Potential Application of Phage Therapy Against Pseudomonas aeruginosa Biofilm Infection in Cystic Fibrosis Patients

Majority of the microbial activity in humans is in the form of biofilms i.e. an Exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotics treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. This has lead to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ, only in the presence of susceptible hosts can be used as natural, self-limiting, and deeply penetrating antibacterial agents. The objective of this study is to identify effective phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAOl and the isogenic constitutively alginate-producing PD0300 strains.These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B and P182). Analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance but none of the sensitive strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the volume required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

THE IMPORTANCE OF MHC-INDEPENDENT NATURAL KILLER CELL RECEPTORS IN THE IMMUNOREGULATION OF MURINE PREGNANCY

Numerous leukocyte populations are essential for pregnancy success. Uterine natural killer (uNK) cells are chief amongst these leukocytes and represent a unique lineage with limited cytotoxicity but abundant angiokine production. They possess a distinct phenotype of activating and inhibitory receptors that recognize major histocompatibility complex (MHC) molecules, such as the killer immunoglobulin like receptors (KIRs; mouse Ly49), and MHC-independent activating receptors, including the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptor 1 (NCR1). While the roles of MHC-dependent receptors are widely addressed in pregnancy, MHC-independent receptors are relatively unstudied. This thesis investigated the roles of MHC-independent receptors in promotion of mouse pregnancy and characterized early leukocyte interactions in the presence and absence of NCR1. It was hypothesized that loss of MHC-independent receptors impairs uNK cell development resulting in aberrations in leukocyte function and decidual vasculature. Implantation sites from Ahr-/- and Ncr1Gfp/Gfp mice were assessed using whole mount in situ immunohistochemistry (WM-IHC) and histochemical techniques. Leukocyte interactions identified during preliminary WM-IHC studies were confirmed as immune synapses. The novel identification of immune synapses in early mouse pregnancy compelled further examination of leukocyte conjugates in wildtype C57BL/6 and Ncr1Gfp/Gfp mice. In Ahr-/- and Ncr1Gfp/Gfp mice, receptor loss resulted in reduced uNK cell diameters, impaired decidual vasculature, and failures in spiral artery remodeling. Ahr-/- mice had severe fertility deficits whereas Ncr1Gfp/Gfp mice had increased fetal resorption indicating differing receptor requirements in pregnancy success. NCR1 loss primarily affected uNK cell maturation and function as identified by alterations in granule ultrastructure, lytic protein expression, and angiokine production. Leukocyte conjugates were frequent in early C57BL/6 decidua basalis and included uNK cells conjugating first with antigen presenting cells and then with T cells. Overall conjugate formation was reduced in the absence of NCR1, but specific uNK cell conjugations were unaffected by receptor loss. While KIR-MHC interactions are associated with numerous pregnancy complications in humans, the role of other uNK cell receptors are not well characterized. These results illustrate the importance of MHC-independent receptors in uNK cell activation during early pregnancy in mice and encourage further studies of pregnancy complications that may occur independently of maternal KIR-MHC contributions.

Intragenus competition between coccolithoviruses: an insight on how a select few can come to dominate many

Viruses are a major cause of coccolithophore bloom demise in both temperate and sub-temperate oceanic regions. Most infection studies on coccolithoviruses have been conducted with a single virus strain, and the effect of intragenus competition by closely related coccolithoviruses has been ignored. Here we conducted combined infection experiments, infecting Emiliania huxleyi CCMP 2090 with two coccolithoviruses: EhV-86 and EhV-207 both simultaneously and independently. EhV-207 displayed a shorter lytic cycle and increased production potential than EhV-86 and was remarkably superior under competitive conditions. Although the viruses displayed identical adsorption kinetics in the first 2 h post infection, EhV-207 gained a numerical advantage as early as 8 h post infection. Quantitative polymerase chain reaction (PCR) revealed that when infecting in combination, EhV-207 was not affected by the presence of EhV-86, whereas EhV-86 was quickly out-competed, and a significant reduction in free and cell-associated EhV-86 was seen as early as 2 days after the initial infection. The observation of such clear phenotypic differences between genetically distinct, yet similar, coccolithovirus strains, by flow cytometry and quantitative real-time PCR allowed tentative links to the burgeoning genomic, transcriptomic and metabolic data to be made and the factors driving their selection, in particular to the de novo coccolithovirus-encoded sphingolipid biosynthesis pathway. This work illustrates that, even within a family, not all viruses are created equally, and the potential exists for relatively small genetic changes to infer disproportionately large competitive advantages for one coccolithovirus over another, ultimately leading to a few viruses dominating the many.

Intragenus competition between coccolithoviruses: an insight on how a select few can come to dominate many

Viruses are a major cause of coccolithophore bloom demise in both temperate and sub-temperate oceanic regions. Most infection studies on coccolithoviruses have been conducted with a single virus strain, and the effect of intragenus competition by closely related coccolithoviruses has been ignored. Here we conducted combined infection experiments, infecting Emiliania huxleyi CCMP 2090 with two coccolithoviruses: EhV-86 and EhV-207 both simultaneously and independently. EhV-207 displayed a shorter lytic cycle and increased production potential than EhV-86 and was remarkably superior under competitive conditions. Although the viruses displayed identical adsorption kinetics in the first 2 h post infection, EhV-207 gained a numerical advantage as early as 8 h post infection. Quantitative polymerase chain reaction (PCR) revealed that when infecting in combination, EhV-207 was not affected by the presence of EhV-86, whereas EhV-86 was quickly out-competed, and a significant reduction in free and cell-associated EhV-86 was seen as early as 2 days after the initial infection. The observation of such clear phenotypic differences between genetically distinct, yet similar, coccolithovirus strains, by flow cytometry and quantitative real-time PCR allowed tentative links to the burgeoning genomic, transcriptomic and metabolic data to be made and the factors driving their selection, in particular to the de novo coccolithovirus-encoded sphingolipid biosynthesis pathway. This work illustrates that, even within a family, not all viruses are created equally, and the potential exists for relatively small genetic changes to infer disproportionately large competitive advantages for one coccolithovirus over another, ultimately leading to a few viruses dominating the many.

Characterisation of algicidal bacterial exometabolites against the lipid-accumulating diatom Skeletonema sp.

Microalgae are of increasing interest due to their occurrence in the environment as harmful algal blooms and as a source of biomass for the production of fine and bulk chemicals. A method for the low cost disruption of algal biomass for environmental remediation or bioprocessing is desirable. Naturally-occurring algal lytic agents from bacteria could provide a cost-effective and environmentally desirable solution. A screen for algal lytic agents against a range of marine microalgae has identified two strains of algicidal bacteria isolated from the coastal region of the Western English Channel. Both strains (designated EC-1 and EC-2) showed significant algicidal activity against Skeletonema sp. and were identified as members of Alteromonas sp. and Maribacter sp. respectively. Characterisation of the two bioactivities revealed that they are small extracellular metabolites displaying thermal and acid stability. Purification of the EC-1 activity to homogeneity and initial structural analysis has identified it as a putative peptide with a mass of 1266. amu.

Characterisation of algicidal bacterial exometabolites against the lipid-accumulating diatom Skeletonema sp.

Microalgae are of increasing interest due to their occurrence in the environment as harmful algal blooms and as a source of biomass for the production of fine and bulk chemicals. A method for the low cost disruption of algal biomass for environmental remediation or bioprocessing is desirable. Naturally-occurring algal lytic agents from bacteria could provide a cost-effective and environmentally desirable solution. A screen for algal lytic agents against a range of marine microalgae has identified two strains of algicidal bacteria isolated from the coastal region of the Western English Channel. Both strains (designated EC-1 and EC-2) showed significant algicidal activity against Skeletonema sp. and were identified as members of Alteromonas sp. and Maribacter sp. respectively. Characterisation of the two bioactivities revealed that they are small extracellular metabolites displaying thermal and acid stability. Purification of the EC-1 activity to homogeneity and initial structural analysis has identified it as a putative peptide with a mass of 1266. amu.

Comparative genomic approaches for investigating evolution in the microbial and viral populations of the rumen

The rumen is home to a diverse population of microorganisms encompassing all three domains of life: Bacteria, Archaea, and Eukarya. Viruses have also been documented to be present in large numbers; however, little is currently known about their role in the dynamics of the rumen ecosystem. This research aimed to use a comparative genomics approach in order to assess the potential evolutionary mechanisms at work in the rumen environment. We proposed to do this by first assessing the diversity and potential for horizontal gene transfer (HGT) of multiple strains of the cellulolytic rumen bacterium, Ruminococcus flavefaciens, and then by conducting a survey of rumen viral metagenome (virome) and subsequent comparison of the virome and microbiome sequences to ascertain if there was genetic information shared between these populations. We hypothesize that the bacteriophages play an integral role in the community dynamics of the rumen, as well as driving the evolution of the rumen microbiome through HGT. In our analysis of the Ruminococcus flavefaciens genomes, there were several mobile elements and clustered regularly interspaced short palindromic repeat (CRISPR) sequences detected, both of which indicate interactions with bacteriophages. The rumen virome sequences revealed a great deal of diversity in the viral populations. Additionally, the microbial and viral populations appeared to be closely associated; the dominant viral types were those that infect the dominant microbial phyla. The correlation between the distribution of taxa in the microbiome and virome sequences as well as the presence of CRISPR loci in the R. flavefaciens genomes, suggested that there is a “kill-the-winner” community dynamic between the viral and microbial populations in the rumen. Additionally, upon comparison of the rumen microbiome and rumen virome sequences, we found that there are many sequence similarities between these populations indicating a potential for phage-mediated HGT. These results suggest that the phages represent a gene pool in the rumen that could potentially contain genes that are important for adaptation and survival in the rumen environment, as well as serving as a molecular ‘fingerprint’ of the rumen ecosystem.

Adaptive Mechanisms of an Estuarine Synechococcus based on Genomics, Transcriptomics, and Proteomics

Picocyanobacteria are important phytoplankton and primary producers in the ocean. Although extensive work has been conducted for picocyanobacteria (i.e. Synechococcus and Prochlorococcus) in coastal and oceanic waters, little is known about those found in estuaries like the Chesapeake Bay. Synechococcus CB0101, an estuarine isolate, is more tolerant to shifts in temperature, salinity, and metal toxicity than coastal and oceanic Synechococcus strains, WH7803 and WH7805. Further, CB0101 has a greater sensitivity to high light intensity, likely due to its adaptation to low light environments. A complete and annotated genome sequence of CB0101 was completed to explore its genetic capacity and to serve as a basis for further molecular analysis. Comparative genomics between CB0101, WH7803, and WH7805 show that CB0101 contains more genes involved in regulation, sensing, and stress response. At the transcript and protein level, CB0101 regulates its metabolic pathways, transport systems, and sensing mechanisms when nitrate and phosphate are limited. Zinc toxicity led to oxidative stress and a global down regulation of photosystems and the translation machinery. From the stress response studies seven chromosomal toxin-antitoxin (TA) genes, were identified in CB0101, which led to the discovery of TA genes in several marine Synechococcus strains. The activation of the relB2/relE1 TA system allows CB0101 to arrest its growth under stressful conditions, but the growth arrest is reversible, once the stressful environment dissipates. The genome of CB0101 contains a relatively large number of genomic island (GI) genes compared to known marine Synechococcus genomes. Interestingly, a massive shutdown (255 out of 343) of GI genes occurred after CB0101 was infected by a lytic phage. On the other hand, phage-encoded host-like proteins (hli, psbA, ThyX) were highly expressed upon phage infection. This research provides new evidence that estuarine Synechococcus like CB0101 have inherited unique genetic machinery, which allows them to be versatile in the estuarine environment.

Análise proteômica de Chromobacterium violaceum: acúmulo estacionário e diferencial após exposição à luz UVC

Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28ºC that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism

Intento de suicidio por intoxicación con organosfosforado bajo administración parenteral (síndrome compartimental): reporte de un caso

INTRODUCCIÓN: La ideación e intento de suicidio constituyen entidades emergentes para el profesional de la salud mental; la ingesta de organofosforados (OF) es una de las maneras más frecuentes de intoxicación debido a su disponibilidad y fácil accesibilidad en nuestro medio (Ecuador); es común que quienes recurren a estas sustancias con fines auto líticos lo hacen por vía oral. Encontrar que alguien utilice la vía parenteral con esta finalidad es poco frecuente y es escasa la literatura al respecto, motivo por el cual se considera de importancia la publicación del siguiente caso. OBJETIVO: Describir los aspectos psicopatológicos del paciente que presentó un intento de suicidio con uso de organofosforados administrados por vía parenteral. MÉTODO: revisión, presentación y análisis de caso clínico. RESULTADOS: El paciente de 32 años fue internado en el Hospital Homero Castanier Crespo de la ciudad de Azogues (Ecuador) tras la severidad de sus lesiones causadas por la administración parenteral de OF; la valoración del estado mental del paciente llevó al diagnóstico de intento de suicidio psicodisplásico con trastorno del estado de ánimo y trastorno límite de la personalidad; fue diagnosticado además de absceso en miembro superior derecho y síndrome compartimental en miembro superior izquierdo, por lo que fue necesaria la realización del drenaje y fasciotomía respectivamente. CONCLUSIÓN: Los envenenamientos por plaguicidas como OF son los métodos más utilizados, se conoce que la ingesta de los OF es común, más no la administración parenteral como en el presente caso, que implicó un tratamiento tanto del área física y de la esfera mental

Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock

UNLABELLED Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

From field to fermentation: characterisation and application of non-dairy cultures in dairy foods

This thesis investigates the phenotypic and genotypic diversity of non-dairy L. lactis strains and their application to dairy fermentations. A bank of non-dairy lactococci were isolated from grass, vegetables and the bovine rumen. Subsequent analysis of these L. lactis strains revealed seven strains to possess cremoris genotypes which did not correlate with their observed phenotypes. Multi-locus sequence typing (MLST) and average nucleotide identity (ANI) highlighted the genetic diversity of lactis and cremoris subspecies. The application of these non-dairy lactococci to cheese production was also assessed. In milk, non-dairy strains formed diverse volatile profiles and selected strains were used as adjuncts in a mini Gouda-type cheese system. Sensory analysis showed non-dairy strains to be strongly associated with the development of off-flavours and bitterness. However, microfluidisation appeared to reduce bitterness. A novel bacteriophage, ɸL47, was isolated using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. The phage, a member of the Siphoviridae, possessed a long tail fiber, previously unseen in dairy lactococcal phages. Genome sequencing revealed ɸL47 to be the largest sequenced lactococcal phage to date and owing to the high % similarity with ɸ949, a second member of the 949 group. Finally, to identify and characterise specific genes which may be important in niche adaptation and for applications to dairy fermentations, comparative genome sequence analysis was performed on L. lactis from corn (DPC6853), the bovine rumen (DPC6853) and grass (DPC6860). This study highlights the contribution of niche specialisation to the intra-species diversity of L. lactis and the adaptation of this organism to different environments. In summary this thesis describes the genetic diversity of L. lactis strains from outside the dairy environment and their potential application in dairy fermentations.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.