediamine tetraacetate (EDTA) and citrate. Perturbation of the outer membrane of gram-negative bacteria via chelation of divalent cations located in the lipopolysaccharide layer is believed to sensitize the cells by providing access to the cytoplasmic membrane where nisin-mediated inactivation occurs.

    The initial focus of this study was to optimize the inhibitory activity of nisin against a NAR skin population, as observed with broiler drumstick skin, were detected following treatment with the four nisin-containing treatments.

     Experiments were also conducted to determine the efficacy of the nisin-based treatments against NAR-infected drumstick skin under varying exposure times and concentrations of nisin. Exposure time significantly influenced the lethality of the treatments and depending on the treatment, nisin concentrations could be reduced from 100 µg/ml to 50 or 25 µg/ml without loss of significant biocidal activity. In other studies, the refrigerated shelf life of broiler drumsticks was extended by 1.5 to 3 days following immersion for 30 minutes in one of the optimized nisin-containing treatments in comparison to drumsticks immersed in distilled, deionized water.

     These findings indicate that treatments containing nisin and varying concentrations of chelating agents and/or surfactant at an acidic pH are capable of significantly inhibiting the population of -free poultry products, the identification and implementation of effective preservation methods could result in several long term benefits including greater public confidence in poultry products, an increased market potential, and increased profits for the poultry industry.