Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.

Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.

Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have caught the attention of federal agencies and researchers due to their great economic impact when illnesses occur. To reduce the presence of these pathogens, different approaches have been used. However, since the global consumer’s demand for natural ingredients is steadily increasing, the investigation of the effectiveness of potential natural antimicrobials is necessary. In this study, the in vitro antimicrobial activity of Hibiscus sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was investigated. Furthermore, H. sabdariffa L and ε-polylysine were evaluated to reduce populations of Salmonella and E. coli O157:H7 in ground beef. The minimum inhibitory concentration (MIC) of H. sabdariffa L extracts against Salmonella and E. coli O157:H7 was 6,489 μg/mL and for L. monocytogenes, 5,309 μg/mL. The minimum bactericidal concentration (MBC) of H. sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was 19,467, 58,400 and 29,200 μg/mL, respectively. The exposure to 58,400 μg/mL of H. sabdariffa extract at 25 oC for 12 h resulted in reductions of more than 6.0 log CFU/mL for any of the 3 pathogens tested. Ground beef inoculated with S. Agona (GFP) and E. coli O157:H7 (RFP) was subjected to 5 decontamination treatments. Three of the treatments were using H. sabdariffa L and the remaining ε-polylysine. S. Agona (GFP) was reduced in 1.1 log cycles using 10% of ground H. sabdariffa L and E. coli O157:H7 (RFP) was reduced 0.9 log cycles using 400 ppm of ε-polylysine. If these natural antimicrobials are combined with current antimicrobial technologies to form a hurdle effect, higher pathogen reductions could be achieved. Reductions in the presence of pathogens in food may lead into reductions in the incidence of foodborne diseases.

Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have caught the attention of federal agencies and researchers due to their great economic impact when illnesses occur. To reduce the presence of these pathogens, different approaches have been used. However, since the global consumer’s demand for natural ingredients is steadily increasing, the investigation of the effectiveness of potential natural antimicrobials is necessary. In this study, the in vitro antimicrobial activity of Hibiscus sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was investigated. Furthermore, H. sabdariffa L and ε-polylysine were evaluated to reduce populations of Salmonella and E. coli O157:H7 in ground beef. The minimum inhibitory concentration (MIC) of H. sabdariffa L extracts against Salmonella and E. coli O157:H7 was 6,489 μg/mL and for L. monocytogenes, 5,309 μg/mL. The minimum bactericidal concentration (MBC) of H. sabdariffa L extracts against Salmonella, E. coli O157:H7 and L. monocytogenes was 19,467, 58,400 and 29,200 μg/mL, respectively. The exposure to 58,400 μg/mL of H. sabdariffa extract at 25 oC for 12 h resulted in reductions of more than 6.0 log CFU/mL for any of the 3 pathogens tested. Ground beef inoculated with S. Agona (GFP) and E. coli O157:H7 (RFP) was subjected to 5 decontamination treatments. Three of the treatments were using H. sabdariffa L and the remaining ε-polylysine. S. Agona (GFP) was reduced in 1.1 log cycles using 10% of ground H. sabdariffa L and E. coli O157:H7 (RFP) was reduced 0.9 log cycles using 400 ppm of ε-polylysine. If these natural antimicrobials are combined with current antimicrobial technologies to form a hurdle effect, higher pathogen reductions could be achieved. Reductions in the presence of pathogens in food may lead into reductions in the incidence of foodborne diseases.

Natural antimicrobials are peptides that are essential components of the innate immune system, providing broad-spectrum protection against bacteria, yeasts and some viruses. In addition to their innate immune activity, they exhibit properties suggesting they interact with the adaptive immune system. These functions imply they may be of particular importance in pregnancy. Intrauterine infection is responsible for approximately one third of cases of preterm labour, and normal labour is considered an inflammatory process, associated with leukocyte invasion of the uterine tissues and increased cytokine production. Little is known, however, about natural antimicrobial expression in pregnant reproductive tract. The aim of this thesis was thus to characterize natural antimicrobial production in pregnancy. The study focused on two main areas - the lower genital tract, comprised of the vagina and cervix; and the innermost fetal membrane, the amnion. In the lower genital tract, levels of natural antimicrobials were determined in samples of cervicovaginal secretions collected from pregnant women, using enzyme linked immunosorbance assay (ELISA). In addition Taqman quantitative PCR and ELISAs were used to investigate natural antimicrobial production by cell lines derived from endocervical, ectocervical and vaginal epithelium. It was found that elafin and secretory leucocyte protease inhibitor (SLPI) were found at high concentrations in cervicovaginal secretions, but levels were diminished in women with the common vaginal infection bacterial vaginosis (p<0.05). Cells derived from the vaginal epithelium expressed greater amounts of elafin than cervically derived cells. However, elafin and SLPI production could be stimulated in endocervical cells by the bacterial product lipopolysaccharide, a response that was not seen in the vaginal cell line. Natural antimicrobial production in the amnion was examined in tissue explants and primary cultured amnion cells, using a combination of Taqman PCR and ELISAs. In addition, cDNA microarray was carried out to investigate factors controlling amniotic antimicrobial production, and the involvement of signalling pathways was studied using specific pathway inhibitors. It was shown that the amnion expressed five antimicrobials: human beta defensins (HBD) 1, 2 and 3, SLPI and elafin. Expression of HBD2 was significantly upregulated following normal labour (p<0.05), with production in primary amnion epithelial cells dramatically increased by IL-1ß. The pattern of HBD2 expression in response to IL-1ß was biphasic, which suggested involvement of a secondary gene product. Several putative influential factors were identified by cDNA micorarray, including the NF-kappaB cofactor NFkappaBinhibitorζ. Its relationship to HBD2 was explored. The involvement of both NF-kappaB and mitogen activated protein (MAP) kinase p38 signalling appeared crucial in the response. This work has shown that natural antimicrobials are expressed by both the lower genital tract, where infections that are associated with preterm labour originate, and in the amnion, which is the fetus last line of defence to infection. They may have an important role in the prevention of infection associated preterm labour. Further characterization of these responses may increase understanding of the physiology, and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Foodborne pathogens are a threat to public health worldwide. Because many consumers prefer natural compounds to synthetic additives, research on safe plant-derived compounds with antimicrobial activity against foodborne pathogens is vital. The aim of this investigation was to evaluate the antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, trans-cinnamaldehyde, citral) and plant-extracts such as green tea, apple skin extract, black and decaffeinated black tea, grapes seed and pomace extracts against foodborne bacteria. Salmonella enterica serotype Typhimurium DT104, and serotype Newport, were selected conducting an antibiotic screening on 23 Salmonella isolates using seven antibiotics to determine antibiotic resistance. Listeria monocytogenes (strain 101M; beef and pork sausage isolate; resistant to antimicrobials in past investigations) was included to represent gram-positive bacteria. Escherichia coli O157:H7 virulent isolates (932- apple juice isolate; ATCC 35150- human isolate; F4637- sprouts isolate; used as a cocktail) were selected after conducting a Multiplex PCR over nine E. coli O157:H7 isolates to detect shiga-toxin 1 and 2 genes. All antimicrobials were evaluated in vitro in phosphate buffered saline. In general, all pathogens were more susceptible to essential oils and their active components, than powder extracts. The most active antimicrobials from each category were directly applied on foods. The activity of oregano oil (0.5%) and green tea (3%) was evaluated against S. Typhimurium on chicken and S. Newport on tomatoes and sprouts, and the results showed that oregano oil was more effective. In addition, baby spinach leaf samples inoculated with green fluorescent protein labeled S. Newport were examined under confocal scanning laser microscope before and after antimicrobial treatments. Antimicrobial experiments against L. monocytogenes on sprouts, ham and bologna, carvacrol at 0.5% and grape seed extract at 3% were used and carvacrol showed better activity. Antimicrobial activity against E. coli O157:H7 was tested on romaine lettuce, spinach and ground beef using oregano oil at 0.5% and green tea at 3%. Both compounds were effective showing no recovery of E. coli O157:H7 from lettuce and spinach; however, was not reduced in ground beef. Antimicrobial plant compounds have the potential for reducing foodborne pathogenic bacteria on/in various foods.

E. coli O157:H7 has been found to survive in dry sausages and cause disease. Isothiocyanates have been studied for their capacity to eliminate pathogens from foods and are attractive from the consumer perspective because of their natural origin. There is a need to better understand how isothiocyanates kill microorganisms and their behaviour in food matrices. It was found that glutathione and cysteine naturally present in meat can react with AIT, forming a conjugate with no or low bactericidal activity against an E. coli O157:H7. In addition, AIT presented higher anti-E. coli activity at lower pH values; therefore, it should be more efficient in acid foods. AIT was also found to inhibit the activity of thioredoxin reductase and acetate kinase; hence, enzymatic inhibition may represent a way in which AIT kills E. coli O157:H7. Mustard powder is used as a spice (active myrosinase) and/or binder (inactive myrosinase) in meat products. Both of these powders killed E. coli O157:H7 in dry fermented sausage. This was not expected since the powder lacking myrosinase is not able to produce isothiocyanates. Starter cultures and E. coli were found to consume significant amounts of glucosinolates. Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M (higher myrosinase-like activity) were compared against P. pentosaceus UM 116P + S. carnosus UM 109M for their ability in reducing E. coli viability in dry sausage. Sausage batches containing powders of hot mustard, cold mustard, autoclaved mustard and no powder were prepared. Both pairs of starters yielded similar results. Reduction >5 log CFU/g of E. coli O157:H7 occurred after 31 d for hot powder and 38 d for cold powder; there was no reduction in the control. E. coli O157:H7 itself has greater effect on glucosinolate degradation than either pair of starters, which may be more important in determining its survival. Autoclaved powder caused >5 log CFU/g reduction after 18 d. This may be the result of synergistic/additive interaction among E. coli O157:H7 myrosinase-like activity, the presence of newly formed/released antimicrobials in the autoclaved powder and the multiple hurdles present in the dry sausage. Autoclaved mustard powder has potential as a novel food ingredient for the meat industry.

E. coli O157:H7 has been found to survive in dry sausages and cause disease. Isothiocyanates have been studied for their capacity to eliminate pathogens from foods and are attractive from the consumer perspective because of their natural origin. There is a need to better understand how isothiocyanates kill microorganisms and their behaviour in food matrices. It was found that glutathione and cysteine naturally present in meat can react with AIT, forming a conjugate with no or low bactericidal activity against an E. coli O157:H7. In addition, AIT presented higher anti-E. coli activity at lower pH values; therefore, it should be more efficient in acid foods. AIT was also found to inhibit the activity of thioredoxin reductase and acetate kinase; hence, enzymatic inhibition may represent a way in which AIT kills E. coli O157:H7. Mustard powder is used as a spice (active myrosinase) and/or binder (inactive myrosinase) in meat products. Both of these powders killed E. coli O157:H7 in dry fermented sausage. This was not expected since the powder lacking myrosinase is not able to produce isothiocyanates. Starter cultures and E. coli were found to consume significant amounts of glucosinolates. Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M (higher myrosinase-like activity) were compared against P. pentosaceus UM 116P + S. carnosus UM 109M for their ability in reducing E. coli viability in dry sausage. Sausage batches containing powders of hot mustard, cold mustard, autoclaved mustard and no powder were prepared. Both pairs of starters yielded similar results. Reduction >5 log CFU/g of E. coli O157:H7 occurred after 31 d for hot powder and 38 d for cold powder; there was no reduction in the control. E. coli O157:H7 itself has greater effect on glucosinolate degradation than either pair of starters, which may be more important in determining its survival. Autoclaved powder caused >5 log CFU/g reduction after 18 d. This may be the result of synergistic/additive interaction among E. coli O157:H7 myrosinase-like activity, the presence of newly formed/released antimicrobials in the autoclaved powder and the multiple hurdles present in the dry sausage. Autoclaved mustard powder has potential as a novel food ingredient for the meat industry.

This study focused on evaluating the antimicrobial activity of chlorine dioxide (ClO2) and different plant-based antimicrobials (carvacrol, thymol, and eugenol as bioactive compounds and muscadine extract- ME and blueberry extract- BBE as plant extracts) against selected foodborne pathogens under in vitro conditions and on produce (spinach and blueberries). In addition, bacterial microbiota associated with blueberries and blueberry farm environments from three different regions: Cundinamarca, Colombia; Mississippi, United States; and Guadalajara, Mexico, was determined. Under in vitro conditions, carvacrol and thymol were more effective (lower MICs and MBCs) than eugenol against Salmonella spp., Escherichia coli O157:H7 (MIC=MBC=0.2 mg/ml), and Listeria monocytogenes (MIC=MBC=0.4 mg/ml). Both plant extracts had the same MIC and MBC for Salmonella spp. while BBE had stronger bactericidal effect on Escherichia coli O157:H7 (MBC=150 mg/ml) and ME on L. monocytogenes (MBC=100 mg/ml). ClO2 had stronger bacteriostatic effect on L. monocytogenes (MIC=1 ppm) than on the Gram-negative bacteria (MIC=3 ppm). For the produce study, 300 mg/ml ME exerted the highest (P ≤ 0.05) E. coli O157:H7 reduction (4.5 log CFU/g at day 1) on spinach, and 3 ppm ClO2 + 300 mg/ml ME had the highest (P ≤ 0.05) L. monocytogenes reduction on both (4.5-5.6 log CFU/g). There was a similar (P>0.05) E. coli O157:H7 reduction on blueberries, regardless of antimicrobial treatment. For the microbiota study, Proteobacteria was the most abundant phylum in blueberries, soil, and water, with the exception of fruits from Mexico. Blueberries grown on the different regions shared two predominant genera: Heliorestis (10.5-47.4%) and Thiomonas (5.0-9.1%). Nonetheless, alpha and beta diversity revealed that blueberry microbiota structures were distinctive. PCoA plots revealed that within regions the microbial composition distribution was different (P ≤0.05) among fruits, soil, and water. Based on the results, ME combined with ClO2, could represent an antimicrobial alternative against foodborne pathogens for the produce industry. Furthermore, the study of the microbiota provided a good understanding on the bacterial community profile in blueberries and the blueberry farming environment across regions.

In recent years, there have been a number of studies looking at inhibition of microorganisms by spices, herbs or their extracts.  Many of these products have been shown to have antimicrobial activity against foodborne pathogens.  The purpose of this research was to evaluate the antimicrobial activity of three essential oil (EO) compounds (thymol, eugenol, and trans-cinnamaldehyde) alone and in combination with three naturally-occurring apple aroma (AA) compounds (hexanal, trans-2-hexenal and 1-hexanol) to identify the minimum inhibitory concentrations necessary to inhibit E. coli O157:H7.  Three commercial apple juice flavoring mixtures (natural apple cinnamon, natural apple spice and natural red apple) were additionally tested alone for antimicrobial activity against E. coli O157:H7. The standard agar dilution method (SAD) and checkerboard assay were used to evaluate the efficacy of the nine compounds, alone and in combination against E. coli O157:H7.  In general, the EO compounds were significantly more effective against E. coli O157:H7 than the AA compounds (P<0.05).  Cinnamaldehye, with an MIC of 0.2 mg/mL, exhibited the highest degree of activity, followed by thymol, eugenol and trans-2-hexenal, which each had individual MIC values of 1.6 mg/mL.  No synergism was found in the combinations of EO compounds with AA compounds. / Master of Science in Life Sciences