An increase in outbreaks caused by zoonotic agents has brought about intensified efforts to address the transmission of infectious organisms in animal settings. In October 2002, the CDC released recommendations for the use of waterless hand sanitizer (WHS) in human healthcare settings. The question arises whether WHS may be as effective in veterinary and animal agricultural settings given some of the dissimilarities in conditions. To address this question, three studies were conducted. The first was a retrospective analysis of a Samonella agona outbreak which occurred in 2001 at the Large Animal Teaching Hospital of the Virginia-Maryland Regional College of Veterinary Medicine (VMRCVM). The second evaluated the pattern of use and efficacy of hand hygiene products in the VMRCVM Large Animal Hospital. The third study assessed the efficacy of WHS among visitors to a children's petting zoo at the 2002 Virginia State Fair. Regarding the Salmonella outbreak, it is thought that a calf from the university owned dairy herd was the index case. A total of 16 equine patients acquired S. agona while hospitalized. The nosocomial disease incidence risk for in-house patients was estimated to be 33% (16/49). The LAH was closed for 7 months for cleaning, disinfection and renovation. The total cost of the outbreak was estimated to be at least $755,000. Waterless hand sanitizer proved useful in the veterinary hospital setting. When measured immediately after use, WHS reduced bacterial loads on the hands of 20 LAH personnel (P < 0.001). Before WHS use, HBC ranged from less than to 20 to 48,800 CFU/ml with a geometric mean of 6,926 CFU/ml. Counts after WHS use ranged from less than 20 to 23,400 with a geometric mean of 1,152 CFU/ml. Differences in before and after ranged from -4,000 to 48,200 CFU/ml with a median of 9,700 CFU/ml. The logarithmic reduction in bacterial load before and after WHS use was 0.78 (79.7%). In the petting zoo study, bacterial counts on the fingers of the children sampled before use of WHS ranged from 40 to 75,200 CFU/ml with a geometric mean of 8,653 CFU/ml. After WHS use, bacterial growth ranged from 19 to 58,400 CFU/ml with a geometric mean of 1,727 CFU/ml. Differences in before and after ranged from -35,600 to 59,400 CFU/ml with a median of 8,190 CFU/ml. The logarithmic reduction in bacterial load before and after WHS use was 0.70 (82.2%; P< 0.001). These data suggest that WHS may be of benefit in veterinary medicine and animal agriculture as a means to reduce nosocomial and zoonotic infections. / Master of Science

The goal of this project is to demonstrate a new hand sanitizer stand. A project template is selected to carry out tasks in six different phases. A preliminary study was carried out aims to gather background information of the thesis topic. The market research was conducted by searching for existing products on the Internet. A set of Design for Environment guidelines have been established aids to develop concepts. The collected information become the basis for subsequent decisions. Three potential concepts are generated. Two concepts are chosen for further development. A number of design techniques such as CAD, FEM analysis and 3D printing are applied in detailed design. Final design solution where all the geometric, functional details of the hand sanitizer stand are developed. Due to the time limitation, costs associated with the design is not estimated. Detail drawings of hand sanitizer stand are provided in the report to describe its dimensions and shape. / Den här rapporten beskriver formgivningsprocessen och konstruktionsarbetet av ett nytt golvstativ för handsprit. En projektmall tillämpas för att skapa en struktur och vägledning i sex olika faser. En förstudie har genomförts för att få förståelse för ämnet. För att samla information gjordes en marknadsundersökning. Design for Environment riktlinjer har skapats för att utveckla koncept. Den valda informationen användes sedan för att ta beslut. Utifrån de tre framställda koncepten valdes två att vidare utveckla. Tekniker såsom CAD-mjukvara, FEM-beräkningar och 3D teknik användas för att lösa problem och sedan uppnå målet. En slutprodukt uppfyllde de definierade produktkraven vad gäller funktionalitet och prestanda. Vi tog inte hänsyn till den ekonomiska kostnaden på grund av tidbegränsningar. Ritningar med måttsättningar finns i rapporten.

Raw vegetables can sometimes be the source of outbreaks of human illness; however the potential for fresh vegetables to serve as a vehicle for antibiotic -resistant bacteria is poorly understood. Antibiotics and antibiotic-resistant bacteria have been shown to persist in manure of animals administered antibiotics, and in compost generated from this manure, where there is the potential for their transfer to produce. The purpose of this study was to determine the survival of antibiotic-resistant bacteria on raw, peeled, carrots after washing with commonly used chemical sanitizers. Multi-drug resistant E. coli O157:H7 and Pseudomonas aeruginosa were inoculated into a compost slurry of composted manure from dairy cattle, with and without prior administration of antibiotics, and used to inoculate carrot surfaces prior to the washing studies. This approach provided defined model antibiotic-resistant pathogens present within a background microbial community simulating potential carry over from manure-derived fertilizer. Carrots (n=3, 25g) were air-dried and stored at 4 °C until washing with tap water, XY-12 (sodium hypochlorite, 50 ppm free chlorine) or Tsunami 100 (peroxyacetic acid/hydrogen peroxide, 40 ppm free paracetic acid), according to manufacturer's directions. A second batch of carrots representing each inoculation x wash condition (n=3) were individually packaged for storage at 2 °C for 1,7, and 14 days, or 10 °C for 7 days and enumerated on those day intervals to recover bacteria from the surfaces of washed carrots. The resulting previously washed and stored carrots were subject to serial dilution and plated onto corresponding agar to enumerate total aerobic bacteria (R2A), aerobic bacteria tolerant or resistant to antibiotics (antibiotic-supplemented R2A), E. coli (Eosin Methylene Blue), and Pseudomonas spp. (Pseudomonas Isolation Agar). In addition, the tetA gene was quantified from the carrot samples as a measure of the effect of sanitizers and storage on an antibiotic resistance gene known to be carried by the inoculated bacteria.Inclusion of sanitizer in the wash water significantly reduced the absolute numbers of inoculated bacteria (E.coli and Pseudomonas) as well as populations of bacteria capable of growth on the R2A media containing cefotaxime (10μg/mL), sulfamethoxazole (100μg/mL), or tetracycline (3μg/mL). Comparable reductions in the inoculated P. aeruginosa resistant to tetracycline (PIA T, 4μg/mL), bacteria resistant to cefotaxime (10μg/mL) and tetracycline (3μg/mL) occurred after washing with XY-12 or Tsunami 100. The sanitizer effectiveness may be bacterial dependent, as evident by larger absolute reductions of the inoculated E. coli (EMB) and bacteria grown on sulfamethoxazole (100μg/mL)-amended plates after washing with Tsunami 100 compared to washing with tap water or XY-12. Re-growth of both the inoculated and native compost-associated bacteria was inhibited by storage at 2 °C, as there were no significant differences in the log CFU/g values on the various media (total aerobic bacteria, bacteria on antibiotic-amended plates, E. coli inoculum, P. aeruginosa inoculum) during the 14-day storage period. However, temperature abuse at 10 °C resulted in significant re-growth of native Pseudomonas, compared to storage at 2 °C. A sanitizer-associated interaction between re-growth and temperature was also observed for bacteria resistant to clindamycin (25μg/mL) and cefotaxime (10μg/mL), with substantial re-growth occurring only on carrots washed with Tsunami 100. There was no significant re-growth of the inoculated E. coli O157:H7 at either temperature. Results indicate that some bacterial populations are reduced by post-harvest washes and that temperature abuse of fresh produce may result in increases in antibiotic-resistant bacterial populations. / Master of Science in Life Sciences

Master of Science / Hospitality Management and Dietetics / Kevin R. Roberts / Many pathogenic microorganisms are spread by contaminated hands and may lead to foodborne illness. The use of hand sanitizers can significantly reduce bacterial contamination and is an efficient and inexpensive method to prevent infections and sickness. Previous researchers have found that the routine use of hand sanitizers allowed the U.S. Army to significantly reduce illness. However, few studies have been conducted within a U.S. Army dining facility, which is considered to be one of the primary sources of foodborne illness within the U.S. Army. Therefore, using the Theory of Planned Behavior, the purpose of this study was to identify the behavioral intention, attitudes, subjective norms, and perceptions of control of using hand sanitizer among military personnel. The study targeted soldiers using a written survey during their lunch hour on the U.S. Army base at Fort Riley, KS. A total of 201 surveys were collected. All data were screened and entered into IBM SPSS for analysis. Results indicated that attitudes, subjective norms, and perceived behavioral control explained 64% of the variance in behavioral intention. Attitude and subjective norms were found to be significant predictors of behavioral intention, with attitude being the strongest predictor. In general, behavioral beliefs were positive among soldiers. Related to normative beliefs, soldiers did perceive negative social pressure from other soldiers not to use hand sanitizers. Analysis of control beliefs found soldiers perceived hand sanitizers were readily available, but disliked their smell and feel after application. Food production managers and Army commanders can use these results to implement hand sanitation behavioral interventions within military dining environments. Practical implications will likely translate to reduced healthcare costs, decreased absenteeism rates, and improved mission readiness. Some of the limitations include commonly perceived social psychology bias. Further, clustered samples were collected within one military installation in a relatively short amount of time.

Food-borne illness outbreaks are occasionally associated with fresh-vegetable consumption, in part due to lack of a microbial inactivation step before consumption. Raw manure or improperly composted manure applied as soil amendments is an established source of pathogenic bacterial contamination. However, less is known about whether such soil amendments could serve as a source of transmission of antibiotic-resistant bacteria (ARB) or antibiotic-resistance genes (ARGs) via fresh produce. As such knowledge is developing, it is useful to identify strategies for mitigating ARGs and ARB on vegetable surfaces, especially those that are synergistic with known benefits in terms of general pathogen reduction on fresh produce. Sanitizers play an important role in post-harvest processing of vegetables, especially in terms of disinfecting the wash water and preventing cross-contamination. Further, temperature and time of storage of vegetables are critical to prevent the growth of microorganisms. To provide a background inoculum representing potential pre-harvest carryover of ARB and ARGs, carrots or romaine lettuce leaves were dipped in a slurry derived from composted manure from dairy cows previously dosed with antibiotics and further inoculated with multi-drug resistant E. coli O157:H7, a human pathogen, and a spoilage-associated and opportunistic pathogenic strain of Pseudomonas aeruginosa. Inoculated carrots (n=3, 25 g) were washed with water containing different sanitizers (sodium hypochlorite or peroxyacetic acid) or unwashed (control), packaged and stored at 10ºC for 7d or 2ºC for up to 60 d. Inoculated lettuce leaves (n=3, 100 g) were washed with sodium hypochlorite, packaged in modified atmosphere conditions (98% nitrogen), irradiated (1.0 kGy) and subsequently stored at 4ºC for 14 d. The effect of post-harvest treatment were compared at various times by enumeration on selective media. In addition, cultureindependent techniques were also performed to determine changes to the surficial carrot and lettuce microbiota by sequencing bacterial 16S rRNA gene amplicons. The effect of post-harvest treatments on the types and relative abundance of ARGs, also known as the “resistome,” were profiled by shotgun metagenomic sequencing and qPCR. Addition of a sanitizer during wash, storage temperature, and duration of storage affected the bacterial community structures on carrots, represented by the weighted Unifrac distance matrices (ANOSIM, R=0.465). Storage of sanitizer-washed carrots at 10ºC was associated with an increase in relative abundance of Pseudomonadaceae compared to 2ºC storage for 7 d (Wilcoxon, p<0.05). Increase in storage temperature from 2ºC (optimum) to 10ºC (temperature abuse) of sanitizer-washed carrots resulted in enrichment of ARGs conferring resistance to the following antibiotic classes: multidrug, peptide, polymyxin, quinolone, triclosan, aminoglycoside, bacitracin, β-lactam, and fosfomycin. Irradiation resulted in significant reductions (~3.5 log CFU/g) of inoculated antibiotic-resistant E. coli O157:H7 and Pseudomonas sp. on lettuce surfaces (ANOVA, p<0.05). The lettuce resistome, represented by the Bray-Curtis similarity of ARG occurrence, was affected by irradiation (ANOSIM, R=0.406). Irradiation of lettuce followed by 14 d of storage at 4ºC resulted in 2-4-fold reductions in relative abundance of ARGs encoding resistance to the following antibiotic classes: triclosan, quinolones, multidrug, polymyxin and β-lactam (Wilcoxon, p<0.05). No additional increase or reduction of the tet(A) gene present on inoculated P. aeruginosa was evident after 14d storage at 4ºC on irradiated samples. Results of this study suggest that inclusion of a sanitizer in wash water, irradiation, and storage at optimum refrigerated temperatures may offer effective strategies to combat proliferation of antibiotic resistant bacteria and antibiotic resistance genes on fresh produce. Further research is needed develop interventions that can mitigate tet(A) and other ARGs on produce that were not significantly reduced by irradiation. This study will guide future research on microbiome and metagenome of processed produce and assessment of critical control points to reduce the risk of antibiotic resistance from farm-to-fork. / PHD / Post-harvest interventions; such as washing, irradiation and cold storage, are employed to provide safe and wholesome fresh vegetables to consumers. Washing of vegetables in water that includes a sanitizing agent, such as chlorine or peroxyacetic acid (POAA), removes soil from the surface, reduces the bacteria in wash water and prevent cross-contamination between vegetables. It has an additional benefit to reduce microorganisms on produce surfaces that may cause the vegetables to spoil or result in illness in humans. Low temperature storage of produce, usually 0-5ºC, decreases the respiration rate of vegetables and reduces growth of microorganisms during storage. Some of the spoilage and/or pathogenic bacteria may also be antibiotic-resistant, which are commonly termed as antibiotic-resistant bacteria (ARB). Antibiotic resistance is a significant public health concern that leads to ineffective medical treatments, prolonged duration of illnesses and increased hospitalization costs. Antibiotic resistance is encoded by genes that confer resistance to wide range of antibiotic classes, including antibiotics used to treat human illnesses. These genes are termed as antibiotic resistance genes (ARGs). In this study we examined the effect of three common post-harvest interventions, washing with sanitizers, gamma irradiation, and cold storage to reduce antibiotic-resistant bacterial pathogens and antibiotic-resistant spoilage bacteria on carrots and lettuce. Storage temperature, inclusion of sanitizer in wash water, and length of chilled storage significantly influenced the diversity of bacteria found on carrot surface. Inclusion of either sanitizer in the wash water significantly reduced the populations of antibiotic-resistant E. coli O157:H7 (a pathogenic bacterium that causes a dangerous form of gastrointestinal illness) and Pseudomonas sp. (a bacterial species that commonly causes food spoilage). Storage at recommended temperature (2ºC) did not allow these bacteria to regrow and also reduced total ARGs on carrot surfaces. Washing of lettuce with sodium hypochlorite followed by irradiation (1.0 kGy) and storage at recommended temperature (4ºC) were effective in reducing the populations of antibiotic-resistant E. coli O157:H7 and Pseudomonas sp., and additionally reduced the number of some ARGs conferring resistance to select classes of antibiotics, including triclosan, quinolones, multidrug, polymyxin and β-lactam antibiotics on the lettuce surface. A novelty of this research is that it employed new, cutting-edge “metagenomic” DNA sequencing technique to identify and track antibiotic resistance through the various post-harvest interventions. Overall results of this research suggest that inclusion of sanitizer in wash water for fresh produce, followed by storage at refrigerated temperatures below 4ºC may reduce the risk posed by antibiotic resistant bacteria and antibiotic resistance genes on produce.

Surtos e casos de listeriose reportados mundialmente e associados a produtos cárneos processados prontos para consumo podem ter sido causados pela contaminação cruzada com Listeria monocytogenes ocorrida durante a etapa de fatiamento destes produtos no varejo. Considerando o impacto da contaminação cruzada para a saúde pública, este trabalho teve por objetivo estudar a transferência de L. monocytogenes durante a etapa de fatiamento de rosbife do tipo \"caseiro\", simulando, em laboratório, cenários observados em estabelecimentos comerciais em relação às práticas adotadas durante o fatiamento. Objetivou-se também avaliar o papel do nível da contaminação do produto (baixo e alto) causador da contaminação experimental do fatiador na contaminação cruzada resultante, bem como avaliar se a exposição da cepa de L. monocytogenes a um sanitizante em concentração insuficiente para a sua eliminação influencia a contaminação cruzada observada. A contaminação do fatiador foi obtida por meio do fatiamento de peças de rosbife experimentalmente contaminadas com o patógeno por imersão em uma suspensão de L. monocytogenes contendo 8 log UFC/mL (alto nível de contaminação) e 4 log UFC/mL (baixo nível de contaminação). Os experimentos foram realizados até a obtenção de 200 fatias. As enumerações de L. monocytogenes nas fatias obtidas foram feitas empregando-se um método cultura-dependente (ISO 11290-2:1998) e um método qPCR, calculando-se também as taxas de transferência. Os resultados mostraram que a contaminação dos fatiadores resultou na transferência do patógeno até pelo menos a 120&#170; fatia de uma nova peça de rosbife fatiada posteriormente. Nos experimentos realizados com L. monocytogenes exposta ao sanitizante Oasis Compac 22 Quat em concentração insuficiente para sua eliminação, foi possível enumerar o patógeno até a 200ª fatia de rosbife obtida após a contaminação experimental do fatiador, independentemente do nível de contaminação da peça de rosbife usada para a contaminação do fatiador. Equações matemáticas resultantes, que descrevem os dados experimentais obtidos, apresentaram R2>0,7 e p<0,05, mostrando bom ajuste. Esses resultados ressaltam a importância de medidas para evitar a ocorrência de contaminação cruzada durante a etapa de fatiamento de produtos cárneos prontos para o consumo, bem como da higienização adequada dos equipamentos utilizados, de forma a fornecer produtos seguros para o consumidor. / Outbreaks and cases of listeriosis reported worldwide and associated to ready-to-eat meat products may have been caused by cross contamination with Listeria monocytogenes occurred during the slicing step of these products at retail. Considering the impact of cross-contamination to public health, this study aimed to study the transfer of L. monocytogenes during the slicing step of homemade type roast-beef simulating in the laboratory scenarios seen in commercial establishments. The study also aimed to evaluate the role of product contamination level (low and high) causing the experimental contamination of the slicer in the resulting cross-contamination and to evaluate if the exposure of the L. monocytogenes strain to a sanitizer in insufficient concentration for the elimination influences the observed cross-contamination. Contamination of the slicer was obtained through the slicing of roast-beef pieces experimentally contaminated with the pathogen by immersion in a suspension of L. monocytogenes containing 8 log CFU/ml (high contamination) and 4 log CFU/mL (low contamination). The experiments were carried out to obtain 200 slices. Enumerations of L. monocytogenes in the slices employed a culture-dependent method (ISO 11290-2: 1998) and qPCR method, also calculating transfer rates. The results showed that contamination of slicers resulted in the transfer of the pathogen to at least the 120th slice of a new piece of roast-beef sliced subsequently. In experiments conducted with L. monocytogenes exposed to the sanitizer Oasis Compac 22 Quat in insufficient concentration for its elimination, the pathogen could be enumerated until the 200th slice obtained after the slicer contamination, regardless of the contamination level of the roast beef used for contamination of the slicer. Mathematical equations describing the experimental data presented R2>0.7 and p<0.05, showing good fit. These results underscore the importance of measures to prevent the occurrence of cross contamination during the slicing step of ready-to-eat meat products, as well as the proper cleaning of the equipment used in order to provide safe products to the consumer.

Surtos e casos de listeriose reportados mundialmente e associados a produtos cárneos processados prontos para consumo podem ter sido causados pela contaminação cruzada com Listeria monocytogenes ocorrida durante a etapa de fatiamento destes produtos no varejo. Considerando o impacto da contaminação cruzada para a saúde pública, este trabalho teve por objetivo estudar a transferência de L. monocytogenes durante a etapa de fatiamento de rosbife do tipo \"caseiro\", simulando, em laboratório, cenários observados em estabelecimentos comerciais em relação às práticas adotadas durante o fatiamento. Objetivou-se também avaliar o papel do nível da contaminação do produto (baixo e alto) causador da contaminação experimental do fatiador na contaminação cruzada resultante, bem como avaliar se a exposição da cepa de L. monocytogenes a um sanitizante em concentração insuficiente para a sua eliminação influencia a contaminação cruzada observada. A contaminação do fatiador foi obtida por meio do fatiamento de peças de rosbife experimentalmente contaminadas com o patógeno por imersão em uma suspensão de L. monocytogenes contendo 8 log UFC/mL (alto nível de contaminação) e 4 log UFC/mL (baixo nível de contaminação). Os experimentos foram realizados até a obtenção de 200 fatias. As enumerações de L. monocytogenes nas fatias obtidas foram feitas empregando-se um método cultura-dependente (ISO 11290-2:1998) e um método qPCR, calculando-se também as taxas de transferência. Os resultados mostraram que a contaminação dos fatiadores resultou na transferência do patógeno até pelo menos a 120&#170; fatia de uma nova peça de rosbife fatiada posteriormente. Nos experimentos realizados com L. monocytogenes exposta ao sanitizante Oasis Compac 22 Quat em concentração insuficiente para sua eliminação, foi possível enumerar o patógeno até a 200ª fatia de rosbife obtida após a contaminação experimental do fatiador, independentemente do nível de contaminação da peça de rosbife usada para a contaminação do fatiador. Equações matemáticas resultantes, que descrevem os dados experimentais obtidos, apresentaram R2>0,7 e p<0,05, mostrando bom ajuste. Esses resultados ressaltam a importância de medidas para evitar a ocorrência de contaminação cruzada durante a etapa de fatiamento de produtos cárneos prontos para o consumo, bem como da higienização adequada dos equipamentos utilizados, de forma a fornecer produtos seguros para o consumidor. / Outbreaks and cases of listeriosis reported worldwide and associated to ready-to-eat meat products may have been caused by cross contamination with Listeria monocytogenes occurred during the slicing step of these products at retail. Considering the impact of cross-contamination to public health, this study aimed to study the transfer of L. monocytogenes during the slicing step of homemade type roast-beef simulating in the laboratory scenarios seen in commercial establishments. The study also aimed to evaluate the role of product contamination level (low and high) causing the experimental contamination of the slicer in the resulting cross-contamination and to evaluate if the exposure of the L. monocytogenes strain to a sanitizer in insufficient concentration for the elimination influences the observed cross-contamination. Contamination of the slicer was obtained through the slicing of roast-beef pieces experimentally contaminated with the pathogen by immersion in a suspension of L. monocytogenes containing 8 log CFU/ml (high contamination) and 4 log CFU/mL (low contamination). The experiments were carried out to obtain 200 slices. Enumerations of L. monocytogenes in the slices employed a culture-dependent method (ISO 11290-2: 1998) and qPCR method, also calculating transfer rates. The results showed that contamination of slicers resulted in the transfer of the pathogen to at least the 120th slice of a new piece of roast-beef sliced subsequently. In experiments conducted with L. monocytogenes exposed to the sanitizer Oasis Compac 22 Quat in insufficient concentration for its elimination, the pathogen could be enumerated until the 200th slice obtained after the slicer contamination, regardless of the contamination level of the roast beef used for contamination of the slicer. Mathematical equations describing the experimental data presented R2>0.7 and p<0.05, showing good fit. These results underscore the importance of measures to prevent the occurrence of cross contamination during the slicing step of ready-to-eat meat products, as well as the proper cleaning of the equipment used in order to provide safe products to the consumer.