Master of Science / Department of Diagnostic Medicine/Pathobiology / Raghavendra Amachawadi / T.G. Nagaraja / Copper is an essential micronutrient that is supplemented in swine diets as a growth promoter. Previous studies suggest a link between copper supplementation and co-selection of antimicrobial resistance (AMR) in Enterococcus, but the data are inconsistent. Therefore, the objective of this study was to assess the impact of copper supplementation, alone or with chlortetracycline (CTC), on prevalence and concentration of copper resistance gene, tcrB, prevalence of tetracycline [tet(M)] and macrolide resistance [erm(B)] genes, and AMR in fecal enterococci of weaned piglets. A total of 320 weaned piglets at 21 days of age were allocated into 64 individual pens distributed equally among two barns. Pen-pair was the experimental unit (n=32). Four treatments were used: a basal diet as the control, a basal diet with 200 ppm of copper as copper sulfate, a basal diet with chlortetracycline (CTC) at 400 g/ton of feed, and a basal diet with 200 ppm of copper and CTC at 400 g/ton of feed. The study period was 35 days with days -7 to -1 as an acclimation period and days 0 to 28 as the treatment period. Direct fecal samples were collected on days 0, 14, and 28. Prevalence of tcrB-positive enterococci was not affected by copper and or CTC supplementation (P > 0.05). Prevalence of tcrB-positive enterococci was higher on day 14 than other sampling days (P = 0.002). Prevalence of tet(M)-positive enterococci was not affected by treatment group or sampling day (P > 0.05). Prevalence of erm(B)-positive enterococci had a significant treatment and sampling day interaction (P = 0.0213). The total copy number of the tcrB gene was quantified as a percent of total bacterial cells in the feces. The median copper MIC of tcrB-negative and -positive isolates was 3 mM and 20 mM, respectively (P < 0.0001). For day 0 (n=64) and day 28 (n=63), all Enterococcus isolates were susceptible to gentamicin, kanamycin, streptomycin, daptomyicin, and tigecycline, with majority of isolates resistant to chloramphenicol, erythromycin, lincomycin, linezolid, tetracycline, tylosin tartrate, and synercid. For day 0 (n=64) and day 28 (n=63), respectively, a total of 61 (95.3%) and 47 (74.6%) isolates were resistant to erythromycin, 51 (79.7%) and 41 (65.1%) to tylosin, and 60 (93.8% and 95.2%) to tetracycline. The results of this study show that supplementing copper, with or without a selection pressure of chlortetracycline, did not increase copper-resistant enterococci and did not co-select resistance to any other antibiotics. Liver abscesses in feedlot cattle have a significant economic impact because of reduction in cattle performance, and carcass yield and liver condemnation at harvest. Fusobacterium necrophorum is the primary causative agent of the liver abscesses. Recently, Salmonella enterica has been isolated from liver abscesses of cattle. Our objectives of this study were to determine prevalence of Salmonella, compare conventional (serological) and commercially available Check &Trace serotyping methods, and to describe the antimicrobial susceptibility patterns of Salmonella isolated from liver abscesses of feedlot cattle. In the 2014 study, the number of liver abscesses positive for Salmonella were higher (P < 0.05) in cattle fed no tylosin in the diet (66/200; 33%) compared to tylosin-fed cattle (31/183; 16.9%). In the 2015 study, Salmonella prevalence tended to be higher in liver abscesses categorized as severe (29/106; 27.4%) compared to mild liver abscesses (38/174; 21.8%), but the difference was not significant. Out of the 164 Salmonella isolated, 152 (92.7%) were used for serotyping and 164 strains were used for antimicrobial susceptibility testing. Serotyping was done by serological method, which is considered as the gold standard, and the commercial Check & Trace method, which is a molecular method based on differences in their DNA sequence. A total of 11 serotypes were identified with Lubbock (66/152; 43.4%) being the predominant serotype, followed by Agona (24/152; 15.8%), Anatum (20/152; 13.2%), and Montevideo (18/152; 11.8%). The commercial identified only a few serotypes correctly suggesting that the method requires further validation. Antimicrobial susceptibility testing was done by microbroth dilution method according to Clinical Laboratory Standards Institute guidelines. A majority of the Salmonella strains were pansusceptible to the antimicrobials included in the panel. Overall, 10 strains (10/164; 6.1%) were resistant to one or more antibiotics and belonged to serotypes Agona, Anatum, Cerro, Lubbock, Mbandaka, and Reading. The top three of nine resistant antibiotics were chloramphenicol (5/10; 50%), streptomycin (5/10; 50%), and tetracycline (6/10; 60%). Whether Salmonella contributes to liver abscess formation or just happen to survive in an abscess initiated by the primary etiologic agent, Fusobacterium necrophorum, remains to be determined.

Enterococos são bactérias que exercem um papel muito importante na produção de vários alimentos fermentados e também podem ser usadas como probióticos. A presença e o crescimento de enterococcos em alimentos fermentados como queijos e lingüiças conferem a esses produtos características organolépticas únicas. Em contrapartida, sua presença nos alimentos também está associada com falta de higiene durante a manipulação. Estes microrganismos também estão relacionados com o desenvolvimento de algumas doenças, como endocardites, septicemia, infecções do trato geniturinário, entre outras. A presença de características de virulência aumenta o potencial de infecção do microrganismo e a severidade da doença a ele relacionada. Com o objetivo de avaliar possíveis modificações fenotípicas e genotípicas de amostras de enterococos isoladas de frango, durante a subcultura destas cepas no laboratório, várias análises foram realizadas como: a presença dos fatores de virulência; proteína de superfície (esp) e gelatinase (gelE), do operon fsr-regulador do gelE, a expressão fenotípica do gelE, a capacidade de formação de biofilme e a resistência a antimicrobianos, desinfetantes e antisépticos. Quarenta isolados de Enterococcus sp. foram avaliados quanto a presença dos genes gelE, esp, operon-fsr, sprE por PCR, a atividade gelatinolítica por testes bioquímicos convencionais, resistência a antimicrobianos, antisépticos e desinfetantes por antibiograma e formação de biofilme pelo método cristal violeta. Todos os testes foram realizados na 1º geração e na 12º geração das cepas. 85% dos isolados produziram gelatinase e em 92,5% dos isolados o gene gelE estava presente na 1º geração. A análise do fsr-operon destes isolados do primeiro cultivo demonstrou que o gene fsrA estava presente em 35 isolados e o fsrC em 37 isolados e a presença destes genes pareceu não ter correlação com a atividade gelationolítica. O gene fsrB estava presente em todos os isolados (35) que apresentaram atividade gelatinolítica sugerindo que a presença deste gene é importante na expressão desta enzima. Após o subcultivo, apenas um isolado perdeu a atividade gelatinolítica e 15 perderam o gene gelE. Doze isolados perderam pelo menos um gene do fsr-operon durante a subcultura, porém nenhum destes perdeu a capacidade de expressar a enzima gelatinase talvez devido à presença do gene fsrB. O gene sprE foi detectado em 34 isolados na primeira geração e na 12º geração em apenas 20 isolados. O gene da proteína de superfície de Enterococcos (Esp), não foi encontrado em nenhum dos isolados. O antibiograma do isolados no primeiro cultivo demonstrou que 100% dos isolados foram sensíveis a ampicilina e a gentamicina, 95% sensíveis a vancomicina, 85% a ciprofloxacina, 5% a tetraciclina, 65% a eritromicina e 52,5% a cloranfenicol tanto na 1º quanto na 12º geração. Após a subcultura a susceptibilidade dos isolados aumentou a eritromicina (67,5%) e ao cloranfenicol (80%). Quanto ao perfil de resistência aos detergentes e anti-sépticos de uso comercial, todos os isolados apresentaram fenótipo de resistentes ao linear alquilbenzeno sulfonato (LAS) e ao triclosan durante a subcultura. Todos isolados foram suscetíveis ao formaldeído, mas se tornaram resistentes ao 8,5% hipoclorito de sódio e a clorexidina durante a subcultura. Em geral, todos os isolados foram formadores de biofilme e a produção de gelatinase parece ser necessária para esta formação. O perfil genético não pareceu ter relação com a formação de biofilme. Tanto o perfil genotípico quanto o fenotípico pode sofrer alterações durante a subcultura das cepas no laboratório. / Enterococci are bacteria that have a very important role in the production of various fermented foods and can also be used as probiotics. The presence and growth of enterococci in fermented foods like cheese and sausages bring to these products unique organoleptic characteristics. However, their presence in foods is also associated with lack of hygiene during handling. These microorganisms are also related to the development of some diseases such as endocarditis, septicemia, genitourinary infections, among others. The presence of virulence characteristics increases the potential infection of the organism and severity of disease related to it. The aim of the present study is analyze the possible changes of phenotypic and genotypic of enterococci isolated from chicken, during the subculture of the strains in the laboratory, the presence of virulence factors: enterococcal surface protein (esp) and gelatinase (gelE), operon-fsr gelE regulator, gelE phenotypic expression, the ability of biofilm formation and antibiotic, disinfectant and antiseptic resistance were determined in samples of enterococci isolated from chicken. The presence of gelE, esp operon-fsr and sprE genes were evaluated by PCR, gelatinase activity were observed by conventional biochemical tests, antibiotics resistance, antiseptics and disinfectants resistance were analyzed by standard disk diffusion method and biofilm formation were detected following the crystal violet staining method in forty enterococci isolates from chicken. All tests were performed in the 1st generation and 12th generation. 85% of the isolates produced gelatinase and in 92.5% of the isolated the gelE gene was present in the 1st generation. The analysis of operon-fsr in the 1st generation of these isolates showed that the fsrA gene was present in 35 isolates and fsrC gene was present in 37 isolates and the presence of these genes seemed to have no correlation with the gelatinase activity. The fsrB gene was present in all isolates (35) with gelatinase activity suggesting that the presence of this gene is important in the expression of this enzyme. After subculture, only one isolate lost the gelatinase activity and 15 isolates lost the gelE gene. Twelve isolates lost at least one gene of the operon-fsr during laboratory subculture, but none of these isolates lost the ability to express the enzyme gelatinase probably due the presence of the fsrB gene. The sprE gene was detected in 34 isolates in the 1st generation and in 12th generation only 20 isolates maintained this gene. The protein surface of enterococci gene (Esp), was not found in any isolate. The antibiogram of the isolates showed that 100% of the isolates were susceptible to ampicillin and gentamicin, 95% susceptible to vancomycin, 85% to ciprofloxacin, tetracycline 5%, 65% to erythromycin and 52.5% to chloramphenicol in the 1st generation. After subculture the susceptibility of isolates to erythromycin (67.5%) and chloramphenicol (80%) increased. As the profile of resistance to detergents and antiseptics for commercial use, all isolates showed resistance phenotype of the linear alkylbenzene sulfonate (LAS) and triclosan during subculture. All isolates were susceptible to formaldehyde, but became resistant to 8.5% sodium hypochlorite and chlorhexidine during the subculture. In general, all isolates were biofilm formers. Gelatinase production appears to be required for biofilme formation. The genetic profile did not appear to have relation with the formation of biofilms. Genotypic and the phenotypic profile may change during the subculture of the strains in the laboratory.

Enterococos são bactérias que exercem um papel muito importante na produção de vários alimentos fermentados e também podem ser usadas como probióticos. A presença e o crescimento de enterococcos em alimentos fermentados como queijos e lingüiças conferem a esses produtos características organolépticas únicas. Em contrapartida, sua presença nos alimentos também está associada com falta de higiene durante a manipulação. Estes microrganismos também estão relacionados com o desenvolvimento de algumas doenças, como endocardites, septicemia, infecções do trato geniturinário, entre outras. A presença de características de virulência aumenta o potencial de infecção do microrganismo e a severidade da doença a ele relacionada. Com o objetivo de avaliar possíveis modificações fenotípicas e genotípicas de amostras de enterococos isoladas de frango, durante a subcultura destas cepas no laboratório, várias análises foram realizadas como: a presença dos fatores de virulência; proteína de superfície (esp) e gelatinase (gelE), do operon fsr-regulador do gelE, a expressão fenotípica do gelE, a capacidade de formação de biofilme e a resistência a antimicrobianos, desinfetantes e antisépticos. Quarenta isolados de Enterococcus sp. foram avaliados quanto a presença dos genes gelE, esp, operon-fsr, sprE por PCR, a atividade gelatinolítica por testes bioquímicos convencionais, resistência a antimicrobianos, antisépticos e desinfetantes por antibiograma e formação de biofilme pelo método cristal violeta. Todos os testes foram realizados na 1º geração e na 12º geração das cepas. 85% dos isolados produziram gelatinase e em 92,5% dos isolados o gene gelE estava presente na 1º geração. A análise do fsr-operon destes isolados do primeiro cultivo demonstrou que o gene fsrA estava presente em 35 isolados e o fsrC em 37 isolados e a presença destes genes pareceu não ter correlação com a atividade gelationolítica. O gene fsrB estava presente em todos os isolados (35) que apresentaram atividade gelatinolítica sugerindo que a presença deste gene é importante na expressão desta enzima. Após o subcultivo, apenas um isolado perdeu a atividade gelatinolítica e 15 perderam o gene gelE. Doze isolados perderam pelo menos um gene do fsr-operon durante a subcultura, porém nenhum destes perdeu a capacidade de expressar a enzima gelatinase talvez devido à presença do gene fsrB. O gene sprE foi detectado em 34 isolados na primeira geração e na 12º geração em apenas 20 isolados. O gene da proteína de superfície de Enterococcos (Esp), não foi encontrado em nenhum dos isolados. O antibiograma do isolados no primeiro cultivo demonstrou que 100% dos isolados foram sensíveis a ampicilina e a gentamicina, 95% sensíveis a vancomicina, 85% a ciprofloxacina, 5% a tetraciclina, 65% a eritromicina e 52,5% a cloranfenicol tanto na 1º quanto na 12º geração. Após a subcultura a susceptibilidade dos isolados aumentou a eritromicina (67,5%) e ao cloranfenicol (80%). Quanto ao perfil de resistência aos detergentes e anti-sépticos de uso comercial, todos os isolados apresentaram fenótipo de resistentes ao linear alquilbenzeno sulfonato (LAS) e ao triclosan durante a subcultura. Todos isolados foram suscetíveis ao formaldeído, mas se tornaram resistentes ao 8,5% hipoclorito de sódio e a clorexidina durante a subcultura. Em geral, todos os isolados foram formadores de biofilme e a produção de gelatinase parece ser necessária para esta formação. O perfil genético não pareceu ter relação com a formação de biofilme. Tanto o perfil genotípico quanto o fenotípico pode sofrer alterações durante a subcultura das cepas no laboratório. / Enterococci are bacteria that have a very important role in the production of various fermented foods and can also be used as probiotics. The presence and growth of enterococci in fermented foods like cheese and sausages bring to these products unique organoleptic characteristics. However, their presence in foods is also associated with lack of hygiene during handling. These microorganisms are also related to the development of some diseases such as endocarditis, septicemia, genitourinary infections, among others. The presence of virulence characteristics increases the potential infection of the organism and severity of disease related to it. The aim of the present study is analyze the possible changes of phenotypic and genotypic of enterococci isolated from chicken, during the subculture of the strains in the laboratory, the presence of virulence factors: enterococcal surface protein (esp) and gelatinase (gelE), operon-fsr gelE regulator, gelE phenotypic expression, the ability of biofilm formation and antibiotic, disinfectant and antiseptic resistance were determined in samples of enterococci isolated from chicken. The presence of gelE, esp operon-fsr and sprE genes were evaluated by PCR, gelatinase activity were observed by conventional biochemical tests, antibiotics resistance, antiseptics and disinfectants resistance were analyzed by standard disk diffusion method and biofilm formation were detected following the crystal violet staining method in forty enterococci isolates from chicken. All tests were performed in the 1st generation and 12th generation. 85% of the isolates produced gelatinase and in 92.5% of the isolated the gelE gene was present in the 1st generation. The analysis of operon-fsr in the 1st generation of these isolates showed that the fsrA gene was present in 35 isolates and fsrC gene was present in 37 isolates and the presence of these genes seemed to have no correlation with the gelatinase activity. The fsrB gene was present in all isolates (35) with gelatinase activity suggesting that the presence of this gene is important in the expression of this enzyme. After subculture, only one isolate lost the gelatinase activity and 15 isolates lost the gelE gene. Twelve isolates lost at least one gene of the operon-fsr during laboratory subculture, but none of these isolates lost the ability to express the enzyme gelatinase probably due the presence of the fsrB gene. The sprE gene was detected in 34 isolates in the 1st generation and in 12th generation only 20 isolates maintained this gene. The protein surface of enterococci gene (Esp), was not found in any isolate. The antibiogram of the isolates showed that 100% of the isolates were susceptible to ampicillin and gentamicin, 95% susceptible to vancomycin, 85% to ciprofloxacin, tetracycline 5%, 65% to erythromycin and 52.5% to chloramphenicol in the 1st generation. After subculture the susceptibility of isolates to erythromycin (67.5%) and chloramphenicol (80%) increased. As the profile of resistance to detergents and antiseptics for commercial use, all isolates showed resistance phenotype of the linear alkylbenzene sulfonate (LAS) and triclosan during subculture. All isolates were susceptible to formaldehyde, but became resistant to 8.5% sodium hypochlorite and chlorhexidine during the subculture. In general, all isolates were biofilm formers. Gelatinase production appears to be required for biofilme formation. The genetic profile did not appear to have relation with the formation of biofilms. Genotypic and the phenotypic profile may change during the subculture of the strains in the laboratory.

Master of Science / Department of Animal Sciences and Industry / Joel M. DeRouchey / Jason C. Woodworth / Two experiments using a total of 720 nursery pigs were used to determine the effects of Elarom SES, in-feed antibiotics, zinc, or copper on nursery pig growth performance and fecal consistency. Two experiments using a total of 1,534 nursery pigs were used to determine the effects of formaldehyde inclusion, lysine level, and synthetic amino acid inclusion on nursery pig performance, amino acid utilization, and gut microbial community. One experiment using a total of 300 nursery pigs were used to determine the effects of chlortetracycline (CTC) or a probiotic inclusion on nursery pig growth performance and antimicrobial susceptibility. Experiment 1 determined the effect of Elarom SES, in-feed antibiotics, or zinc on nursery pig performance and fecal consistency. The addition of Elarom SES or ZnO alone reduced ADG, but G:F was poorest when all three additives were fed in combination. Addition of in-feed antibiotics increased ADG and G:F throughout the study. Experiment 2 determined the effects of Elarom SES or copper inclusion on nursery pig performance and fecal consistency. The addition of Elarom SES or increasing copper did not provide consistent benefits in performance. In both experiments, there were no individual or overall treatment effects or treatment × day interactions observed for fecal consistency. Experiment 3 compared the effects of formaldehyde source and lysine level on nursery pig growth performance. Regardless of source or lys level, the inclusion of formaldehyde in nursery pig diets marginally reduced ADG and resulted in poorer G:F. Experiment 4 compared the effects of formaldehyde and synthetic amino acid inclusion level on nursery pig growth performance, amino acid utilization, and gut microbial community. The inclusion of Sal CURB in diets reduced ADG and ending BW while inclusion decreased ADFI. ADFI response was dependent on synthetic amino acid level in the diet. Sal CURB inclusion in diets reduced total and available lysine, but reduced bacterial microflora in treatment feed. Experiment 5 determined the effects of CTC or a probiotic on nursery pig performance and antimicrobial susceptibility. The addition of CTC to diets improved ADG, ADFI, and ending BW. The addition of Poultry Star improved ADFI and d 14 BW, but benefits did not carry throughout the study.

Staphylococcus aureus (S. aureus) is a bacterium that commonly colonises the skin and nares of around one third of otherwise healthy individuals. While colonisation is benign, S. aureus can cross skin and mucosal barriers to cause infections that manifest as clinical disease. Clinical outcomes are diverse and range from mild, non-complicated and often self-limiting skin and soft tissue infections (including boils, abscesses and cellulitis) to more severe and life-threatening conditions including pneumonia, toxic shock syndrome and bacteraemia. Medication isn’t always needed for mild S. aureus infections as often they resolve with time but, for severe or persistent cases, antimicrobial treatment is generally required. Following decades of widespread and intensive usage of topical, enteral and parenteral antimicrobials to treat S. aureus infections; AMR has become an established and ubiquitous problem in the treatment of infections caused by this microorganism, especially when in the methicillin resistant form (i.e. MRSA). The aim of this thesis was to examine aspects of S. aureus epidemiology (including MRSA and methicillin-sensitive S. aureus (MSSA)) in Scotland using statistical methods and data from several large public health databases. More specifically this involved: descriptions of spatial and temporal trends of morbidity and mortality; comparisons of epidemiological and molecular attributes (including antimicrobial resistance) of (1) MSSA and MRSA, and (2) the dominant clones of MRSA (i.e. EMRSA-15 and EMRSA-16); descriptions of spatial and temporal trends of antimicrobial prescribing in primary and secondary care and any associations between prescribing rates and MRSA antimicrobial resistance; and carrying out a hospital-level risk factor analysis of MRSA, testing hypotheses that hospital size, hospital connectivity (through shared transfer patients) and hospital category have an effect on hospital-level incidences of MRSA in mainland Scottish hospitals. Results showed that total S .aureus bacteraemia and MRSA bacteraemia in Scotland statistically declined over time (p < 0.0001), but MSSA bacteraemia did not (p > 0.05). While combined mortality rates (i.e. all MSSA deaths (both primary and secondary cause), or all MRSA deaths (both primary and secondary cause)) mirror these findings; case-fatality ratios (CFR) show no declines over time for either MRSA or MSSA. Results also show that several epidemiological factors point towards a predominant community source for MSSA isolates (i.e. outside healthcare) and hospital source for MRSA. Evidence for this included: (1) the lack of resistance genes in the MSSA population, (2) MRSA was more associated with long-term care and high-risk patients in the specialties care of the elderly, high dependency units/intensive care units (HDU/ICU), and surgery and conversely MSSA with specialties that commonly served outpatients, and (3) the abundance of non-EMRSA-15/non- EMRSA-16 ‘other’ clones in the MSSA population as compared with the hospital-associated CC22 (EMRSA-15) and CC30 (EMRSA-16) clones. EMRSA-15 was by far the most dominant MRSA clone in Scotland with EMRSA-16 declining significantly and non-EMRSA-15/non-EMRSA-16 clones causing an increasing number of infections (over the time period 2003-2013). EMRSA-16 was resistant to a larger number of antimicrobials than EMRSA-15, typically 9 versus 5, and while resistance varied for EMRSA-16 over the study period, resistance remained stable for EMRSA-15. There was little difference between clinical and screening MRSA isolates. Analyses of antimicrobial prescribing showed that prescribing rates of several drugs increased over time (2003-2013). Prescribing was far higher in primary care settings than in secondary care, although this differed between antimicrobials. Significant positive associations between prescribing and resistance rates were found for gentamicin (pr - p<0.0001, se - p<0.0001) and trimethoprim (pr - p<0.01, se - p<0.0001) in both primary (pr) and secondary (se) care, and clindamycin (p<0.0001) in primary care only. Finally, in Scotland there is a threshold of connectivity above which the majority of hospitals, regardless of size, are positive for MRSA. Higher levels of MRSA are associated with the large, highly connected teaching hospitals with high ratios of patients to domestic staff. While there were a number of data limitations, this body of work provides a better understanding of the epidemiology of S. aureus including MRSA in Scotland.

Antibiotics are nedůležitějším means that the company has in the fight against bacterial infections. The discovery of penicillin by Sir A.Flemingem in 1928 , its isolation Chaine and Floreym in 1940 and subsequent clinical use have meant that until serious or fatal bacterial infectious diseases are today curable . In the following years there have been many more antibiotics or chemotherapeutics , which expands treatment options microbial infections . The high popularity of antibiotics in clinical practice over the course of a few decades taken their toll. Substantial consumption of antimicrobial agents and their misuse has led to the emergence of bacterial resistance with which the discovery of antibiotics planned. Adverse the result is a reduced capacity in the selection of antibiotic therapy in the causal infection. This poses a huge risk to the current population , but especially for the next generation . It may even occur to the fact that the antibiotics used today lost due to bacterial resistance efficiency, and we will not be able to fight against bacteria . One of the branches of medicine where there permanently increased consumption of antibiotics , pediatrics . Children have limited immune mechanisms , and therefore constitute a population that is more susceptible to infections. Most often ,...

Enterococos são bactérias que exercem um papel muito importante na produção de vários alimentos fermentados e também podem ser usadas como probióticos. A presença e o crescimento de enterococcos em alimentos fermentados como queijos e lingüiças conferem a esses produtos características organolépticas únicas. Em contrapartida, sua presença nos alimentos também está associada com falta de higiene durante a manipulação. Estes microrganismos também estão relacionados com o desenvolvimento de algumas doenças, como endocardites, septicemia, infecções do trato geniturinário, entre outras. A presença de características de virulência aumenta o potencial de infecção do microrganismo e a severidade da doença a ele relacionada. Com o objetivo de avaliar possíveis modificações fenotípicas e genotípicas de amostras de enterococos isoladas de frango, durante a subcultura destas cepas no laboratório, várias análises foram realizadas como: a presença dos fatores de virulência; proteína de superfície (esp) e gelatinase (gelE), do operon fsr-regulador do gelE, a expressão fenotípica do gelE, a capacidade de formação de biofilme e a resistência a antimicrobianos, desinfetantes e antisépticos. Quarenta isolados de Enterococcus sp. foram avaliados quanto a presença dos genes gelE, esp, operon-fsr, sprE por PCR, a atividade gelatinolítica por testes bioquímicos convencionais, resistência a antimicrobianos, antisépticos e desinfetantes por antibiograma e formação de biofilme pelo método cristal violeta. Todos os testes foram realizados na 1º geração e na 12º geração das cepas. 85% dos isolados produziram gelatinase e em 92,5% dos isolados o gene gelE estava presente na 1º geração. A análise do fsr-operon destes isolados do primeiro cultivo demonstrou que o gene fsrA estava presente em 35 isolados e o fsrC em 37 isolados e a presença destes genes pareceu não ter correlação com a atividade gelationolítica. O gene fsrB estava presente em todos os isolados (35) que apresentaram atividade gelatinolítica sugerindo que a presença deste gene é importante na expressão desta enzima. Após o subcultivo, apenas um isolado perdeu a atividade gelatinolítica e 15 perderam o gene gelE. Doze isolados perderam pelo menos um gene do fsr-operon durante a subcultura, porém nenhum destes perdeu a capacidade de expressar a enzima gelatinase talvez devido à presença do gene fsrB. O gene sprE foi detectado em 34 isolados na primeira geração e na 12º geração em apenas 20 isolados. O gene da proteína de superfície de Enterococcos (Esp), não foi encontrado em nenhum dos isolados. O antibiograma do isolados no primeiro cultivo demonstrou que 100% dos isolados foram sensíveis a ampicilina e a gentamicina, 95% sensíveis a vancomicina, 85% a ciprofloxacina, 5% a tetraciclina, 65% a eritromicina e 52,5% a cloranfenicol tanto na 1º quanto na 12º geração. Após a subcultura a susceptibilidade dos isolados aumentou a eritromicina (67,5%) e ao cloranfenicol (80%). Quanto ao perfil de resistência aos detergentes e anti-sépticos de uso comercial, todos os isolados apresentaram fenótipo de resistentes ao linear alquilbenzeno sulfonato (LAS) e ao triclosan durante a subcultura. Todos isolados foram suscetíveis ao formaldeído, mas se tornaram resistentes ao 8,5% hipoclorito de sódio e a clorexidina durante a subcultura. Em geral, todos os isolados foram formadores de biofilme e a produção de gelatinase parece ser necessária para esta formação. O perfil genético não pareceu ter relação com a formação de biofilme. Tanto o perfil genotípico quanto o fenotípico pode sofrer alterações durante a subcultura das cepas no laboratório. / Enterococci are bacteria that have a very important role in the production of various fermented foods and can also be used as probiotics. The presence and growth of enterococci in fermented foods like cheese and sausages bring to these products unique organoleptic characteristics. However, their presence in foods is also associated with lack of hygiene during handling. These microorganisms are also related to the development of some diseases such as endocarditis, septicemia, genitourinary infections, among others. The presence of virulence characteristics increases the potential infection of the organism and severity of disease related to it. The aim of the present study is analyze the possible changes of phenotypic and genotypic of enterococci isolated from chicken, during the subculture of the strains in the laboratory, the presence of virulence factors: enterococcal surface protein (esp) and gelatinase (gelE), operon-fsr gelE regulator, gelE phenotypic expression, the ability of biofilm formation and antibiotic, disinfectant and antiseptic resistance were determined in samples of enterococci isolated from chicken. The presence of gelE, esp operon-fsr and sprE genes were evaluated by PCR, gelatinase activity were observed by conventional biochemical tests, antibiotics resistance, antiseptics and disinfectants resistance were analyzed by standard disk diffusion method and biofilm formation were detected following the crystal violet staining method in forty enterococci isolates from chicken. All tests were performed in the 1st generation and 12th generation. 85% of the isolates produced gelatinase and in 92.5% of the isolated the gelE gene was present in the 1st generation. The analysis of operon-fsr in the 1st generation of these isolates showed that the fsrA gene was present in 35 isolates and fsrC gene was present in 37 isolates and the presence of these genes seemed to have no correlation with the gelatinase activity. The fsrB gene was present in all isolates (35) with gelatinase activity suggesting that the presence of this gene is important in the expression of this enzyme. After subculture, only one isolate lost the gelatinase activity and 15 isolates lost the gelE gene. Twelve isolates lost at least one gene of the operon-fsr during laboratory subculture, but none of these isolates lost the ability to express the enzyme gelatinase probably due the presence of the fsrB gene. The sprE gene was detected in 34 isolates in the 1st generation and in 12th generation only 20 isolates maintained this gene. The protein surface of enterococci gene (Esp), was not found in any isolate. The antibiogram of the isolates showed that 100% of the isolates were susceptible to ampicillin and gentamicin, 95% susceptible to vancomycin, 85% to ciprofloxacin, tetracycline 5%, 65% to erythromycin and 52.5% to chloramphenicol in the 1st generation. After subculture the susceptibility of isolates to erythromycin (67.5%) and chloramphenicol (80%) increased. As the profile of resistance to detergents and antiseptics for commercial use, all isolates showed resistance phenotype of the linear alkylbenzene sulfonate (LAS) and triclosan during subculture. All isolates were susceptible to formaldehyde, but became resistant to 8.5% sodium hypochlorite and chlorhexidine during the subculture. In general, all isolates were biofilm formers. Gelatinase production appears to be required for biofilme formation. The genetic profile did not appear to have relation with the formation of biofilms. Genotypic and the phenotypic profile may change during the subculture of the strains in the laboratory.

Colistin, also known as polymyxin E, is antibiotics active against most of Gram-negative bacteria. In the pas decade, emergency of multidrug-resistant bacteria led to increase of colistin administration as a last resort antibiotic for human infections. The first plasmid-mediated colistin resistance gene mcr-1 was identified in 2015 in animals in China and after first detection, additional mcr genes: mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7 a mcr-8 were described throughout the world. The aim of this thesis was to clarify whether there is horizontal transmission colistin resistance encoded by the mcr genes in gram-negative bacteria isolated from the environment, animals and their breeding and food. The mcr-1 gene was detected in 2 strains Escherichia coli isolated from waste water. The mcr-4 gene was detected in 1 strain Shewanella putrefaciens isolate obtained from the lake. The environment is the most important source and way of spreading this type of resistance in the Czech Republic.

Antibiotics are commonly prescribed and requested for viral illnesses despite evidence-based research studies and societal guidelines that advise against this practice. Literature has indicated that antibiotic decision-making comes from a provider's experience or exposure to illness, uncertainty of illness, or from being pressured by the patient. Nurses and advanced practice nurses are important participants in the antibiotic stewardship initiative. The purpose of this project was to examine potential knowledge deficits responsible for inappropriate antibiotic prescribing at a rural urgent care clinic in the southeastern United States, which when addressed could promote an educational in-service to decrease the number of antibiotics prescribed during a high-volume cough, cold, and flu months. The health belief model was used as a foundational model and a knowledge, attitude, and practice survey to collect data. Antibiotic prescribing habits were evaluated in the preintervention group (n = 250) and a year later in the postintervention group (n = 265). Antibiotic prescribing decreased positively from 80% to 70% and watchful waiting also increased positively from 4% to 30%; X-² (1) = 12.302, p = .000. The increase in educational awareness from these results can support a decrease in inappropriate antibiotic prescriptions, which prevents the emergence of antibiotic-resistant bacteria, contributing to positive social change.

Antimicrobial resistance (AMR) is a threat to global health, food security, and economic productivity. Infections caused by drug resistant Gram-negative pathogens, such as Escherichia coli, Pseudomonas aeruginosa, and Neisseria gonorrhoeae, are continuously becoming harder to treat due to limited treatment options and long turnaround times for culture-based phenotypic diagnosis. Alternatively, genotypic approaches that exploit whole genome sequencing have the potential to be faster and more accurate. Genotypic approaches rely on using bacterial genomes to predict AMR phenotypes. I generated a rules-based algorithm and machine learning models using known resistance determinants from bacterial genomes to predict resistance or susceptibility. I showed that machine learning was superior to a rules-based algorithm and achieved an average accuracy of 94% and 89% for E. coli and P. aeruginosa, respectively. These machine learning models identified novel AMR genotype-phenotype relationships between known resistance determinants and resistance phenotypes, which were experimentally validated. To identify the parameters that can improve machine learning models, I tested a variety of genetic features, algorithms, and evaluation metrics. I observed an intricate dependency between parameters for AMR prediction performance, illustrating that careful selection of parameters is required to generate accurate AMR prediction models. A limitation of this work was its prediction of resistance and susceptibility categories, as these are interpretations of minimum inhibitory concentrations defined by clinical breakpoint guidelines. Since multiple guidelines exist, these prediction models are not generalizable, so prediction of MIC values was explored. The average accuracy of my MIC prediction models was 86%, 41%, and 98% for E. coli, P. aeruginosa, and N. gonorrhoea, respectively. Despite the multifactorial and intricate nature of the resistome, I was able to accurately predict AMR phenotypes for many antibiotics for these pathogens. This is a step towards advanced diagnostic microbiology methods driven by genomics. / Thesis / Doctor of Philosophy (PhD) / Many surgeries, chemotherapy, and transplantation will be impossible if antibiotic resistance is not addressed. Antibiotic misuse, overuse, and time to definitive therapy exacerbate this global health problem. Phenotypic testing determines definitive therapy, but bacterial culturing is slow. A potentially faster and more accurate approach relies on sequencing the pathogen’s genome. I used machine learning to generate antibiotic resistance prediction models that achieved average accuracies of 94% and 89% for Escherichia coli and Pseudomonas aeruginosa, respectively. These models identified novel relationships between known resistance genes and resistance phenotypes, which were experimentally validated. Resistance and susceptibility are interpretations of a minimum inhibitory concentration (MIC) using a clinical breakpoint guideline. Since there are different guidelines, I generated MIC prediction models with average accuracies of 86%, 41%, and 98% for E. coli, P. aeruginosa, and Neisseria gonorrhoea, respectively. My findings work towards a world where clinical sequencing and genomics-based diagnostics are the gold standard.