Master of Science / Department of Grain Science and Industry / Bhadriraju Subramanyam / Hulya Dogan / Stored-product insects and stored products from feed mills and swine farms contain antibiotic and potentially virulent Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus hirae. Stored-product insects can serve as potential vectors of these enterococci which possess antibiotic resistance genes that can be spread by horizontal transfer to more serious human pathogens. In the present study, the species and concentration of enterococci from adults and larvae of key stored-product insects and insect diets and their antibiotic resistance profile were characterized. Adults of five species out of the 15 stored-product insects were tested positive for enterococci, and these included Callosobruchus maculatus (F.), Sitophilus granarius (L.), Stegobium paniceum (L.), Lasioderma serricorne (F.), and Sitophilus zeamais Motschulsky. Three enterococcal species (E. casseliflavus, E. faecalis, and E. faecium) were found in 53 to 97% of the 30 adults screened for each insect species, and the enterococcal concentrations ranged from 1.4 x 10³ to 3.1 x 10⁶ CFU/adult. About 10 to 100% of the mature larvae of the respective five insect species had these three enterococcal species with concentrations ranging from 0.3 x 10¹ to 1.4 x 10⁵ CFU/larvae. Only three of the eight insect diets screened had the same three enterococci species in addition to E. gallinarum and E. hirae at concentrations of 0.2 x 10¹ to 5.9 x 10³ CFU/g. The greatest enterococcal concentration was found in C. maculatus adults but not in their larvae or diet (cowpeas). In C. maculatus during a nine-day period after adult eclosion, the enterococcal concentrations increased exponentially from 0.6 x 10¹ to a maximum of 4.1 x 10⁷ CFU/adult. Enterococci were detected in the fecal material of C. maculatus during a four-day period with a maximum concentration of 3.3 x 10³ CFU/adult on the fourth day. A total of 298 enterococcal isolates from adults, larvae, and diets were represented by E. faecalis (51.7% of the total), E. faecium (19.1%), E. casseliflavus (18.8%), E. gallinarum (5.7%), and E. hirae (4.7%). Enterococci were phenotypically resistant to quinupristin (51.3% of the total), erythromycin (38.9%), tetracycline (30.1%), enrofloxacin (29.2%), doxycycline (11.5%), and tigecycline (2.7%). All isolates were susceptible to ampicillin and vancomycin.

No description available.

Beach advisories are issued at recreational beaches when the water quality exceeds regulatory limits for the indicator organism, enterococci. Elevated levels of enterococci have been observed at Hobie Cat Beach, the study marine beach site, located on Virginia Key, Florida. The study site represents a classic non-point source sub/tropical marine recreational beach area with high human and animal use, representative of many beaches worldwide in sub/tropical areas. The dissertation consisted of two separate but related studies: the first to identify environmental and geographic factors, and the second to evaluate the impact of known animal sources of enterococci. The first efforts were made to identify the geographic location of the source of enterococci to the beach waters and to assess the environmental factors that impact the variation in concentrations observed at the site. These environmental factors and conditions include: proximity to shoreline, tidal changes, impacts of runoff, and sunlight intensity. Enterococci were enumerated by traditional membrane filtration or the chromogenic substrate method. Overall, results showed that the source of enterococci to the study beach was geographically located within the inter-tidal zone. These results suggest that the wash-in of sediments and accompanying pore waters (where the pore water is the water filling the spaces between grains of sediment) from the inter-tidal zone play a major role in controlling enterococci levels within the water column. Wash-in occurs through both tidal fluctuations and runoff. The second effort evaluated non-point sources, including animals, which are known to contribute to elevated levels of enterococci in recreational marine beach waters. Specifically, feces from dogs, birds, and shrimp mounds were collected from the beach; additional bird fecal samples were collected from both a local zoo and bird rehabilitation center. Fecal samples were weighed gravimetrically, and enumerated for enterococci using traditional membrane filtration method. The total numbers of animals which frequented the site were obtained through camera image analysis and in-field visual counting surveys. The highest enterococci concentrations were observed in dog feces (avg. 7.4 x 10^6 CFU/g dry feces), then birds (avg. 3.3 x 10^5 CFU/g dry feces) and the lowest measured levels of enterococci were observed in shrimp fecal mounds (2.0 CFU/g dry feces on average). A comparison of the microbial load (CFU per fecal event) showed that 1 dog fecal event was equivalent to 6,940 bird fecal events or 3.2 x 10^8 shrimp fecal events. Given the abundance of animals observed on the beach, these study results suggest that dogs are the largest contributing source of enterococci to the beach site (6.3 x 10^11 CFU per day during weekends and 2.9 x 10^11 CFU per day during weekdays), with humans (4.6 x 10^9 CFU per day during weekends and 4.8 x 10^8 CFU per day during weekdays) and birds (2.7 x 10^8 CFU per day) serving as secondary contributors. Shrimp served as an insignificant source (1.9 x 10^4 CFU per day). When maximum daily contributions were considered, dogs contributed the highest proportion of enterococci (99.2%) compared to humans (0.72%), birds (0.04%), and shrimp (<0.04%). Beach management efforts at the study site should thus focus on requiring dog owners to properly dispose of dog feces deposited at the beach.

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.

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.

Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically relevant microorganisms. E. faecium possesses two distinct aaPGSs: aaPGS1 and aaPGS2. In addition, another gene coding for a putative hydrolase (pHyd) is located in the same operon as aaPGS2, and has no known function. To investigate the physiological relevance of aa-PG formation, and the function of aaPGS1, aaPGS2, and pHyd in E. faecium, we generated individual knockouts of these genes using a markerless deletion strategy. Deletion of aaPGS1 did not noticeably alter lipid aminoacylation, whereas deletion of aaPGS2 led to a loss of aa-PG synthesis. Deletion of pHyd also led to a loss of lipid aminoacylation; however, additional experiments are needed to verify that expression of aaPGS2 (which resides just downstream in the same operon) is unaffected in the pHyd-deletion strain. Development of the mutant strains described here will enable us to investigate additional phenotypes associated with these genes, and to determine whether aa-PG formation contributes to antibiotic resistance in E. faecium as in several other pathogenic microorganisms.

No description available.

The U.S. EPA has identified pathogen indicators as a leading cause of impairments in rivers and streams in the U.S. Elevated levels of bacteria in streams draining the agricultural watersheds cause concern because they indicate the potential presence of pathogenic organisms. Limited understanding of how bacteria survive in the environment and are released from fecal matter and transported along overland flow pathways results in high uncertainty in the design and selection of appropriate best management practices (BMPs) and in the bacterial fate and transport models used to identify sources of pathogens. The overall goal of this study was to improve understanding of the fate and transport mechanisms of two pathogen indicators, E. coli and enterococci, from grazed pasturelands. This goal was addressed by monitoring pathogen indicator concentrations in fresh fecal deposits for an extended period of time. Transport mechanisms of pathogen indicators were examined by developing a method to partition between the attached and unattached phases and then applying this method to analyze runoff samples collected from small box plots and large transport plots. The box plot experiments examined the partitioning of pathogen indicators in runoff from three different soil types while the transport plot experiments examined partitioning at the edge-of-the-field from well-managed and poorly-managed pasturelands. A variety of techniques have been previously used to assess bacterial attachment to particulates including filtration, fractional filtration and centrifugation. In addition, a variety of chemical and physical dispersion techniques are employed to release attached and bioflocculated cells from particulates. This research developed and validated an easy-to-replicate laboratory procedure for separation of unattached from attached E. coli with the ability to identify particle sizes to which indicators preferentially attach. Testing of physical and chemical dispersion techniques identified a hand shaker treatment for 10 minutes followed by dilutions in 1,000 mg L-1 of Tween-85 as increasing total E. coli concentrations by 31% (P value = 0.0028) and enterococci concentrations by 17% (P value = 0.3425) when compared to a control. Separation of the unattached and attached fractions was achieved by fractional filtration followed by centrifugation. Samples receiving the filtration and centrifugation treatments did not produce statistically different E. coli (P value = 0.97) or enterococci (P value = 0.83) concentrations when compared to a control, indicating that damage was not inflicted upon the cells during the separation procedure. In-field monitoring of E. coli and enterococci re-growth and decay patterns in cowpats applied to pasturelands was conducted during the spring, summer, fall and winter seasons. First order approximations were used to determine die-off rate coefficients and decimal reduction times (D-values). Higher order approximations and weather parameters were evaluated by multiple regression analysis to identify environmental parameters impacting in-field E. coli and enterococci decay. First order kinetics approximated E. coli and enterococci decay rates with regression coefficients ranging from 0.70 to 0.90. Die-off rate constants were greatest in cowpats applied to pasture during late winter and monitored into summer months for E. coli (k = 0.0995 d-1) and applied to the field during the summer and monitored until December for enterococci (k = 0.0978 d-1). Decay rates were lowest in cowpats applied to the pasture during the fall and monitored over the winter (k = 0.0581 d-1 for E. coli and k = 0.0557 d-1 for enterococci). Higher order approximations and the addition of weather variables improved regression coefficients (R2) to values ranging from 0.81 to 0.97. Statistically significant variables used in the models for predicting bacterial decay included temperature, solar radiation, rainfall and relative humidity. Attachment of E. coli and enterococci to particulates present in runoff from highly erodible soils was evaluated through the application of rainfall to small box plots containing different soil types. Partitioning varied by indicator and by soil type. In general, enterococci had a higher percent attached to the silty loam (49%) and silty clay loam (43%) soils while E. coli had a higher percent attached to the loamy fine sand soils (43%). At least 50% of all attached E. coli and enterococci were associated with sediment and organic particles ranging from 8 – 62 μm in diameter. Much lower attachment rates were observed from runoff samples collected at the edge-of-the-field, regardless of pastureland management strategy. On average, 4.8% of E. coli and 13% of enterococci were attached to particulates in runoff from well-managed pasturelands. A second transport plot study found that on average only 0.06% of E. coli PC and 0.98% of enterococci were attached to particulates in runoff from well-managed pasturelands, but percent attachment increased slightly in runoff from poorly-managed pasture with 2.8% of E. coli and 1.23% of enterococci attached to particulates. Equations to predict E. coli and enterococci loading rates in the attached and unattached forms as a function of total suspended solids (TSS), phosphorous and organic carbon loading rates appeared to be a promising tool for improving prediction of bacterial loading rates from grazed pasturelands (R2 values ranged from 0.61 to 0.99). This study provides field-based seasonal die-off rate coefficients and higher order approximations to improve predictions of indicator re-growth and decay patterns. The transport studies provide partitioning coefficients that can be implemented into NPS models to improve predictions of bacterial concentrations in surface waters and regression equations to predict bacterial partitioning and loading based on TSS and nutrient data. Best management practices to reduce bacterial loadings to the edge-of-the-field from pasturelands (regardless of management strategy) should focus on retention of pathogen indicators moving through overland flow pathways in the unattached state. Settling of particulates prior to release of runoff to surface waters might be an appropriate method of reducing bacterial loadings by as much as 50% from highly erodible soils. / Ph. D.

Sources of fecal pollution were evaluated as a function of sampling frequency with stream samples from Mill Creek, Montgomery County, VA. Samples were collected monthly for one year, plus weekly for four consecutive weeks during seasonal high flows (March), and seasonal low flows (September-October), plus daily for seven consecutive days within the weekly schedules. Thirty stream samples were collected from each of two sites (60 total) in Mill Creek, and 48 isolates of E. coli per sample (total of 2,880 stream isolates) were classified by source using antibiotic resistance analysis (ARA) and comparing the resulting patterns against a known-source E. coli library (1,158 isolates). The same process was performed with enterococci isolates against an enterococci library (1,182 isolates). The average rate of correct classification (ARCC) for the E. coli library with a three-way split (human, livestock, and wildlife) was 89.0%, and the ARCC of the species-specific E. coli library (cattle, deer, goose, human, misc. wildlife) was 88.9%. The ARCC of the enterococci library with a three-way split was 85.3%, and the ARCC of the species-specific enterococci library was 88.1%. The results did not justify the need for daily or weekly sampling, but indicated that monthly was adequate (quarterly and every-other-month were not). There was a seasonal effect as the human signature was highest during high flow while the livestock signature dominated during low flow. The results also indicated that sampling should be done over a period of time that includes both seasonal wettest and driest periods (at least 8 months). / Ph. D.