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Enterococci bacteria that cannot be treated eectively with the antibiotic vancomycin are termed Vancomycin-Resistant Enterococci (VRE). In this thesis, we develop a mathematical framework for determining optimal strategies for prevention and treatment of VRE in an Intensive Care Unit (ICU). A system of ve ordinary dierential equations describes the movement of ICU patients in and out of dierent states related to VRE infection. Two control variables representing the prevention and treatment of VRE are incorporated into the system. An optimal control problem is formulated to minimize the VRE-related deaths and costs associated with controls over a nite time period. Pontryagin's Minimum Principle is used to characterize optimal controls by deriving a Hamiltonian expression and dierential equations for ve adjoint variables. Numerical solutions to the optimal control problem illustrate how hospital policy makers can use our mathematical framework to investigate optimal cost-eective prevention and treatment schedules during a VRE outbreak. / McAnulty College and Graduate School of Liberal Arts; / Computational Mathematics / MS; / Thesis;

Enterococci are opportunistic pathogens that are frequently a source of nosocomial infections and it is their resistance to antibiotics and their ability to form biofilms that represent important virulence traits. Normally, in healthy individuals it is a harmless commensal that is usually found in the intestine. This thesis firstly studies signal peptidases (SPases), which play an essential role in protein translocation. Interestingly, E. faecium was found to contain three type I SPases. Many proteins that are secreted are virulence factors, and the aim was to delete one or more of the SPases and study the effect of its removal on virulence. Unfortunately no mutants were obtained suggesting that the genes were essential. To establish if the genes were essential an inducible integration vector was constructed, but due to time constraints this could not be tested further. Biofilm formation was studied in both E. faecium and E. faecalis. The presence of the Enterococcal Surface Protein (Esp) in E. faecium was shown to increase hydrophobicity, and therefore also increase biofilm formation. Similarly, E. faecalis isolates that were good biofilm formers were also more hydrophobic in nature. The expression of Esp in E. faecium was studied under different conditions; these studies indicated that the highest level of Esp expression was found in biofilms cells. This growth-dependent manner Esp expression was not observed in E. faecalis BS12297. Surprisingly, Esp in E. faecium was also shown to have a role in ampicillin resistance, which was identified using calorimetry. This method proved to be a sensitive and rapid method to analyse antibiotic resistance. In the gut, bacteria encounter various adverse conditions, such as low pH and the presence of bile salts. Here we investigated the effects of bile salts on biofilm formation in E. faecium and E. faecalis and demonstrated that biofilm formation is induced at physiological concentrations of bile salts. In E. faecium the presence of bile salts caused an increase in initial attachment, microcolony formation and EPS production. Various factors were investigated, including hydrophobicity, cell growth, cell morphology, Esp expression and the production of extracellular polymeric substance (EPS). In E. faecium, only EPS production appeared to play a role, but the stimulation of biofilm formation due to bile salts is still to be fully explained.

Enterococci are intestinal facultative anaerobic strains which recognized as opportunistic pathogens. The ability to form biofilms is an important virulence trait that has been reported for Enterococci. Biofilm formation showed differences between E. faecalis strains. However, several factors were involved in this process e.g. the presence of virulence factors, hydrophobicity and heterogeneity. Interestingly, we demonstrated for the first time a biochemical test for a cell surface protein in biofilm formation: addition of the purified N domain of Esp (EspN) to E. faecium E1162Δesp resulted in the restoration of biofilm formation. Streptococcus bovis also, is an intestinal facultative anaerobic bacterium. This organism also has been reported as an opportunistic pathogen causing multiple diseases such as septicemia and endocarditis associated with colorectal cancer (CRC). Although the association of S. gallolyticus infection with CRC is a major issue, the mechanisms behind this link are still unclear. This link between CRC and the virulence of S. bovis strains was studied in more detail in a collaborative project with Dr Harold Tjalsma. The Tjalsma group mainly focussed on host-pathogen interactions, whereas we analysed biofilm formation of S. bovis strains as well as their pathogenicity using the in vivo C. elegans infection model. Our biofilm showed that S. bovis strains form biofilms particularly well on collagen-rich surfaces at least indicate why there is this association. C. elegans experiments also showed that pathogenicity of S. bovis strains is more similar to E. faecalis than to E. faecium in which both S. bovis and E. faecalis have a slow mode of killing that is absent in E. faecium. Full genome sequences of S. gallolyticus UCN34 strain have revealed the presence of a number of potential collagen-binding proteins (e.g., gallo_2179) that are related to the MSCRAMMs family. However, we successfully cloned the gallo_2179 gene in an enterococcal expression vector, and demonstrated transcribed in E. faecalis. Unfortunately, this strain did not form better biofilms on a collagen surface, suggesting either that not sufficient amount of the protein was made, or that the protein is not functional in E. faecalis. In addition, a bioinformatics analysis was performed to identify putatively secreted proteins in S. gallolyticus. Proteins that were expected to be found include for instance three collagen-binding proteins, amylase, tannase and beta lactamase.

The genus Enterococcus has an exceptional ability to acquire and transmit antibiotic resistance genes and is considered to be a major vector in their dissemination. Enterococci are part of the normal gut microbiota of humans and animals and are frequently encountered in food products including dry fermented sausage. Since fermented sausages are not heat-treated before consumption they might be a vehicle for transmitting resistance and virulence traits of enterococci by conjugation with commensal bacteria present in the human gut and pathogenic bacteria that might be present, such as Listeria species. A PCR-based assay was developed to detect enterococci in dry fermented sausage meat at the generic level by targeting a 16S rRNA sequence and a total of 29 Enterococccus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. The susceptibility of these enterococci to antibiotics was tested and it was found that 27/29 were resistant to more than one antibiotic and possessed antibiotic resistance determinants. All strains were positive for at least one virulence gene. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci and probably contributed to their survival in the harsh conditions experienced during dry sausage fermentation and drying. SmaI pulsed-field gel electrophoresis (PFGE) patterns exhibited genomic heterogeneity within and between the two larger groups of isolates. In spite of this heterogeneity, the phenotypic similarities observed suggested that food could still be a vehicle for distribution of antibiotic resistant bacteria among humans. In vitro conjugation experiments demonstrated transfer of the tetracycline resistant determinant, tet(M), from E. faecium S27 isolated from fermented sausage to clinical isolates of both E. faecium and E. faecalis. The streptomycin resistance of E. faecium S27 was also transferred to a clinical strain, E. faecalis 82916, which was confirmed by the presence of the streptomycin resistance gene, aadA, in the donor and transconjugant strains. E. faecium S27 also transferred tet(M) and streptomycin resistance to Listeria monocytogenes GLM-2 by in vitro mating. Evidence suggests that enterococci in fermented meats may contribute to the spread of resistance determinants. / October 2015

Swimming in ocean water, including beaches not impacted by known point sources of pollution, continues to pose a rising health concern. Studies which evaluate the relationship between indicator microbes, pathogens, and human health at recreational beaches are rare, especially at non-point beaches, although such studies are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this dissertation was to better understand the non-point source beach system, and hence determine possible directions that should be taken from a beach regulatory perspective in order to protect bather health and minimize economic burdens. This objective was accomplished through two studies. The first study included an initial evaluation of the presence of and associations among indicator microbes, pathogens and environmental conditions at a subtropical, recreational marine beach in South Florida (USA) impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events conducted at high or low tide and during elevated or reduced solar insolation conditions. Analyses included fecal indicator bacteria (FIB) (fecal coliform, Escherichia coli, enterococci, Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp. and Giardia spp.). The objective of the second study was to capture a series of "snapshots" of the study beach with respect to pathogens and indicator microbe levels, environmental conditions, and cumulative human health effects. Two sets of daily composite samples were collected during the BEACHES epidemiology study conducted in Miami (FL) at the same non-point source subtropical marine beach. These samples were analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters similar to those in the first study. Enterococci concentrations in water and sand assessed by qPCR were greater than membrane filtration measurements in both studies. FIB concentrations in water were below recreational water quality standards for three of four sampling events; pathogens and MST markers were also generally undetectable during the first study. FIB levels exceeded regulatory guidelines during one event, accompanied by the detection of HPyVs and pathogens, including the autochthonous bacterium Vibrio vulnificus in both sand and water, the allochthonous protozoan Giardia spp. in water, and Cryptosporidium spp. in sand samples. These elevated microbial levels were detected at high tide, low solar insolation conditions, and after elevated 2 day antecedent rainfall. Analyses of the second study demonstrated that rainfall and tide were also significant factors in determining the presence of both indicator microbes and pathogens. Additional potential associations between environmental factors, microbes and human health effects were also identified. However, more analyses should be conducted as the limited number of samples renders it difficult to make conclusive statements about the relationship between these different factors. The results of this research demonstrate the complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches will be needed to assess beach water quality for the purpose of protecting bather health. A recommended approach "The comprehensive tool box with approval process approach" to address the beach complexity is presented and applied to the study beach.

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Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Bioanalytical laboratory diagnostics in health care Candidate: Bc. Martina Malíková Thesis supervisor: PharmDr. Ondřej Janďourek, Ph.D. Consultant: RNDr. Irena Hanovcová, CSc. Title of diploma thesis: Effectiveness of disinfectants for hygienic hand disinfection against clinically important strains of enterococci Alcohol-based hand disinfectants for hygienic hand disinfection are a key tool for the control of nosocomial infections worldwide. The aim of this study was to verify the effectiveness of these products, which are used in healthcare facilities, against vancomycin-sensitive and vancomycin-resistant enterococci isolated from clinical materials. Testing of the efficacy of disinfectants was performed according to the Czech technical standard ČSN EN 1040 - Chemical disinfectants and antiseptics - Quantitative test using suspension to determine the basic bactericidal effect of chemical disinfectants and antiseptics - Test method and requirements (phase 1). We used the dilution method with a neutraliser for our testing. There were used a total of 35 strains of enterococci to determine efficacy, 12 of which were vancomycin sensitive (four E. faecium strains and eight E. faecalis...

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ludek Zurek / Transfer of bacteria, including antibiotic resistant strains between companion animals and people is likely due to close physical contacts. However, surveillance programs on prevalence of antibiotic resistance are focused mainly on food animals and very little is known about the role of companion animals in the development and spread of antibiotic resistant bacteria. For this study, enterococci were chosen as model organism due to intrinsic and acquired antibiotic resistance and several virulence traits that make them the 3rd most important nosocomial pathogens. In addition, increased fecal shedding of antibiotic resistant bacteria from stressed animals has been reported from studies on food animals. To determine whether the gut microbiota of pet animals serves as a reservoir of clinically important enterococci, 360 enterococcal isolates from two groups: healthy group and pyoderma (stressed) group with 9 dogs in each were identified and screened for resistance to 10 antibiotics and 4 virulence traits. The transferability of resistance determinants and clonality of selected isolates were assessed by horizontal gene transfer assays and pulsed-field gel electrophoresis, respectively. In addition, overall diversity of bacteria as well as antibiotic and metal resistance genes in feces of healthy dogs was assessed by tag-encoded parallel pyrosequencing and microarray analysis, respectively. The most prevalent enterococcal species identified was E. faecalis: healthy group (70.5%); pyoderma group (44.0%). In the pyoderma group, antibiotic resistance and virulence traits (esp, gelE) were more frequent than in the healthy group; however, the overall prevalence of antibiotic resistant strains was low (< 37%) in both groups. The most prevalent resistance genes were tet(M)and tet(S). The antibiotic resistance traits were transferable in-vitro in E. faecalis (tetracycline, erythromycin, doxycycline) and E. faecium (tetracycline). Genotyping revealed less diverse E. faecalis community in pyoderma infected dogs. Pyrosequencing (~7,500 sequences per dog) revealed Firmicutes as the dominant phylum and most common genera included Turicibacter, Lactobacillus, Ruminococcus, Clostridium, and Fusobacterium. Two phyla Lentisphaerae (<1%) and Fibrobacteres (<1%) are reported for the first time from healthy dogs. Microarray data revealed the presence of several tetracycline, erythromycin, aminoglycoside, and copper resistance genes; however, most of these originated from one animal with history of chronic skin infection two year prior to our sampling. Higher prevalence of antimicrobial resistance in pyoderma infected dogs may be related to stress; however, this requires further investigation. In conclusion, based on our data, healthy and pyoderma infected dogs do not represent an important reservoir of clinically significant antibiotic resistant microbiota.

Context: Daptomycin is the only antibiotic available with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). Its increased use has resulted in cases of decreased daptomycin efficacy. Recent in vitro studies have shown effective use of beta (beta)-lactam and daptomycin antibiotics, as a combination therapy, in the treatment of VRE. We describe a case of effective treatment in a patient with VRE infection using dual ampicillin and daptomycin therapy that shows bench-to-bedside application of the abovementioned finding. Case Report: A 76-year-old gentleman with a history of bilateral arthroplasty was admitted with a swollen left knee. Blood cultures were positive for Enterococcus faecium. Left knee joint aspiration showed leukocytosis and alpha defensins. Extensive imaging did not show any other source of infection. Culture sensitivity results showed multidrug-resistant enterococci sensitive to daptomycin. The patient was started on intravenous (IV) daptomycin. His left knee prosthesis was explanted and a spacer was placed. The patient continued to be bacteremic for 10 days after removing the knee prosthesis. The patient was trialed on combination IV ampicillin and daptomycin. His blood culture turned negative 2 days later. The patient was discharged home to continue 6 weeks of IV ampicillin and daptomycin. Conclusion: The exact mechanism of the daptomycin/ampicillin synergy effect is unclear. Current hypothesis suggests that ampicillin causes a reduction in the net positive charge of the bacterial surface, possibly by releasing lipoteichoic acid (LTA) from the cell wall. This process increases the ability of the cationic daptomycin/calcium complex to bind to the cell wall more effectively. Our case shows the clinical application of the same. A prospective randomized control trial to explore the effectiveness of dual antibiotic therapy in vivo is needed. If proven, daptomycin/-lactam can become a standard of care to treat VRE and decrease daptomycin nonsusceptibility.