The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe. The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria. Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed. Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host. Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results. Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Virtually all interacting species (such as hosts and parasites) are embedded within diverse communities. However, evolutionary interactions are typically considered in a pairwise species framework. Although coevolutionary theory suggests that multiple species interactions may provide greater opportunities for diversification, the impacts of community diversity on coevolution have not been directly tested. In this thesis I synthesize the findings from recent experimental work to assess the effects of increased species diversity on the patterns and processes of host and parasite evolution. I then investigate the effects of parasite diversity on host-parasite population dynamics and evolution using the pathogen Pseudomonas aeruginosa and five lytic bacteriophage parasites in a brief evolution experiment. Parasite diversity was manipulated by assembling phage communities with different number of species. Phage communities suppressed host populations more rapidly but also showed reduced phage density, likely due to inter-phage competition. The evolution of resistance allowed rapid bacterial recovery that was greater in magnitude with increases in phage diversity. These results were then followed up via longer term experimental coevolution of the same host and parasite communities. Here the data showed that greater parasite diversity accelerates coevolutionary arms races and drives more diversification among lineages. Coevolution between hosts and parasite communities drove more successive increases in host resistance coupled with increasingly frequent selective sweeps at the genomic level. Consistent with this, the most rapidly evolving host genes under coevolution with parasite communities were those involved in various host resistance strategies. These results demonstrate, at phenotypic and genomic levels, how areas of high community diversity may be hotspots for rapid evolution in interacting, antagonistic species. Finally, In the face of escalating antibiotic resistance, there is now an urgent need to develop alternative antimicrobials, these results may be relevant to the application of phages as therapeutics and they are discussed in that context.

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

As antibiotic resistant bacterial strains are becoming more prevalent in healthcare settings, it is necessary to find alternative methods of detecting and treating these infections. One of the antibiotic resistant strains of interest is the carbapenem-resistant Enterobacteriaceae (CRE). CREs have the ability to evade some of the most potent antibiotics currently in use and employ carbapenemases to negate the effect of antibiotics. The three most common carbapenemase genes, found in carbapenem-resistant Enterobacteriaceae along with a gene found only in Escherichia coli were chosen to create a qPCR assay for rapid detection of resistant infections. The carbapenemase genes are KPC, VIM and NDM and the E. coli gene is uidA, a β-glucuronidase gene. Consensus sequences were obtained from each of the genes to account for the many variants of each gene. We were able to triplex the assay and test it against a library for twenty isolates varying by which gene they contain. Additional research has been conducted on the library of carbapenem-resistant Enterobacteriaceae using bacteriophages or phage. The Phage Hunters class isolated and identified twenty phage that infect K. pneumoniae. Out of the twenty phage, seven phage were able to effectively infect carbapenem-resistant K. pneumoniae.

No description available.

According to CDC, every year at least 2 million people are affected and 23,000 dies as a result of antibiotic resistance in U.S. It is considered one of the biggest threats to global health. More and more bacterial infections are becoming harder to treat. One such infection is fire blight, one of the most destructive disease of apple and pear trees. It is caused by bacteria Erwinia amylovora and its outbreaks have been known to destroy entire orchards in a single season. The conventional method of treatments includes use of antibiotics like streptomycin and oxytetracycline but the incidences like presence of multi-drug resistant bacteria in the mammals grazing in the fields have raised concerns. Phage therapy is considered one of the few ways available to combat bacterial resistance and prevent fire blight. In this method, a cocktail of highly lytic bacteriophages is prepared and sprayed on the trees at different time intervals. Bacteriophages are an “intelligent” drug. They multiply at the site of the infection until there are no more bacteria and then they are excreted back into the nature. These phenomena make them more efficient than an antibiotic, which kills all kind of bacteria including good bacteria and can be maintained in the environment for long periods of time. These qualities of bacteriophage have resulted in many commercially available phage therapies. The initial part of this research focuses on isolation, characterization and genomic comparison of bacteriophages that infect a plant pathogen E.amylovora of Erwiniaceae family of Enterobacteriales order. In this study, 28 novel bacteriophages were isolated, fully sequenced, characterized and grouped into seven families based on phage homology. To take this further, we characterized a novel jumbo family of bacteriophages that has a small burst size of 4.6-4.9 and are most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity. These bacteriophages are shown to infect Erwinia and Pantoea bacterial strains, but no infection of 9 other bacterial strains tested, was seen, under laboratory conditions. The results of this work provide an insight on special characteristics that makes bacteriophage so unique and adaptable. The final part of this research explores the enormous diversity of bacteriophages. In 2014 Grose and Casjens grouped 337 fully sequenced tailed phages into 56 diverse clusters (32 lytic and 24 temperate). We further expanded our current understanding of these clusters by performing the comprehensive analysis of genomes and proteomes of 1037 tailed bacteriophages, posted on GenBank. The results of this work provide insights into diversity and relatedness of bacteriophages and the data is posted on GenBank.

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

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.