Detecta la presencia y determina la prevalencia de E. coli O157:H7 en Latuca sativa (lechuga) y Spinacea oleracea (espinaca), obtenidas de fundos agrícolas de la periferia de la ciudad de Lima. Se analizan 120 muestras de hortalizas, proveniente de 4 fundos de Lima; se utiliza la técnica del número más probable para la enumeración de E. coli, para la caracterización de E. coli O157:H7 se emplea el enriquecimiento selectivo, el análisis bioquímico y pruebas serológicas. Para determinar la presencia de los factores de virulencia shigatoxina (stx) e intimina (eaeA) se emplea el método de PCR en tiempo real. Para la entero hemolisina se realiza la prueba de hemólisis. Del total de muestras recolectadas (120), el 13,33 %(16) resulta positivo para E. coli O157:H7, el 1,67 %(2) presenta E. coli O157 no H7. De las 16 cepas (13,33 %) de E. coli O157:H7 se obtienen las siguientes secuencias de factores de virulencia: 3 (2.50 %) stx - / eaeA + y Hem - ; 8 (6,65 %) stx +/ eaeA + y Hem -; 2 (1,67 %) stx + / eaeA + y Hem +; 2 (1,67 %) stx + / eaeA - y Hem -; y 1 (0,83 %) stx - / eaeA + y Hem +. Así mismo el 86,67 % resulta negativo para E. coli O157:H7. El estudio revela la presencia de E. coli O157:H7 con una prevalencia del 13,33 % en hortalizas de fundos agrícolas de la periferia de la ciudad de Lima. / Tesis

With modern culture moving towards convenience in terms of fresh produce, especially with meat products, science needs to constantly evolve to serve these customer needs. These needs however can sometimes be implemented too hastily without the proper assessment done for factors such as food safety. One such improvement is the use of modified atmosphere packaged minced meat. This form allows the minced meat to be kept for much longer than normal without freezing the product, providing the fast pasted consumer both the convenience of having fresh, unfrozen meat as well as an added shelf life. MAP works by disrupting the atmosphere within these packages, retarding the growth of the spoilage micro-organisms, thus causing them to require a longer time span to reach spoilage numbers. The problem however arises that although most of these techniques are tried and trusted on the products’ spoilage organisms, it does not take into consideration the effect MAP and the altered spoilage organism communities would have on a pathogen that might be present on the products. This study thus aimed to assess the effect of both factors on Escherichia coli O157:H7 (phase 1) and Staphylococcus aureus (phase 2) in minced meat kept at 5°C as well as trying to identify the major affecting factors. The two subject organisms were each inoculated into 2 different types of MAP packs and a non-atmosphere modified PVC overlaid minced meat sample at two differing concentrations of 105 and 102 to also assess the impact of high andlow initial pathogen presence. These packs were then analysed over a time period of 16 days to track the changing minced meat environment. APC, Pseudomonads, LAB and Enterobacteriaceae counts were all investigated along with the pathogenic counts. Apart from colony enumeration, the colour of the minced meat samples were also taken to determine the effect that these parameters have on the appearance of the product, as colour is often the first sensorial characteristic that determines the purchase of fresh meat products. pH was determined to ascertain the environmental changes occurring in the product and whether groups such as the LAB would change the environment to better suit their needs. Finally the atmospheric makeup was also measured to determine the effect of the MAP system and the change occurring in a closed system that could be attributed to the growth and respiration of the bacterial communities present. Apart from the main aim of the study, two additional studies were performed that arose during the planning and analyses of the two primary phases. Firstly the use of a quarter versus a full plate enumeration was studied to determine its accuracy as well as possibility of use in full studies to aid enumeration and decrease time and financial input. Here a direct comparison was done between the two techniques after which they were compared and assessed in their functionality for both homogenous and heterogeneous community enumeration on selective and non-selective media. The other secondary study focussed on the use of new technology for both the enumeration and tracking of genetically modified organisms in a variety of different environments. Here a bioluminescent imaging system was used on a genetically modified strain of E. coli to track its spread through minced meat, packaged either in a MAP or PVC overlaid pack, over 48 hours in an accelerated shelf life study. Enumeration of said organism was also undertaken whereby the intensity of emitted light would correspond to a defined count, enabling rapid enumeration of samples, whether overgrown or not. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Food Science / unrestricted

Escherichia coli 0157:H7 is classified as a member of the enterohaemorrhagic E. coli (EHEC) family. These organisms are responsible for a variety of clinical manifestations ranging from non-bloody diarrhoea to gross bloody diarrhoea with complications that include haemorrhagic colitis (HC), haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP). Infection occurs by the ingestion of faecally contaminated food products, water sources and through person-to-person contact. Outbreaks of E. coli O157:H7 have been reported worldwide although most outbreaks seem to be from countries in the Northern hemisphere. Very little information is available on the prevalence of E. coli O157:H7 in South Africa. The only data available on E. coli O157:H7 were from a 1992 outbreak in Swaziland with some cases spreading to the adjacent provinces of South Africa. Selective methods were assessed and optimised to identify and isolate E. coli O157:H7 from food, water and faeces. These methods included culture techniques, immunomagnetic separation, immunoassays and molecular confirmation techniques. The methods optimised and assessed in this study proved to be suitable for the detection and isolation of E. coli O157:H7 from environmental water, food and faecal samples. In addition to the isolation of E. coli O157:H7 from these sources, methods were also optimised for the characterisation of E. coli O157:H7 using repetitive sequence analysis and induction of shiga toxin (Stx)¬converting phages which carry the genes coding for Stx from strains of E. coli O157:H7. The prevalence of E. coli O157:H7 in human-, bovine- and porcine faeces, sewage and recreational waters was investigated in a selected region of South Africa. Data suggested a low prevalence in sewage (0.76%), recreational waters (0%) and human faecal (0%) samples with a higher prevalence among carriers such as cattle (12.5%) and pigs (14.29%). UV-induced Stx-converting phages were examined and found to have different phage morphologies to the previously described lambdoid structure. In order to establish the host range susceptibility of these phages, all induced phages were subjected to conditions favourable for infecting E. coli O157:H7, non-O157 E. coli and other members of the enterobacteriaceae family including Salmonella, Shigella, Enterobacter, Klebsiella, and Proteus. These results have shown that Stx-phages were able to infect Salmonella cholerasuis and produce infectious progeny from these strains. Stx-converting phages propagated in Salmonella cholerasuis were able to re-infect strains of E. coli O157:H7. This study has shown that IMS in combination with molecular techniques was a sensitive tool for the isolation, identification and characterisation of E. coli O157:H7 from different sources. Results indicated that Stx-phages induced from E. coli O157:H7 demonstrated lambdoid structure as well as phages with long hexagonal heads and long non-contractile tails. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2005. / Medical Virology / unrestricted

Escherichia coli O157:H7 detection in food is conducted mainly by DNA/PCR, immunoassay or conventional methods. However, all the methods require multiple incubation steps. Antibiotic and isolation agars were found as the main factors that lead to false-positive results. An improved rapid detection method was developed by decreasing detection time and enhancing easiness of detection without the need for any analytical instrumentation. A combination of selective ingredients and temperature was utilized to allow the growth of Escherichia coli O157:H7 in the detection. The detection method minimized the effects of the main false positive bacteria, Pseudomonas spp. and Enterobacter spp. The sensitivity, specificity and accuracy of the 24h detection method in foodstuffs were 96.2%, 99.6% and 97.0%, respectively when the original inoculation was 10-100cfu/g in food. This method can be utilized to detect Escherichia coli O157:H7 in foodstuffs more rapidly, economically and conveniently when compared to the methods that are currently used.

Three studies regarding Escherichia coli O157:H7 in apple cider were conducted. The objectives were: to evaluate the effectiveness of wash and sanitizers for removing E. coli O157:H7 from apples; to survey cider producer practices; and to determine the efficacy of ultraviolet light for reducing E. coli O157:H7 in cider. Apples with a five-strain acid resistant mixture of E. coli O157:H7 were treated with 200 ppm hypochlorite, a phosphoric acid-based fruit wash, 5% acetic acid, 5% acetic acid followed by 3% hydrogen peroxide, a peroxyacetic acid-based solution, and distilled water. The water wash caused insignificant reductions. All other treatments caused significant reductions. Acetic acid and peroxyacetic acid were the most effective with reductions of 3.1 and 2.6 logs, respectively. The survey determined that most producers are small, seasonal operations. Most use sound orchard management practices, clean and sanitize daily, sort and wash apples, use refrigeration, and try to prevent contamination. However, some use drop and damaged apples. Few use chemical sanitizers on apples, preservatives, pasteurize cider, or have HACCP programs. Cider inoculated with the same mixture of E. coli O157:H7 was processed using a thin- film ultraviolet disinfection unit operating at 254 nm. Dosages ranged from 9,402 to 61,005 æW- sec/cm2. Treatment significantly reduced E. coli O157:H7 (pó 0.0001) with a mean reduction of 3.81 log CFU/ml. Reduction was also affected by the level of background microflora in cider. Results indicate that ultraviolet light can reduce this pathogen in cider. However, additional reduction measures are necessary to achieve the required 5 log reduction. / Master of Science

The number of food borne illnesses associated with the consumption of fresh fruits and vegetables and their minimally processed products (juices) has increased over the past years. Of particular interest is the ability of microbial pathogens to internalize and survive in fresh produce that are commonly used for juices. This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple. Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production. / Master of Science

The main objective of this project is to gain an understanding of the internalization of Escherichia coli in the tissues of apples. This broad statement includes the rate of internalization in young versus mature apples as well as injured versus non-injured apples. Five apple varieties, Redfree, Red Delicious, Golden Delicious, Rome Beauty and York Imperial, were used to compare differences and similarities in structure and ability to internalize the pathogen. Both the surrogate species, E. coli ATCC 25922, and the pathogen, E. coli O157:H7, were used for field and lab studies, respectively. Internalization of E. coli in apples under natural environmental conditions was addressed in the first study using a controlled outdoor setting. Escherichia coli species (ATCC 25922) was used as an alternative to the pathogenic species. The bacterial culture was applied to topsoil and spread evenly on a 6x6-foot area. Red Delicious, Golden Delicious, and Rome Beauty apples were placed randomly on the soil much like a drop or windfall apple. The position was noted as to whether the apple fell calyx up, down or on its side. Apples were examined for the presence of E. coli and sampled on days 1, 3, 8, and 10. Skin, flesh, inner, and outer core samples were plated on MacConkey agar supplemented with cycloheximide and MUG to ease in identification. Escherichia coli was found in the inner core and flesh samples of all apple varieties, indicating the potential for infiltration by the organism outside laboratory conditions. The second study determined the rate of internalization in immature apples. Redfree was used in a long-term study in which individual apples were spray inoculated at the beginning of the growing season with E. coli ATCC 25922 at 104 cfu/apple. The apples were picked on days 1, 30 and 60, and sectioned into skin, flesh, inner and outer cores. The remaining four apples species were used in an intensive, two-week study. In the long-term study, apples were inoculated two weeks prior to harvest and picked every other day until harvest. The surrogate E. coli was not found in the apples after day 1. Other coliforms, such as E. vulneris, Klebsiella pneumoniae and Kl. ozaenae were present in each pick. The two-week study showed higher rates of internalization in Red and Golden Delicious than in Rome and York, with the E. coli present in all four sections of the apples. Red Delicious apples showed a trend of increasing counts of bacteria over the two-week period with initial counts ranging from less than one cfu/ml to final counts as high as 2.64±1.90 log cfu/ml. Again Klebsiella species and E. vulneris were found in the apples. Microscopy was used for imaging of the apples tissues. Morphological differences were found in the skin, where lenticel presence or absence may affect internalization. Differences were also shown in the flesh where cell wall thickness was shown to vary depending on variety. Imaging thick sections of skin showed cuticle cracks and thickness, which also vary depending on the apple variety. This study indicates that internalization occurs at a high degree in drop apples and to a limited extent in tree apples. However, with the low infective dose required for illness, it is necessary to instate strict regulations to ensure safety. The most effective treatment involves the inclusion of a five-log reduction of the target organism, E. coli O157:H7. This reduction can be obtained through one step or the combination of two or more steps. / Master of Science

Escherichia coli O157:H7 is an enteric pathogen that can cause severe gastrointestinal disease, sometimes leading to hospitalization and death. These bacteria have a variety of virulence factors that can be encoded for on pathogenicity islands (PAIs). The goal of this study was to characterize specific E. coli O157:H7 PAI deletion mutants using three methods: Phentotype Microarrays (PM), growth curves and survival curves were used to elucidate possible roles for the PAIs. Results from the PM study suggest that PAIs have a role in carbon substrate utilization; i.e., four of the O-island (OI) deletion mutants (OI-87, 98, 102 and 172) and an S-Loop (SL-72) deletion mutant exhibited differences in substrate utilization (gains and losses in utilization) compared to parental O157:H7 strains EDL933 (OI) and Sakai (SL), respectively. All of the mutants with the exception of the OI-135 mutant exhibited differences in level of substrate utilization for substrates shown to have important roles in the bacterium. Cell growth results showed that three OI deletion mutants (OI-55, 87 and 102) and the SL (SL-72) mutant exhibited a difference in rate of growth compared to the parental strains. Cell viability results showed that seven of the OI deletion mutants (OI-51, 55, 98, 108, 135, 172 and 176) exhibited different rates of decline in cell number when transferred to sterile water compared to the parental strain. The results show that removal of PAIs from E. coli O157:H7 can affect carbon utilization, growth and survival demonstrating the importance of PAIs in the ecology of these bacteria. / xx, 208 leaves : ill. (some col.) ; 29 cm

[Truncated abstract] The applications of biosensors are rapidly expanding with the increased emphasis placed on the use of technology in the evaluation of food safety and also in military use. The United States food industry carried out 144.3 million microbiological tests in 1999 (Alocilja and Radke, 2003). These numbers are expected to rise with the recently implemented regulatory measures for food safety in the United States. In fact, similar trends in food safety are occurring on a global scale. Furthermore, with the recognition and establishment of Microbial Forensics as a new field of forensics, the interest in biosensor development for the detection of microbes will thrive. Moreover, the recent spate of biocrimes, notably the anthrax scares, has called for newer and improved techniques for the sensitive, rapid and reproducible detection of microbes. Biosensors have the capability to fill this role as an efficient device for microbial detection. There is a wide range of biosensors available for different purposes. In addition, their versatility allows for their overlap in many fields. The quartz crystal microbalance (QCM) is a biosensor that is cost-efficient, sensitive, field-deployable with the ability to perform automated, real-time assays within minutes. The QCM is a mass sensitive device that works on the principle where a change in mass deposited on the crystal is inversely proportional to the change in the resonant frequency of the crystal. Therefore, frequency decreases with increasing mass deposited. The QCM has been used in several studies as a biosensor for the detection of a number of viral and bacterial species. ... High antibody incubation concentration required a shorter antibody incubation duration. Conversely, low antibody incubation concentration required a longer antibody incubation duration. Furthermore, regardless of antibody incubation concentration, a distinct pattern in the rate of antibody binding with time was observed. One hour antigen incubation at ambient room temperature (22.5oC) was sufficient for the efficient binding of the antigens to the immobilized antibody layer. Extension of antigen binding time to 15 hours produced inconsequential differences in readings. The binding efficiency of the quartz crystals after a storage period of 2 to 4 weeks at ambient room temperature (22.5oC) fared better than the crystals that were refrigerated at 4oC. Results showed that 0.2M glycine hydrochloride is a poor reagent for the removal of the antigen layer on the quartz crystals for repeated assay use. The 16-mercaptohexadecanoic acid (MHDA) layer and adsorbed proteins on the quartz crystals can be removed by a mixture of sulphuric acid and hydrogen peroxide, known as a piranha process. This allows the crystals to be repeatedly recoated and reused. Overall, this research provides new insights into the preparation process of the quartz crystals for the specific detection of E. coli O157:H7. Conclusive results have been obtained for several tested parameters and suggestions have been raised for further studies in the optimization of the QCM for the E. coli O157:H7 detection process. With improved knowledge and recognition in the capability of the QCM as a biosensor, the QCM may soon be used in conjunction with conventional techniques for the rapid detection of E. coli O157:H7.

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