Dissertationen zum Thema „Bacteriophage, Staphylococcic“

Staphylococcus aureus est l’un des principaux agents étiologiques des infections suppuratives superficielles et profondes ainsi que des syndromes liés à l’action de toxines. Paradoxalement, cette bactérie est un agent commensal qui est présent sur la peau ainsi que dans les cavités nasales notamment. Cela permet de considérer cette bactérie comme un organisme colonisant commensale. Les bases génétiques expliquant la différence entre une bactérie pathogène et une bactérie commensale reste inconnues. En utilisant la technique Optical Maps sur des souches de S. aureus isolées de plaies de pieds diabétiques avec différents niveau de virulence, nous avons pu montrer l’existence d’un prophage insérés dans le génome des souches colonisantes et absent des souches infectantes. Le phage, nommé ROSA, est localisé dans un hotspot d’insertion de phage NM2. Il est aussi localisé en amont du locus isd qui est requis pour l’assimilation du fer essentiel à la bactérie dans sa phase pathogène. Le phage ROSA inactive la voie isd en dérégulant l’activité du régulateur transcriptionnel majeur Fur en absence de fer. Il réduit aussi la virulence de ces souches sur les 2 modèles de virulence (Le ver C. elegans et le Zebrafish). L’expulsion du phage ROSA restaure la régulation du locus isd par Fur et la production de sidérophores en absence de Fer, la formation du biofilm et la virulence des souches. La mutation du gène Fur nous a permis de déduire que le phage ROSA affectait les bactéries de manière indépendante de Fur. Enfin, nous avons étudié la prévalence des souches colonisantes sur les plaies de pieds diabétiques. Nous avons observé que 20% des souches présentait l’insertion ROSA et 89% appartenait au complexe clonal CC8. Les souches colonisantes, avec leur niveau bas de virulence, devraient faire l’objet de détection dans le but de rationnaliser l’utilisation des antibiotiques et ainsi lutter contre l’apparition de bactéries multirésistantes aux antibiotiques
Staphylococcus aureus is an opportunistic bacterium capable of causing a wide range of severe diseases when it gains access to underlying tissues. Paradoxically, this causative pathogen is a common inhabitant of the skin microflora and colonizes the nares and other human mucosa, and as such, may be considered as a commensal colonizing organism. The genetic basis for the differences in pathogenic/colonizing potential is unknown. By performing optical maps comparisons of a collection of S. aureus strains of defined virulence potential isolated from diabetic foot ulcers at different stages, we brought to light a prophage present in colonizing-causing bacteria. The phage, namely ROSA, was localized in a hotspot region NM2 near the locus isd, the main iron surface determinant that transport iron across the bacterial wall. It induces a deregulation of the activity of the transcriptional regulator Fur involving the biofilm formation of the bacteria in response to low iron environment. It reduced also significantly the virulence of the strain in two in vivo models (the nematode C. elegans and the zebrafish). The expulsion of the phage restored the regulation of the locus isd, the siderophore production, the biofilm formation and the virulence of the strain. The mutation of the fur gene within the colonizing strain enabled us to determine that the phage ROSA affect the the bacteria in a Fur-independent manner. Finally we determined the prevalence of these colonizing strains in skin and soft tissue infections (diabetic foot ulcers). We observed that 20% (39/195) of the strains harboured this insertion and 89% belonged to the clonal complex CC8. This colonizing strain by its low virulence potential must be detected in the aim to contribute to a sounder use of antibiotic treatment, an important point in front of the increase of multidrug resistant bacteria

Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Acquisition of a superantigen pathogenicity island (SaPI) significantly increases virulence in Staphylococcus aureus. Horizontal transfer of SaPIs occurs at high frequency and depends upon a helper bacteriophage, either through direct infection or SOS-mediated induction of a lysogen. SaPIs hijack the packaging machinery of the helper phage, leading to the formation of SaPI-containing transducing particles that can introduce the pathogenicity island into neighboring SaPI-negative cells. All SaPIs contain a conserved core of genes, some of which are co-transcribed as an operon and encode functions involved in helper exploitation. The goal of this study was to more fully understand the intricate relationships between the SaPI elements and their helper bacteriophages, specifically any regulatory crosstalk that might occur between them. We demonstrated phage-host interactions in 80 and 80α, and SaPI1 and SaPIbov1-mediated crosstalk with helper phage 80α. The phage Sri protein was shown to be a bi-functional protein that both derepresses SaPI1 and interferes with host chromosome replication. Incoming SaPI1 experiments showed that SaPI1 modulates the levels of the N-terminal part of orf14 mRNA. Induction experiments using the 80α ΔrinA phage as a genetic tool, reveal several new phage genes that SaPI1 targets for expression modulation. Finally, a novel SaPI1 interference mechanism was identified. In an 80α ΔrinA mutant, which cannot activate its late operon, SaPI1 can directly turn on expression of the packaging and structural genes in a noncanonical manner, initiating from the 2nd gene in the operon, the large terminase subunit.

The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009.
Title from PDF title page (viewed Jan. 26, 2010). Additional advisors: Asim K. Bej, Gail E. Christie, Peter E. Prevelige, Jr., R. Douglas Watson. Includes bibliographical references.

Staphylococcus aureus is a common bacterium found on the skin and mucosal membranes of about 20% of the population. S. aureus growth on the skin is harmless, but if it bypasses the skin it can causes life-threatening diseases such as pneumonia, meningitis, bacteremia, and sepsis. Antibiotic-resistant strains of S. aureus, called Methicillin Resistant S. aureus (MRSA), are resistant to most antibiotics except vancomycin. However, vancomycin resistant strains of MRSA are becoming more common. In this study, 12 phages were isolated capable of infecting human S. aureus and/or MRSA strains. Five phages were discovered through mitomycin C induction of prophages and seven phages were found through enrichment of environmental samples. Primary S. aureus strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates. S. aureus isolates were tested for susceptibility to oxacillin in order to determine methicillin-resistance. Experiments were performed to assess the host range and killing potential of newly discovered phage. The M1M4 phage had the broadest host range and lysed 12% of the S. aureus strains that were tested. The host ranges were reinforced by spectrophotometric assay data which showed a reduction in bacterial optical density of 1.3 OD600. The phages were used to decontaminate MRSA from fomites (glass and cloth) and successfully reduced colony forming units by 1-2 logs, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

Thesis (Ph. D.)--Illinois State University, 1995.
Title from title page screen, viewed May 12, 2006. Dissertation Committee: Radheshyam K. Jayaswal (chair), Brian J. Wilkinson, Alan J. Katz, Herman E. Brockman, Anthony J. Otsuka. Includes bibliographical references (leaves 112-118) and abstract. Also available in print.

Staphylococcal phage 80α can serve as a helper bacteriophage for a family of mobile genetic elements called Staphylococcus aureus pathogenicity islands (SaPIs). The prototype island, SaPI1, is able to hijack the 80α capsid assembly process and redirect capsid formation to yield smaller, phage-like transducing particles carrying SaPI DNA. Capsid size redirection is accomplished through two SaPI1-encoded gene products, CpmA and an alternate scaffold protein, CpmB. The normal 80α scaffold and the SaPI1 CpmB scaffold share a small block of conserved residues at their C-termini, several of which had been shown to be essential for CpmB function. This led to the hypothesis that the C-termini of both the phage and SaPI scaffolds interact in similar ways with the major capsid protein. The goal of this study was to test this hypothesis and to identify the amino acid residues at the capsid-scaffold interface, using a genetic approach.

The growing threat of drug- resistant Staphylococcus aureus (S. aureus) infections mandates the need to develop novel, effective and alternative antibacterial therapeutics. Despite infection prevention and control measures, methicillin resistant S. aureus (MRSA)-associated deaths reached 11,285 in 2011 in the USA (CDC, 2013). To counteract the threat of drug resistant S. aureus, we sought to construct and characterize a novel therapeutic based on the display of lytic antibacterial enzymes, termed endolysins. These endolysins were displayed on the surface of a specific bacterial virus, bacteriophage (phage), to generate lytic antibacterial nanoparticles. Endolysins are encoded individually by a variety of double-stranded DNA phage and act to direct host lysis and escape. These lytic enzymes confer a high degree of host specificity that could potentially substitute for, or be combined with, antibiotics in the treatment of gram-positive drug resistant bacterial infections such as MRSA. In this study, modular domains of the phage-encoded endolysin K enzyme, specific to S. aureus, were displayed on the capsid surface of phage lambda () via fusion with the λ major head (capsid) protein, gpD. The constructs of displayed endolysins were prepared in various combinations to maximize the functional display of gpD::X fusions on the phage. Phage lysates were generated, collected and purified and lysis was investigated by adding to fresh lawns of MRSA, vancomycin resistant S. aureus (VRSA) and bovine S. aureus. Phage preparations did not readily confer cell lysis, likely due to poor incorporation of the fusions onto the functional phage capsid. We purified the fusion proteins (gpD::X) and tested them for their lytic activity. We noted that the activity of the gpD::LysK protein was not impaired by the fusion and demonstrated lysis on live and dead (autoclaved) bovine S. aureus. In contrast to gpD::LysK, the gpD::CHAP protein fusion, expressing only the CHAP catalytic domain of endolysin K showed variable results in the lysis assays that we performed. In the zymogram assay, gpD::CHAP did not elicit any observable lysis on live bovine S. aureus cells, but did effectively lyse dead cells of the same S. aureus species; however, it was highly lytic in the inhibition assay on bovine S. aureus. The CHAP::gpD protein fusion, which is the CHAP domain fused to the N terminus of gpD only showed its ability to inhibit bovine S. aureus growth on the inhibition assay. The fusion of endolysin K or its CHAP domain to gpD protein does not seem to interfere with lytic activity, but may result in recalcitrant gpD fusions that compromise the ability to efficiently decorate the phage capsid. Suggestions for improved fusion capsid integration are discussed.

Tese de doutoramento em Engenharia Biomédica
The increasing numbers of healthcare-associated infections are alarming and have resulted in higher morbidity and mortality rates, which ultimately are a burden to the public health systems. Remarkably, it was reported that around 65% of these infections were caused by biofilms. Biofilms are microbial communities attached to a surface and surrounded by exopolymeric substances that form a complex structure. Nowadays it is known that the majority of microbes live within a structured biofilm and not as free-floating organisms. Staphylococcus epidermidis is the most predominant species isolated in central line-associated bloodstream infections and central venous catheter-associated bloodstream infections, and second in surgical site infections. Due to the high tolerance of S. epidermidis biofilms to antibiotics, alternative strategies are required to treat these infections. Currently, the use of bacteriophages (phages) is seen as an important strategy to combat pathogenic organisms. Phages are viruses that specifically infect bacteria and are the most abundant entities on our planet, possessing several advantages for therapy, in comparison with antibiotics. Due to the current advances in phage therapy and the almost absence of virulent phages described that infect S. epidermidis, the purpose of this study was the isolation and characterization of new S. epidermidis-specific phages and the assessment of their suitability for controlling S. epidermidis biofilms. Two different phages were isolated from raw effluents of wastewater treatment plants, phiIBB-SEP1 (SEP1) and vB_SepS_SEP9 (SEP9). Phage SEP1 was shown to have typical features of the Myoviridae genus, Twortlikevirus. This phage was shown to be S. epidermidis-specific, being able to kill all the 41 strains tested and in 4 hours, was able to reduce cell viability around 6-log. Phage SEP9 was shown to possess a set of morphological and genomic features that highlights its uniqueness in comparison to all phages described to date. In vitro experiments showed that besides the presence of an integrase, this phage was unable to lysogenize, presenting a virulent behavior. In addition, SEP9 is characterized by a high lytic spectrum being able to infect 28 out of the 38 S. epidermidis strains tested. Furthermore, the presence of a depolymerase suggests its use for therapeutical purposes. As Twortlikevirus are referred as the best candidates for phage therapy, and SEP1 demonstrated good infective properties, this phage was further used in this study. The efficacy of SEP1 in controlling planktonic and biofilm cells of S. epidermidis was assessed. This phage was highly effective in killing planktonic stationary-phase cells. This rare feature makes it a promising biofilm controller due to the heterogeneous physiology of cells within a biofilm. However, this phage was not able to reduce the number of viable cells of 24 h biofilms neither reduce its total biomass. In order to determine if this inefficacy was a consequence of the metabolic activity of the cells within the biofilm and/or a matter of the biofilm matrix, the efficacy of SEP1 was tested against modulated biofilms with distinct metabolic activities (higher or less proportions of dormant cells). The results showed that distinct metabolic activities did not affect phage efficacy. The reduction of activity against planktonic cells upon addition of biofilm matrix, and the efficacy demonstrated by SEP1 against scrapped biofilm cells suggested that the biofilm matrix hinders phage efficacy. Thus, it was hypothesized that S. epidermidis biofilm matrix confers a protective effect against phage predation most probably due to the interaction of phages with PNAG, limiting their access to the cells. Another goal of this thesis was to provide a deeper knowledge about phage endolysins (lysins), which are phage-encoded peptidoglycan hydrolases that are expressed at the end of the phage infection and are promising antimicrobial agents. For that, a database containing all derived lysins from fully sequenced phages were organized and characterized in silico. New catalytic domains and cell binding domains were encountered and described and a new nomenclature was provided, in order to standardize the annotation of endolysins. Finally, the cloning and expression of the first specific endolysins derived from S. epidermidisspecific phages were attempted. Although several approaches to clone these proteins were tested, they were shown to be difficult to express, having solubility or folding problems. In conclusion, the work described in this thesis contributed significantly to increase the knowledge about S. epidermidis phages and endolysins. Furthermore, new insights on phagebiofilm interactions were obtained, which opens new challenges for this field of research.
O numero crescente de infecções associadas à prestação de cuidados de saúde é alarmante e tem resultado em grandes taxas de morbilidade e mortalidade com avultados custos associados. Uma grande percentagem destas infeções, cerca de 65%, são causadas por biofilmes. Biofilmes são definidos como comunidades microbianas aderidas a uma superfície e envoltas numa matriz exopolimérica, formado uma estrutura tridimensional e complexa. Atualmente reconhece-se que a maioria dos microrganismos vive em biofilmes estruturados e não como células livres. Staphylococcus epidermidis é o principal agente etiológico de infecções da corrente sanguínea associadas ao uso de cateteres e em particular ao cateter venoso central, e a segunda espécie mais isolada em infecções no local cirúrgico. Devido ao facto dos biofilmes desta espécie terem elevada tolerância aos antibióticos, é necessário o desenvolvimento de formas alternativas para tratamento destas infecções. Atualmente, o uso de bacteriófagos (fagos) é visto como uma importante estratégia para combater bactérias patogénicas. Os fagos são vírus que infectam especificamente bactérias e são considerados as entidades mais abundantes no nosso planeta. Estes agentes apresentam várias vantagens terapêuticas quando comparados com antibióticos. Devido aos avanços atuais na terapia fágica e à quase ausência de fagos virulentos descritos que infectam S. epidermidis, o objetivo deste estudo foi o isolamento e caracterização de novos fagos específicos para S. epidermidis e à avaliação da sua adequação para o controlo de biofilmes. Dois fagos diferentes foram isolados a partir de efluentes brutos de estações de tratamento de águas residuais – phiIBB-SEP1 (SEP1) e vB_SepS_SEP9 (SEP9). O fago SEP1 mostrou ter características típicas do género Twortlikevirus pertencente à família Myoviridae. Este fago demonstrou ser específico para S. epidermidis, sendo capaz lisar todas as 41 estirpes testadas. Adicionalmente apresentou a capacidade de, em 4 horas, reduzir a viabilidade das células em cerca de 6 log. O outro fago isolado, SEP9, possui características morfológicas e genómicas que destacam a sua singularidade em relação a todos os fagos descritos até ao momento. Experiências in vitro mostraram que este fago tem um comportamento virulento, não obstante apresentar uma integrase no seu genoma. O espectro lítico alargado deste fago (infecta 28 das 38 estirpes de S. epidermidis testadas), bem como a presença de uma depolimerase sugerem a sua utilização para fins terapêuticos. Por outro lado, como os fagos pertencentes ao género Twortlikevirus são referidos como os melhores candidatos para a terapia fágica, o fago SEP1 foi seccionado para ensaios de infecção em biofilmes. A eficácia do fago SEP1 foi avaliada no controlo de células de S. epidermidis quer em fase planctónica quer em biofilmes. Este fago foi altamente eficaz em matar células em fase estacionária. Esta característica rara faz com que seja um fago promissor para controlo de biofilmes, devido à heterogeneidade fisiológica das células dentro de um biofilme. No entanto, este fago foi incapaz de reduzir a biomassa total de biofilmes de 24 horas e de reduzir o seu número de células viáveis. De modo a determinar se esta ineficácia foi uma consequência da atividade metabólica das células dentro do biofilme e/ou resultado da influência da matriz do biofilme, a eficácia do fago SEP1 foi testada em biofilmes modulados com atividades metabólicas diferentes (maior ou menor proporção de células dormentes). Os resultados mostraram que a eficácia fago não é afetada por atividades metabólicas distintas. A redução da atividade detectada contra as células planctónicas, após adição de matriz de biofilme, bem como a eficácia demonstrada contra as células de biofilmes raspados, sugerem que a matriz do biofilme dificulta a eficácia fago. Assim, admitiu-se a hipótese de que a matriz do biofilme de S. epidermidis confere um efeito protetor contra a predação fágica provavelmente devido à interação de fagos com o PNAG, limitando o seu acesso para as células. Outro objetivo deste trabalho foi aprofundar o conhecimento sobre endolisinas (lisinas) fágicas que são hidrolases do peptidoglicano, expressas no final da infecção fágica e que estão descritas como agentes antimicrobianos promissores. Para isso, foi construída uma base de dados contendo todas as lisinas derivadas de fagos totalmente sequenciados através de dados in silico e todas as lisinas foram anotadas. Este trabalho permitiu a identificação de novos domínios catalíticos e domínios de ligação à parede celular. Adicionalmente, com base nos resultados obtidos foi proposta uma nova nomenclatura para uniformizar a anotação de endolisinas. Finalmente, fizeram-se várias tentativas de clonagem e expressão das primeiras lisinas e depolimerases provenientes de fagos específicos para S. epidermidis. Apesar das várias abordagens utilizadas, as proteínas demonstraram ser de difícil expressão, pela baixa solubilidade ou provavelmente pela dificuldade em adquirir a conformação correta. Em conclusão, o trabalho descrito nesta tese contribuiu significativamente para aumentar o conhecimento sobre fagos de S. epidermidis fagos e endolisinas. Além disso, foram obtidos novos conhecimentos sobre interações fago-biofilme, o que poderá abrir novos desafios para esta área de investigação.
FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project “BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER.
The project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, Ref. FCOMP-01-0124-FEDER-027462 and the FCT grant SFRH/BD/66166/2009.

Chronic rhinosinusitis (CRS) is a debilitating condition characterised by critical inflammation of the mucosa of the nose and paranasal sinuses. Effecting up to 14% of the world’s population CRS severely impacts a patient’s quality of life. The aetiology of CRS is complex and relatively undefined encompassing a multitude of contributing factors. Bacterial infection is one factor thought to play a role in the pathogenesis of CRS. More specifically biofilm forms of the bacterial species Staphylococcus aureus have been shown to negatively influence post-operative progression. Current practice treatment strategies often fail to remove biofilms from the mucosa of the nose. It is therefore of import to develop novel anti-biofilm therapeutics. Our understanding of the epidemiology of S. aureus infections and biofilms in CRS is also limited. Increasing our epidemiological knowledge would help in the development of effective treatment strategies against recurrent infections. Investigation into the epidemiology of S. aureus infections was undertaken by collecting S. aureus isolates from mucous and biofilm structures of CRS patients. The clonal type of each isolate was then compared to the other isolates using pulse field gel-electrophoresis. Results of this study indicated that the majority of patients experiencing recurrent infections maintained the same clonal type. Furthermore the study suggested that long-term antibiotic therapy in some patients can lead to the development of bacterial antibiotic resistance. Therefore development of a novel antibacterial therapy outside of antibiotics is required. A potential anti-biofilm therapy both eliminating and preventative in nature is the application of bacteriophage. Bacteriophage (phage) are viruses that specifically target, infect and destroy bacterial cells. Initially in vitro study was undertaken to assess the anti-biofilm activity of a phage cocktail specific for S. aureus (CT-SA) using a minimal biofilm eradication assay plate. S. aureus isolates from CRS patients were grown to mature biofilm form and treated with CT-SA for 48hrs. Following treatment biofilm biomass was determined by staining bacteria with a Live/Dead BacLight stain, imaging the biofilm using confocal scanning laser microscopy and determining biofilm biomass using software COMSTAT2. Results showed CT-SA significantly reduced S. aureus biofilms of susceptible strains. Results also indicated that a cocktail of phage was superior to use of a single phage as it reduced the frequency of bacterial resistant to the phage treatment. Following on from in vitro work, the safety and efficacy of CT-SA was assessed in vivo using a sheep model of frontal sinusitis associated with S. aureus infections. CT-SA was also combined with ethylenediaminetetraaceticacid (EDTA) to observe if these therapies would synergise. Results indicated both CT-SA and EDTA were safe for short term topical application to the sinus regions. Furthermore both CT-SA and EDTA individually significantly reduced S. aureus biofilm levels in the frontal sinus, but were not seen to synergise. Work conducted in this thesis has helped lead towards development of a novel anti-S. aureus biofilm agent. Future translation of CT-SA to a clinical trial setting may not only reduce or remove S. aureus biofilm from CRS patient noses but also improve their symptomatology and quality of life.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015.

Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

With the global increasing and spreading of antibiotic-resistant microorganisms, there is a need to develop strategies capable of inactivating plantonic and biofilm-forms of pathogenic microorganisms that causes untreatable and mortal infections. Antimicrobial photodynamic therapy (aPDT) is an alternative approach capable of combating microorganisms independently of their resistance profile. Althought this technique presents great results and advantages, the neutral and monocationic photosensitizers (PS) do not usually kill efficiently gram-negative bacteria and fungi, and their synthetic preparation are usually expensive and laborious. In this The results of these experiments demonstrated that FORM is efficient on inactivating planktonic forms of bacteria, fungi and viruses and that when combined with KI was clearly more effective to inactivate all the microorganisms. This combination allows also to destroy efficiently the preformed biofilms of bacteria and fungi and avoided also the formation of E. coli and S. aureus biofilms, contrarily to that observed with FORM but without KI. The use of FORM combined with KI allowed to reduce PS concentration and the treatment time which will promote to transpose the aPDT to the clinic or environment fields.context, it is needed to develop new approaches that can improve the antimicrobial effect of a PS and simultaneously to allow the decrease of the applied PS concentration and also of the treatment time. Recent studies have reported a enhancer effect on antimicrobial photoinactivation by the combined used of some PS and potassium iodide (KI), an inorganic salt. These studies have always shown potentiating effect of KI for the tested PS. The main goal of this work was, in a first phase, to achieve an insight into the KI potentiation effect on diferent groups of PS; tetraarylporphyrins positively charged at meso (including a formulation consisting of five cationic porphyrins - FORM) or β-pyrrolic positions and non-porphyrinic dyes, using a bioluminescent Escherichia coli as bacterial model. The results of these studies pointing out that the presence of KI can enhance the aPDT killing effect of some PS, but this enhancement is not general for all PS. The comparison of the obtained results with the ones from the literature allowed to confirm that the enhance effect of KI is related to the generation of 1O2 by PS, PS structure (charge number and charge position), aggregation behavior and its affinity for the external structures of the microorganisms. In a second phase, the aPDT effect of the FORM (easy to prepare when compared with their corresponding porphyrins that constitute the mixture in the pure form) and of its combined effect with KI (100mM) on planktonic and biofilm forms of a broad-spectrum of microorganisms. Therefore, this study was performed on the free and biofilms forms of gram-negative and gram-positive bacteria: E. coli resistant to chloramphenicol and ampicillin, and Staphylococcus aureus resistant to methicillin (MRSA), of the fungi Candida albicans as well as on the free-form of a T4 like bacteriophage as a model of human viruses.
O aumento e disseminação da resistência a antimicrobianos torna necessário o procura de novas estratégias para combater infeções não tratáveis e muitas vezes mortais, causadas por microrganismos quer na forma planctónica quer organizados em biofilmes. A terapia fotodinâmica antimicrobiana tem-se revelado uma alternativa capaz de inativar microrganismos, independentemente do seu perfil de resistência aos antimicrobianos convencionais. Contudo, a baixa eficácia de fotosensibilizadores (PS) neutros e aniónicos na inactivação de bactérias de gram-negativo e fungos, bem como os elevados custos de produção e purificação associados, tem exigido à comunidade científica encontrar moleculas ou coadjuvantes que permitam aumentar a fotoinactivação microbiana (aPDT) e com diminuição dos custos associados quer pela diminuição da quantidade de composto aplicado quer pela diminuição do tempo de tratamento. Estudos recentes, demonstraram que o iodeto de potássio (KI), um sal inorgânico, é capaz de potenciar o efeito de alguns PS porfirínicos e não porfirínicos catiónicos e não catiónicos. Estes estudos mostraram sempre nos PS testados um efeito potenciador do KI. Assim, o presente trabalho teve como objectivo determinar as características dos PS que influenciam o efeito potenciador do KI, usando para tal PS com características diferentes. Para tal, foram realizados ensaios de aPDT usando como modelo uma estirpe de Escherichia coli bioluminescente. Nestes ensaios o KI (50 mM e 100 mM) foi testado na presença de diferentes PS porfirínicos catiónicos substituídos quer em posições meso (incluindo uma formulação constituída por uma mistura de cinco porfirinas - FORM) quer nas posições ‐pirrólicas, e PS não porfirínicos [Azul de metileno (MB), Rosa Bengal (RB), Azul de Toluidina (TBO), Violeta Cristal (CV) e Verde de Malachita (MG)]. Os resultados evidenciam que o KI é capaz de potenciar o efeito antibacteriano da maior parte dos PS testados, permitindo reduzir ainda o tempo de irradiação necessário para produzir o efeito fotodinãmico desejado. Contudo, não se observa o efeito potenciador do KI quando combinado com todos os PS porfirínicos e não-porfirínicos. Uma comparação dos resultados obtidos com os da literatura permite confirmar que o efeito potenciador quando o sal KI é combinado com um PS depende da produção de oxigénio singleto (1O2) por parte do PS, da sua estrutura (número de cargas e a sua posição espacial), da sua tendência para agregar e da sua afinidade para com as estruturas externas dos microrganismos. Na segunda fase do trabalho pretendeu-se avaliar a capacidade da formulação porfirínica (FORM, de fácil preparação e obtenção comparativamente com as respectivos constituintes puros), e da combinação da FORM com KI (100 mM) na inativação de vários microrganismos (bactérias e fungos) quer na forma planctónica quer em biofilmes. Para tal, foram realizados ensaios com os seguintes microrganismos: E. coli resistente ao clorofenicol e à ampicilina (bactéria de gram-negativo), Staphyloccocus aureus resistente à meticilina (bactéria de gram-positivo), o fungo Candida albicans; quer na forma livre quer em biofilmes; bem como um bacteriófago tipo T4, utilizado como modelo de vírus humano. Os resultados obtidos mostraram que a FORM isoladamente é eficaz na inativação de bactéias, fungos e vírus na sua forma planctónica, e quando usada em combinação com o KI o seu efeito antimicrobiano é intensificado. A combinação da FORM com o KI foi também eficaz na destruição de biofilmes bacterianos e fúngicos, e evitou a formação de biofilmes bacterianos, o que não se verifica quando a FORM foi utilizada isoladamente. A utilização da FORM combinada com KI em aPDT permitiu reduzir a concentração de PS e o tempo de tratamento o que facilitará possíveis aplicações quer na clínica quer no ambiente.
Mestrado em Microbiologia