O uso de diferentes tipos de medicação intracanal para o controle do processo infeccioso, principalmente nos casos em que há presença de microrganismos resistentes às manobras de desinfecção, tem sido alvo de muitas pesquisas. A proposta deste estudo foi avaliar, in vitro, o efeito antimicrobiano de bacteriófagos específicos diante de cinco cepas de Enterococcus faecalis e a ação de um lisado híbrido polivalente na eliminação da infecção causada por essas cinco cepas da mesma espécie. Foram utilizados 37 dentes unirradiculares humanos, recentemente extraídos e de proporções aproximadas. As coroas foram removidas e os canais instrumentados até a lima tipo K de número 45. Os espécimes foram, então, esterilizados e utilizados em dois experimentos distintos. O primeiro experimento utilizou 25 raízes divididas em cinco grupos de cinco espécimes. Três espécimes de cada grupo foram inoculados com uma das culturas bacterianas e seus fagos correspondentes na proporção 1:1, por um período de três horas a 37 °C, enquanto os outros dois, receberam a cultura de microrganismos ou somente meio de cultura (controle positivo e negativo, respectivamente). No segundo experimento, 11 espécimes receberam um inóculo formado pelas cinco cepas por um período de 10 dias de incubação a 37 °C, com o propósito de manter condições apropriadas para a penetração das bactérias no interior dos túbulos dentinários, e um outro espécime recebeu apenas meio de cultura (controle negativo). Essa penetração foi confirmada empregando-se microscopia ótica e eletrônica realizada em dois espécimes. Após o período de incubação, o lisado polivalente, preparado com os cinco fagos, foi aplicado por 24 horas a 37 °C em 8 espécimes, e os demais preenchidos com meio de cultura (controle positivo e negativo). Alíquotas do interior de todos os canais foram colhidas antes e depois do contato com os fagos e no segundo experimento, também 24 e 48 horas depois, para semeadura e contagem de unidades formadoras de colônia. Os resultados do primeiro experimento mostraram 100% de redução do crescimento bacteriano nos espécimes que receberam a suspensão de fagos específicos, em comparação a seus respectivos controles positivos, em todos os grupos. No segundo experimento, foi comparado o crescimento obtido após os 10 dias de infecção com aquele posterior a aplicação dos fagos, redução que variou entre 50% e 100%. Diante desses resultados, conclui-se que os bacteriófagos foram eficazes na diminuição dos microrganismos presentes no interior de canais radiculares e nos túbulos dentinários de dentes humanos. / Many studies have investigated different intracanal medications to control infection processes, especially in cases of microbial resistance to disinfection procedures. The purpose of this study was to evaluate the in vitro antimicrobial effect of specific bacteriophages on five isolates of Enterococcus faecalis, as well as the activity of a lysate cocktail in eliminating the infection caused by these bacteria. Thirty-seven recently extracted human teeth of approximately equal size and with single roots were used. The crowns were removed and each canal was prepared using K files,up to # 45, and sterile physiological saline. Specimens were then sterilized and used in two separate studies. The first study utilized 25 individual roots divided into five groups of five specimens each. Three specimens of each group were inoculated with one of the bacterial cultures and the corresponding bacteriophage in a proportion of 1:1, and incubated for three hours at 37°C; the other two specimens were inoculated with only the bacterial culture or only the culture medium (positive and negative controls, respectively). In the second study, 11 specimens were inoculated with all five strains and incubated for ten days at 37°C in order to allow bacteria to penetrate the interior of the dental tubules, and another one, received just the culture medium (negative control). Penetration into the tubules was confirmed by optical and electron microscopy of two specimens. Following incubation, the lysate cocktail prepared using all five bacteriophages was applied to the other 8 specimens for 24 hours at 37°C, and 2 specimens were filled with the culture medium (positive and negative controls). In the first study, samples were taken from the lumen of all canals before and after contact with bacteriophages; in the second, aliquots were also taken 24 and 48 hours after the bacteria were exposed to the phages. All samples were diluted and plated and the number of colony forming units was counted. In the first study, there was a 100% reduction in bacterial growth in specimens that received the specific bacteriophage suspension compared to the positive controls within each group. In the second study, after ten days the number of bacteria was reduced by 50% to 100% following the bacteriophage application. These results suggest that bacteriophages are effective in reducing the number of bacteria inside the root canal and in the dental tubules of human teeth.

O uso de diferentes tipos de medicação intracanal para o controle do processo infeccioso, principalmente nos casos em que há presença de microrganismos resistentes às manobras de desinfecção, tem sido alvo de muitas pesquisas. A proposta deste estudo foi avaliar, in vitro, o efeito antimicrobiano de bacteriófagos específicos diante de cinco cepas de Enterococcus faecalis e a ação de um lisado híbrido polivalente na eliminação da infecção causada por essas cinco cepas da mesma espécie. Foram utilizados 37 dentes unirradiculares humanos, recentemente extraídos e de proporções aproximadas. As coroas foram removidas e os canais instrumentados até a lima tipo K de número 45. Os espécimes foram, então, esterilizados e utilizados em dois experimentos distintos. O primeiro experimento utilizou 25 raízes divididas em cinco grupos de cinco espécimes. Três espécimes de cada grupo foram inoculados com uma das culturas bacterianas e seus fagos correspondentes na proporção 1:1, por um período de três horas a 37 °C, enquanto os outros dois, receberam a cultura de microrganismos ou somente meio de cultura (controle positivo e negativo, respectivamente). No segundo experimento, 11 espécimes receberam um inóculo formado pelas cinco cepas por um período de 10 dias de incubação a 37 °C, com o propósito de manter condições apropriadas para a penetração das bactérias no interior dos túbulos dentinários, e um outro espécime recebeu apenas meio de cultura (controle negativo). Essa penetração foi confirmada empregando-se microscopia ótica e eletrônica realizada em dois espécimes. Após o período de incubação, o lisado polivalente, preparado com os cinco fagos, foi aplicado por 24 horas a 37 °C em 8 espécimes, e os demais preenchidos com meio de cultura (controle positivo e negativo). Alíquotas do interior de todos os canais foram colhidas antes e depois do contato com os fagos e no segundo experimento, também 24 e 48 horas depois, para semeadura e contagem de unidades formadoras de colônia. Os resultados do primeiro experimento mostraram 100% de redução do crescimento bacteriano nos espécimes que receberam a suspensão de fagos específicos, em comparação a seus respectivos controles positivos, em todos os grupos. No segundo experimento, foi comparado o crescimento obtido após os 10 dias de infecção com aquele posterior a aplicação dos fagos, redução que variou entre 50% e 100%. Diante desses resultados, conclui-se que os bacteriófagos foram eficazes na diminuição dos microrganismos presentes no interior de canais radiculares e nos túbulos dentinários de dentes humanos. / Many studies have investigated different intracanal medications to control infection processes, especially in cases of microbial resistance to disinfection procedures. The purpose of this study was to evaluate the in vitro antimicrobial effect of specific bacteriophages on five isolates of Enterococcus faecalis, as well as the activity of a lysate cocktail in eliminating the infection caused by these bacteria. Thirty-seven recently extracted human teeth of approximately equal size and with single roots were used. The crowns were removed and each canal was prepared using K files,up to # 45, and sterile physiological saline. Specimens were then sterilized and used in two separate studies. The first study utilized 25 individual roots divided into five groups of five specimens each. Three specimens of each group were inoculated with one of the bacterial cultures and the corresponding bacteriophage in a proportion of 1:1, and incubated for three hours at 37°C; the other two specimens were inoculated with only the bacterial culture or only the culture medium (positive and negative controls, respectively). In the second study, 11 specimens were inoculated with all five strains and incubated for ten days at 37°C in order to allow bacteria to penetrate the interior of the dental tubules, and another one, received just the culture medium (negative control). Penetration into the tubules was confirmed by optical and electron microscopy of two specimens. Following incubation, the lysate cocktail prepared using all five bacteriophages was applied to the other 8 specimens for 24 hours at 37°C, and 2 specimens were filled with the culture medium (positive and negative controls). In the first study, samples were taken from the lumen of all canals before and after contact with bacteriophages; in the second, aliquots were also taken 24 and 48 hours after the bacteria were exposed to the phages. All samples were diluted and plated and the number of colony forming units was counted. In the first study, there was a 100% reduction in bacterial growth in specimens that received the specific bacteriophage suspension compared to the positive controls within each group. In the second study, after ten days the number of bacteria was reduced by 50% to 100% following the bacteriophage application. These results suggest that bacteriophages are effective in reducing the number of bacteria inside the root canal and in the dental tubules of human teeth.

<p>The  Pakistan  poultry  industry  has  developed  into  the  11thlargest  poultry  industry  in  the world  and  poultry  products  provide  high-quality  and  affordable  protein  sources  to  communities throughout the country. However, <em>Salmonella </em>Gallinarum, the etiological agent for fowl typhoid, is  endemic  in  Pakistan  with  infections  leading  to  high  mortality  and  substantial  economic  loss. Currently, <em>Salmonella </em>Gallinarum  infectionsin  Pakistan  poultry  are  controlled  with  antibiotics. The continued emergence of antibiotic resistance, however, has led to global initiatives to reduce the  use  of  antibiotics  in  both  human  and  veterinary  medicine.  Concurrently,  the  Pakistan government  recently  introduced  new  national  policies  that  limit  the  use  of  antibiotics  for performance  in  livestock  and  poultry  production.  As  such,  controlling  bacterial  infections  in poultry  without  increasing  the  likelihood  of  antibiotic use could  ensure  the  sustainability  of Pakistan  poultry  production  without  posing  risks  to  public  health.  Toward  this  end,  we hypothesized that <em>Salmonella</em> Gallinarum infections inchickens could be prevented or otherwise controlled through the use of phages. To test this hypothesis, wastewater samples were collected from Lahore, Pakistan and different cities of Indiana, US and processed to isolate bacteriophages. The  phages  were  characterized  in  terms  of  morphology,  host  spectra,  lytic  capacity,  genomic sequencing,  and  survivability  in  different  environments. Transmission  electron  microscopy showed these phages belonged to myoviridae (n = 5) and podoviridae (n = 1) families. Spectrum analysis  revealed  that  each  phage  lysed  at  least  8  out  of  10  different  strains  of <em>Salmonella </em>Gallinarum and significantly reduced (P < 0.05) <em>Salmonella </em>Gallinarum when co-cultured in liquid medium with the bacterium. Stability of the phages was tested insimulated gastric fluid (SGF; pH= 2.5) andsimulated intestinal fluid (SIF; pH~6.8). Results showed that phage concentrationswere reduced to undetectable levels when exposed to SGF for more than 5 minutes. However, exposure to SIF did not result in appreciable reductions in phage concentrations. To mitigate potential effects of  gastric  environments,  phages  were  encapsulated  using  a  sodium  alginate-based  method.  In contrast  to  unprotected  phages,  encapsulated  phages  remained  viable  (~100%)  after  30  minutes exposure to SGF. Additionally, encapsulation efficiencies ranged between 90-99%. Encapsulated phages were sequentially incubated in SGF (30 minutes) and SIF(120 minutes) to determine the rate  of  release  of  the  phages  from  capsules. All  phages  were  released from  capsules after  60 minutes  of  exposureto  SIF. To  determine  if  the  phages  effectively  controlled <em>Salmonella </em>Gallinarum infections in chickens, 100, day-old Jumbo Cornish Rock Cross birds were randomly assigned  to  one  of  four  treatments:  1)  Control 1  (bacterial  challenge,  no  phage  treatment);  2) Control 2 (no phage or bacterial challenge); 3) challenged with SalmonellaGallinarum and treated with  unprotected  phages;  and  4)  challenged  with <em>Salmonella</em> Gallinarum  and  treated  with encapsulated phages. At7 d of age, chicks receiving the bacterial challenge were administered 5 X106CFU (500 μL) of <em>Salmonella</em> Gallinarum. For birds in phage treatment groups, the phages were administered (500 uL; 5 X108 PFU/mL or g) at 0, 12, and 24 hours post-challenge. Six birds from each group were euthanized at 1, 2, and 4 days post-challenge (dpc) and cecal SalmonellaGallinarum  concentrations  were  quantified.  At 1  dpc, birds  treated  with  unprotected  and encapsulated  phages  had significantly lower (P  <  0.05) SalmonellaGallinarum concentrations(4.36 ± 0.20and 5.05 ± 0.22 logCFU/g, respectively) than those found in untreated birds (5.71 ± 0.13). Likewise,  at4  dpc, <em>Salmonella </em>Gallinarum concentrationsin  ceca  of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 3.26 ± 0.62 and 4.02 ± 0.15 log  CFU/g,  respectively)  than  those  found  in untreated  birds(4.65  ±  0.08log  CFU/g). A second trial was conducted with higher challenge doses (1 mL at 1× 109CFU) and an additional treatment including a mixture (1:1) of unprotected and encapsulated phages. At1dpc, <em>Salmonella</em> Gallinarum concentrations  in the ceca  of  birds  treated  with unprotected  phages,  encapsulated phages, and a mixture of unprotected  and encapsulated phages  were significantly lower(4.28 ± 0.11, 3.72 ± 0.40, and 3.81 ± 0.36log CFU/g, respectively) than found in those of untreated birds (5.26 ± 0.19log CFU/g). At 2 dpc, concentrations of<em> Salmonella </em>Gallinarumin the ceca of birds treated  with  unprotected,  encapsulated,  and  a mixture  of  unprotected  and  encapsulated  phages were significantly  lower  (P  <  0.05; 4.31  ±0.53, 3.96  ±0.61,  and 4.38  ±  0.44logCFU/g, respectively) than those found in the ceca of untreated birds (5.72 ± 0.27logCFU/g).However, no significant differences were found in concentrations of <em>Salmonella</em> Gallinarum in the ceca of birds treated with encapsulated phages versus those treated with unprotected phagesor a mixture of   encapsulated   and   unprotected   phages.   Similarly,   at   4   dpc, <em>Salmonella </em>Gallinarum concentrations in the ceca  of  birds  treated  with unprotected  phages, encapsulated  phages,  and  a mixture of unprotected and encapsulated phages were significantly lower (3.17 ± 0.45, 3.56 ± 0.51, and 3.81 ± 0.54log CFU/g, respectively) than found in those of untreated birds (5.79 ± 0.08log CFU/g). At  7  d  post-challenge,  concentrations of <em>Salmonella</em> Gallinarum in  the  ceca  of  birds treated  with mixture  of  unprotected  and  encapsulated phages(2.40  ±  0.55log  CFU/g)  were significantly lower (P  <  0.05) than  those  found  in the ceca  of  untreated  birds(7.08  ±  0.19log CFU/g). Similarly,  concentrations of<em> Salmonella</em> Gallinarum  in the  ceca of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 4.29 ± 0.39and 4.60 ± 0.37 log  CFU/g,  respectively)  than  those  found  in  untreated  birds.  Taken  together,  these  data indicate that <em>Salmonella </em>Gallinarum infections could be controlled with phage-based treatments. Additionally, the use of a mixture of unprotected and encapsulated phages may be more effective, presumably  by  allowing  unprotected  phages  to  act  immediately  in  the  proximal  gastrointestinal tract  (GIT;  e.g.,  crop)  with  encapsulated  phages  having  greater  activity  once  released  from capsules in the distal small intestine. While no deleterious effects of the phages were observed on the chickens themselves, continuing studies should more comprehensively assess host-response to phage treatment including potential impact on microbial communities throughout the chicken GIT.</p>

Tato práce se zabývá strukturní studií dvou proteinů: proteinu Tail morphogenetic protein H (TmpH) bakteriofága 812, který napadá Zlatého stafylokoka (Staphylococcus aureus) a hexamerinu z potemníka (Tribolium castaneum). S. aureus je jedním z nejvíce rezistentních patogenů způsobující onemocnění s vysokou morbiditou a mortalitou. Bakteriofág 812 je schopen infikovat a lyzovat 95 % kmenů S. aureus a má potenciální využití ve fágové terapii. Protein TmpH je součástí virionu tohoto fága. V rámci této práce bylo připraveno několik plazmidů nesoucích gen TmpH, které byly použity pro rekombinantní expresi proteinu v buňkách E. coli BL21(DE3). Protein byl vyčištěn afinitní a gelovou chromatografií. Pro čistý protein byly optimalizovány krystalizační podmínky. Hexamerin je nejhojnějším proteinem larev a kukel hmyzu s dokonalou proměnou. V průběhu metamorfózy hexamerin slouží jako zdroj aminokyselin. V rámci této práce byl hexamerin izolován z kukel potemníka T. castaneum. Pro stanovení struktury hexamerinu byly použity dvě metody: rentgenová krystalografie a kryo-elektronová mikroskopie. Byly optimalizovány podmínky pro růst krystalů a vypěstovány krystaly vhodné pro sběr difrakčních dat. Nicméně struktura hexamerinu byla rychleji vyřešena kryo-elektronovou mikroskopií s rozlišením 3.2 . Znalost struktury hexamerinu umožní pochopení jeho funkce v regulaci vývoje hmyzu s dokonalou proměnou.

The evolution and adaptation of microorganisms is so rapid it can be seen in the time frame of days. The root cause for their evolution comes from selective environmental pressures that see organisms with beneficial mutations survive otherwise deadly encounters or outperform members of its population who fail to adapt. This does not always result in strict improvement of the individual as in the case of antibiotic resistant bacteria who often display fitness tradeoffs to avoid death (see Reviews [1-3]). For example, when an ampicillin resistance gene (ampC) containing plasmid that is occasionally found in the wild was transformed into S. typhimurium the bacteria had slower growth and impaired invasiveness [4]. In another example, capreomycin use with mycobacteria resulted in lower binding of the drug to the ribosome through mutations in rRNA methylase TlyA 16S rRNA, which decreases the overall fitness of the mycobacteria [5]. The evolutionary interactomes between bacteria and antibiotics do not end there, as antibiotic resistant bacteria often accumulate compensatory mechanisms to regain fitness. These range in effect with some altering individual cellular pathways and others having systemic affects [1]. My work has focused on the intersection of diabetes and related antibiotic resistant bacterial infections. Diabetes is one of the leading health issues in the United States, with over 10% of the adult population and over 26% of the elderly diagnosed (American Diabetes Association) [6]. Herein I further characterize the molecular pathways involved in diabetes, through the study of PAS kinase (PASK) function. PAS kinase is a serine-threonine protein kinase which regulates the pathways disrupted in diabetes, namely triglyceride accumulation, metabolic rate (respiration), adiposity and insulin production and sensitivity [7-9]. In this study I specifically focus on the effects of PAS kinase and its substrate, USF1/Cbf1p, and how their altered metabolic deficiencies can be suppressed using yeast cells. Through this study I further characterized the molecular function of USF1/Cbf1p through the identification of putative co-transcriptional regulators, identify novel genes involved in the regulation of respiration, and uncover a function or a previous uncharacterized protein, Pal1p. Part of the diabetes healthcare challenge results from the wide range of diseases that are associated with diabetes, including obesity [10, 11], renal failure [12, 13], neuropathies and neurodegeneration [14, 15], endocrine dysfunctions [16, 17], and cancers [18]. In addition, diabetes is a leading cause of lower limb amputations, due to poor circulation and the prevalence of ulcers [19-21], many of which are antibiotic resistant [22-25]. Phage therapy, based on the administration of bacterial viruses, is a viable option for the treatment of these diseases, with our lab recently isolating bacteriophages for several clinical cases. In the second half of my thesis, I present the study of the adaptation of bacteriophages to their hosts as well as report contributions of local ecology to their evolution.

No description available.

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No description available.

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.