This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

A collaborative pilot study of the microflora on biological activated charcoal (BAC) filters employed in the tertiary treatment of drinking water revealed the principle bacterial genera to be Pseudomonas, Alcaligenes, Achromobacter, Bacillus, Micrococcus, Corynebacterium, Chromobacterium, Microcyclus and Paracoccus. The microbial population of the filters paralleled seasonal carbon dioxide production. Of particular interest were the effects of the BAC miroorganisms upon precursors of trihalomethanes (THMs). Mixed populations of BAC microorganisms were cultivated for 50 days in a mineral salts-humic acid medium. It was concluded that (1) the BAC microflora enhances the absorptive capacity of the filters; (2) chemico-physical and biological processes operate in concert to lower the concentration of precursors of THMs; and (3) few bacterial pathogens establish themselves on the filters.

This dissertation examined the microbial ecology of Listeria monocytogenes in three distinctly different environments: a cheese microbial community; a farm environment; and a soil microbial community. The aim of the first study was to investigate the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu cm⁻² of a L. monocytogenes six strain cocktail were conducted. The first trial had to be terminated early (day 28) as contamination of Pseudomonas spp. from the initial brine did not produce the expected characteristics of the cheese during the aging period. For the second trial, cheese samples were aged in the lab for 60 days according to the cheesemakers specifications. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms found on the surface of the rind while L. monocytogenes had little effect. In addition, although the concentration of L. monocytogenes increased to levels of 10⁶ cfu cm⁻² based on the enumeration data, the genetic data was not able to identify it in the flora due to the fact that other genera were found at much higher concentrations, which is a limitation of molecular methods used for identification of pathogens in foods. For the second study the presence and incidence of L. monocytogenes on farms that either produce raw milk cheese or supply the milk for raw milk cheese production was investigated. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested with 10 isolates found in the barn samples, 5 from environmental sites and 1 from milking equipment. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks, which emphasizes the adaptability and persistence of highly pathogenic stains in food manufacturing environments. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data and field observations we identified that drinking water sources for the animals is also an important reservoir of L. monocytogenes in farm environments. More importantly this study has shown the importance of continuous monitoring of environmental sites for the presence of the pathogen, particularly in silage. Lastly manure amended soils in the northeastern U.S. were tested for the presence and survival of rifampicin resistant Escherichia coli (rE. coli), generic E. coli (gE. coli) and Listeria spp.. Both gE.coli and rE.coli samples were processed using either direct enumeration, MPN or bag enrichment methods. Samples were taken from both tilled and surface dairy solid manure-amended plots. Listeria samples were processed using a modified BAM method. Listeria presence was constant throughout the study. In contrast, rE. coli and gE. coli levels declined with time. The main conclusions of this study were that soil type, location and physical characteristics have a significant role in the survival of bacterial populations of rE. coli, gE. coli and Listeria spp. in soil. Dairy solids application does not seem to have a long term effect on the natural microbial population of soils. Tilling of soils results in increased survival of the bacterial population due to the fact that it increases soil pore size and facilitates moisture entry, which in turn has been shown to increase bacterial survival rates. Data from this research will assist in the creation of preventative measures that lead to the elimination of pathogen reservoirs. It will be further used to verify that a 120 day interval following manure application should be sufficient to ensure food safety of edible crops subsequently planted on these soils.

The increasing prevalence of allergic diseases in affluent societies has been associated with changes in microbial exposure early in life and a less diverse gut flora. The objective of this thesis was to assess the development of allergic sensitisation and symptoms during the first four years of life in a non-selected birth cohort in relation to environmental factors, family history, gut microbiota and salivary IgA antibodies. The cohort comprised all 1,228 infants living in a Swedish county who were born over a one-year period. The parents replied to questionnaires, and 817 children (67 %) were skin prick tested both at 1 and 4 years of age. Saliva (n=279), faecal (n=139) and blood (n=253) samples were collected at 1 year of age from children with a positive skin prick test at 1 year and from a sample of children with a negative skin prick test. Faecal samples were also obtained from 53 children at 4 years of age. Dog keeping during infancy was associated with a decreased risk of sensitisation to pollen and late-onset wheezing at age 4, and the reduced odds ratios persisted after adjustment for heredity and avoidance measures, OR 0.3, 95% CI 0.1-0.9 and OR 0.5, 95% CI 0.2-1.0, respectively. In contrast, early dog keeping was associated with an increased risk of earlyonset transient wheezing but only in children with parental asthma (OR 2,8, 95% CI 1.3-6.4). Levels of short chain fatty acids (SCFAs) in faeces were assessed both at 1 and 4 years of age and related to the development of sensitisation and symptoms. The levels of acetic (p&lt;.01) and propionic (p&lt;.01) acids decreased from one to four years of age, whereas valeric acid (p&lt;.001) increased which is in line with a more complex gut microbiota with age. Allergic children, compared with non-allergic children, had lower levels of i-butyric, i-valeric and valeric acid in faeces both at 1 and 4 years of age. Low levels of secretory IgA (SIgA) in saliva were associated with wheezing but only in sensitised children. In children with positive SPT to at least one allergen both at 1 and 4 years of age and in children with circulating IgE antibodies to egg or cat at one year of age, those who developed late-onset wheezing had lower levels of SIgA than those who did not, p=.04 and p=.02 respectively. Of 9 children with levels of SIgA in the upper quartile and persistent sensitisation, none developed wheezing, compared with 10/20 children with lower levels, (p=. 01). Having older siblings, more than three infections during infancy, at least one smoking parent and male gender were all associated with high levels (in the upper quartile) of total IgA and SIgA. The findings in this thesis indicate that the microbial load early in life could affect the development of allergy. A functional assessment of the gut flora demonstrated differences between allergic and non-allergic children both at 1 and 4 years of age. Salivary IgA was associated with infections during infancy, and high levels of secretory IgA protected from symptoms in sensitised children. Finally, dog keeping in infancy may offer protection from allergy, but the mechanism is uncertain.

Different techniques were studied to evaluate the presence of different microorganisms that played important roles in decay processes of historic stones. In that scope, limestones and sandstones from Nemrut Mount Monument, and marbles and andesites from Temple of Augustus were studied. For measurement of enzymatic activity, fluorescein diacetate (FDA) hydrolysis method previously applied to assess soil microbial activity was carried out. Total microflora method based on countings of colony number was conducted for determination of the level of bacterial and fungal activity of stones. ATP bioluminescence method, developed for the field of hygiene monitoring, was carried out in order to detect global metabolic activity degree in historic stones. Most probable number (MPN) method was carried out to detect the number of microbial cells, namely nitrifying and sulphur oxidising bacteria which could take part in the decay processes. Moreover, fungi identification was done for determining occurance of detrimental species. Presence of lichenic and algal zones existed on stones of Nemrut Mount Monument and the presence of black discolorations on stones of Temple of Augustus was common. Results have shown that the bacterial and fungal activity was low, however considerable quantity of FDA hydrolyses has shown the importance of algal population in the stones of two studied historical sites. This study has proved that FDA hydrolyses, total microflora and MPN method were efficient for the evaluation of biodeterioration in historic stones.

<p>Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases.</p><p>The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of <em>Lactobacillus spp</em>. in fecal samples retrieved from a study of the probiotic strain<em> L. reuteri</em> ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained <em>L. reuteri</em> belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.</p>

1. Ištirtos registruotos Lactobacillus plantarum U-14, kuri išskirta iš sveikos karvės makšties ir Lactobacillus fermentum U-5, kuri išskirta iš sveiko veršelių fekalijų, padermės, nustatytos jų optimalios kultivavimo temperatūros, įvertintos jų probiotinės savybės, nustatytas antagonistinis aktyvumas, atsparumas antibiotikams bei įvertinta liofilizacijos įtaka šioms laktobacilų padermėms. 2. Liofilizacijos būdu pagamintas probiotikas, sudarytas iš Lactobacillus plantarum U-14 ir Lactobacillus fermentum U-5 padermių, nustatytas laktobacilų gyvybingumas preparate laikant vienus metus +4oC temperatūroje. 3. Nustatyta probiotiko profilaktinė dozė naujagimiams veršeliams girdant su krekenomis arba pienu pirmas 10 gyvenimo dienų, stebint veršelių klinikinius požymius, įvertinant paros priesvorį, įtaką bendram enterobakterijų ir laktobacilų kiekiui bei nustatant laktobacilų rūšinę sudėtį fekalijose įvertinant kraujo morfologinius ir biocheminius parametrus.4. Nustatyta probiotiko profilaktinė dozė veršeliams, kuri buvo – 4g/d. ir ji palyginta su probiotiko Yeasture profilaktine doze. / 1.The registered strains of Lactobacillus plantarum U-14 isolated from the vagina of healthy cow and Lactobacillus fermentum U-5 isolated from the faeces of healthy calve were studied, the optimal temperature for the cultivation was defined, were evaluated their probiotic properties, the antagonistic activity, resistance to antibiotics and the effect of lyophilization on these strains of lactobacillus were studied. 2.The probiotic preparation, consisting of Lactobacillus plantarum U-14 and Lactobacillus fermentum U-5 strains was produced by the method of lyophilization and the viability of lactobacillus during the period of 1 year storage at +4oC temperature was defined. 3.The preventive dose of probiotic preparation for neonate calves given with colostrum or milk was defined during the first 10 days, the clinical signs of calves, daily weight gain, effect on the total number of enterobacteria and lactobacillus were studied and the lactobacillus species composition in the faeces together evaluating morphological and biochemical indicators of blood was investigated. 4.The defined dose of probiotic preparation for calves was – 4g/d. and it was compared to the preventive dose of the preparation Yeasture.

Stomatitas yra dažna roplių burnos infekcinė liga. Didėjant namie auginamų roplių skaičiui, daugės ir klinikinių atvejų veterinarijos gydyklose. Svarbu identifikuoti sukėlėjus ir paskirti tikslingą antimikrobinę terapiją. Tyrimui burnos gleivinės tepinėliai paimti iš: 8 kukurūzinių žalčių, 2 karališkųjų gyvačių ir 13 stepinių vėžlių, auginamų Lietuvos zoologijos sode. Išskirta burnos mikroflora: kukurūzinių žalčių-Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Bacillus spp.; karališkųjų gyvačių: Staphylococcus spp., Streptoccocus spp.; stepinių vėžlių: Staphylococcus spp., Pseudomonas spp., Bacillus spp., Klebsiella spp. padermės. Jų atsparumui antimikrobinėms medžiagoms nustatyti atliktas testas pagal modifikuotą Kirby-Bauer (indikatorinių diskų) metodą. Panaudotos antimikrobinės medžiagos buvo: penicilinų, cefalosporinų, aminoglikozidų, sulfonamidų grupių. Atlikus sveikų kukurūzinių žalčių burnos mikrofloros tyrimus, nustatytas atsparumas antimikrobinėms medžiagoms- atspariausia sulfonamidui. Sergančio kukurūzinio žalčio išskirta burnos mikroflora atspari cefaklorui, oksitetraciklinui, amoksicilinui. Atlikus sveikų stepinių vėžlių burnos mėginių mikrofloros tyrimą, nustatytas atsparumas antimikrobinėms medžiagoms, atspariausia amoksicilinui ir klavulano rūgščiai. / Stomatitis (mouth infection) is common disease in reptiles. The number of reptiles kept as house pets is increasing so it is likely that clinical cases in veterinary clinics will also increase. It is necessary to identify infective agents and start optimal antimicrobial therapy. In this study microflora was isolated from reptiles kept at Lithuanian zoo: 8 corn snakes, 2 royal snakes, 13 russian tortoises. Isolated microflora was: corn snakes- Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Bacillus spp.; royal snakes: Staphylococcus spp., Streptoccocus spp.; russian tortoises: Staphylococcus spp., Pseudomonas spp., Bacillus spp., Klebsiella spp. Resistance to antimicrobial substances was identified using modified Kirby-Bauer (indicatory discs) method. Antimicrobial substances were: penicillins, cephalosporins, aminoglycosides, sulphonamides. Resistance to antimicrobial substances of healthy corn snakes mouth microflora was: highest resistance to sulphonamides. Mouth microflora of corn snake with stomatitis was most resistive to cephaclor, oxitetracyclin, amoxicillin. Russian tortoise mouth microflora was most resistive to amoxicillin and clavulanic acid.

Ausies uždegimas ne retai pasitaikanti liga porakanopiniams gyvūnams. Dėl racionalaus antimikrobinių vaistų vartojimo, svarbu išskirti ir identifikuoti sukelėjus ir jų jautrumą antimikrobinėms medžiagoms. Mėginiai imti iš dvylikos sveikų kamerūninių ožių, keturių šiaurinių elnių ir keturių lamų ausų landų. Visi gyvūnai yra laikomi Lietuvos zoologijos sode. Išskirti mikroorganizmai (Staphylococcus spp., Streptococcus spp., Escherichia coli, Bacillus spp., Enterococcus spp., Micrococcus spp.) ir nustatytas jų jautrumas antimikrobinėms medžiagoms. Kamerūniniams ožiams iš ausų landų išskirtos mikroorganizmų padermės Bacillus spp. ir Staphylococcus spp. Mikroflora jautriausia antimikrobinėms medžiagoms – amoksicilinui, cefaleksinui ir gentamicinui. Šiauriniams elniams ir lamoms iš ausų landų išskirtos mikroorganizmų padermės Bacillus spp. ir Staphylococcus spp. Mikroflora jautriausia antimikrobinei medžiagai - amoksicilinui. / Otitis is a common disease in artiodactyla animals. Identification the microorganism and begin the antibacterial treatment is important for rational use of antimicrobials. Samples were collected from twelve cameroons goats, four reindeers and four llamas external ear canal. They all are kept in Lithuanian zoo. Staphylococcus spp., Streptococcus spp., Escherichia coli, Bacillus spp., Enterococcus spp., Micrococcus spp. were identified in the samples and set their sensitivity to antimicrobials. In all twelve samples from healthy cameroon goats were identified Bacillus spp. and Staphylococcus spp. Our data showed that healthy cameroon goats sample's microflora were most susceptible to amoxicillini, gentamicini and cephalexini. In all eight samples from healthy llamas anr reinderrs Bacillus spp. and Staphylococcus spp. were identified. Reindeer and llamas external ear canal microflora samples were most susceptible to amoxicillin.

Mucositis manifesting as diarrhoea is a common side effect of chemotherapy which remains poorly understood. It is one of a number of manifestations of alimentary mucositis, which affects the entire gastrointestinal tract. The exact number of patients that are affected by diarrhoea as a result of treatment is uncertain, although it is believed that approximately 10% of patients with advanced cancer will be afflicted. Despite advances in the understanding of oral and small intestinal mucositis over recent years, large intestinal mucositis, including diarrhoea, has not been well defined and the underlying mechanisms of the condition are yet to be established. The majority of the literature available concerning diarrhoea is based on clinical observations, with very little basic research existing. However, from the research conducted, it is likely that the intestinal microflora and mucins play a role in the development of chemotherapy-induced diarrhoea. This thesis will examine in detail what is known about the mechanisms of chemotherapy-induced diarrhoea (CID). Furthermore it will explore the potentially important relationship between intestinal microflora, the luminal environment and the subsequent development of chemotherapy-induced mucositis and diarrhoea. 5-Fluorouracil (5-FU) is a commonly used chemotherapy agent in clinical oncology practice. Two of its major side effects are mucositis and diarrhoea. The structure of mucins offers mucosal protection, and allows maintenance of intestinal flora by providing attachment sites and preventing bacterial overgrowth and/or penetration. Following treatment with 5-FU, we showed decreases in Clostridium spp., Lactobacillus spp. and Streptococcus spp., and an increase in Escherichia spp. in the jejunum. In the colon, 5-FU caused decreases in Enterococcus spp., Lactobacillus spp. and Streptococcus spp. Real time PCR of faecal samples showed decreasing trends in Lactobacillus spp. and Bacteroides spp., and an increasing trend in E. coli. Significant increases (p<0.05) were seen in Clostridium spp. and Staphylococcus spp. at 24 h. Goblet cell numbers decreased significantly in the jejunum from 24-72 h, with a significant increase in the percentage of cavitated goblet cells, suggesting 5-FU treatment causes significant changes in intestinal flora and mucin secretion in rats. These changes could result in systemic effects, and in particular may contribute to the development of chemotherapy-induced mucositis. Irinotecan causes cholinergic and delayed onset diarrhoea in patients, in which β-glucuronidase produced by gut bacteria is thought to be involved. Diarrhoea was observed in treated rats, as expected, following irinotecan treatment. β-glucuronidase expression increased in the jejunum and colon. Faecal flora changed quantitatively after treatment also, with increases in E. coli, Staphylococcus spp., and Clostridium spp. (all β-glucuronidase producing), and decreases in Lactobacillus spp., Bifidobacterium spp. (both beneficial bacteria), and Bacteroides spp. (β-glucuronidase producing, major component of intestinal flora), suggesting that irinotecan-induced diarrhoea may be caused by an increase in β-glucuronidase producing bacteria. However, the increase in bacteria may also be caused by irinotecan, further exaggerating the toxicity of the drug, and emphasising the need for these specific bacteria to be therapeutically targeted for successful treatment regimens to be accomplished. Mucus production appears to be increased after irinotecan treatment, which may contribute to the development of diarrhoea. Goblet cells were demonstrated to decrease significantly after irinotecan treatment. However, mucin secretion increased. Mucin expression changed significantly after treatment. Muc2 and Muc4 decreased significantly in the villi of the jejunum after treatment, Muc2 and Muc4 decreased significantly in the crypts. Muc2 decreased significantly in the colon. This indicates that irinotecan causes an increase in mucin secretion and a net decrease in mucin-producing goblet cells, and the expression of Muc2 and Muc4 in the gastrointestinal tract is altered following treatment. Increased mucin secretion is likely to be related to altered mucin expression, and may contribute to chemotherapy-induced diarrhoea. To determine if the changes to the intestinal microflora caused by chemotherapy could be translated to the clinic, a pilot clinical study was carried out. Sixteen patients experiencing CID were recruited to the study with two control subjects. A large proportion of patients (75%) demonstrated a reduced anaerobic component of their faecal microflora. A reduced diversity of species was also observed in patients. The majority of patients exhibited decreases in Clostridium spp., Lactobacillus spp. and Bifidobacterium spp., whilst all patients exhibited decreases in Bacteroides spp. and Enterococcus spp. Patients receiving antibiotics did not exhibit any marked differences to patients not receiving antibiotics. This indicates that the results observed in the animal studies are clinically relevant, and further research into this area should be undertaken. CID is associated with marked changes in the intestinal microflora. These changes may result in diminished bacterial functions within the gut, altering gut function and initiating intestinal damage, resulting in the onset of diarrhoea. In conclusion, there is clear evidence demonstrating chemotherapy treatment results in changes to the intestinal microflora and mucin secretion, which may be responsible in part for the development of severe mucositis and diarrhoea. Irinotecan toxicity may be compounded by the increase in β-glucuronidase producing bacteria. The intestinal flora of cancer patients experiencing CID is also noticeably different to that of healthy subjects. Irinotecan causes changes to mucin secretion, and the specific expression of Muc2, Muc4 and Klf4, suggesting that secretory control by the enteric nervous system may also be affected by chemotherapy. This research has extended the understanding of chemotherapy-induced mucositis and diarrhoea, complex side effects of chemotherapy. However, new areas for future research have also been identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1352119 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2009