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No description available.

Methicillin-resistant Staphylococcus aureus (MRSA) are opportunistic commensal bacteria that are evolving to become increasingly resistant to antimicrobial agents. Resistance is due to acquisition of resistance genes encoded by mobile genetic elements (MGEs) but little is known about the gene transfer and conditions influencing this process in clinically important MRSA isolates. In this thesis I showed that populations of MRSA colonizing isolates vary phenotypically in antimicrobial resistance (AMR) and genotypically in MGE profiles. A potential mechanism of horizontal gene transfer (HGT) in colonized patients is generalized transducing bacteriophage and we detected free bacteriophage in nasal swabs. AMR and genetic variability have implications for diagnostics, epidemiology, antimicrobial stewardship and selective pressures driving evolution of MRSA populations. All clinical strains harbour at least one prophage which are potentially capable of HGT. Bacteriophage are thought to be induced by environmental conditions including exposure to antibiotics, and 30% of hospitalized patients who are carriers are prescribed antibiotics. Exposure of clinical MRSA isolates to sub-inhibitory concentration of range of anti microbials resulted in increased phage induction and resistance gene transfer, however the ratio of virulent particles to transducing particles differed for each antimicrobial using the new technology of droplet digital peR. Exposure of colonizing MRSA strains to specific antimicrobials may lead to enhanced horizontal transfer of antimicrobial resistance potentially leading to fully resistant MRSA. MRSA isolates collected from patient admitted to St Georges NHS Healthcare Trust between 1999-2003 versus 2012-2013 showed that the recent isolates more easily induced phages and transferred resistance genes more effectively, especially upon induction with ciprofloxacin, suggesting that MRSA is evolving over time to transfer AMR genes more effectively. I developed a new in vitro model mimicking in vivo colonization conditions in the human host by co-culturing two distinct isolates from MRSA carriers in human plasma. Transfer of DNA via HGT occurred at high frequency in plasmas from all 8 donors. However, the presence of certain resistance genes had a fitness cost in some plasma but not others. Frequent horizontal transfer of antimicrobial resistance and other genes between isolates during colonization may play an important role in host-pathogen adaptation and evolution.

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The globally increasing risk of acquiring hospital-acquired infections (HAIs) has attracted much attention in recent years. In this study, an in-house designed kilohertz (kHz)-driven atmospheric pressure non-thermal plasma (APNTP) jet has been investigated for its potential application as a new and promising alternative decontamination and sterilization method for several potential clinical applications. APNTP has been shown to exert a significant antibiofilm activity against wide range of clinical Pseudomonas aeruginosa isolates. Phenotypic and genotypic factors mediate an elevated tolerance of Pseudomonas aeruginosa biofilms to APNTP, evident in observed variation in susceptibility profiles between mucoid and nonmucoid isolates following plasma exposure. In addition, APNTP significantly inactivated several biofilm forming persister Pseudomonas aeruginosa strains. APNTP has also shown to have marked antibiofilm activity against Burkholderia cenocepacia biofilms. This variability in sensitivity was correlated with biofilm biomass as well as catalase activity in each case. Isolates with greater biofilm biomass and higher catalase activity exhibited significantly enhanced tolerance to plasma exposure. A preliminary study was conducted to assess the potential of APNTP as adjuvant therapy with other conventional antimicrobial agents that frequently used for control of Pseudomonas aeruginosa infection, and whose activity are known to be attenuated in the presence of biofilm matrix components. Pre-exposure of a 48 hour biofilm to APNTP increased the sensitivity of Pseudomonas aeruginosa biofilm to the tested antimicrobial agents. Finally, Non-thermal plasma has a significant virucidal activity against MS2 bacteriophages which are frequently used as a surrogate for human Noroviruses. More than seven log10 reductions were achieved in nine minutes exposure to 0.75% oxygen/He APNTP. Virucidal activity was affected by percentage oxygen concentration in the helium (He) carrier gas where activity increased with increasing oxygen concentration up to 0.75% indicating a role for reactive oxygen species (ROS) in viral inactivation

Lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria, is implicated as the key factor in the development of the Systemic Inflammatory Response Syndrome (SIRS). LPS can arise from an underlying bacteraemia, but given that the majority of patients with SIRS have no detectable bacteraemia, then LPS derived from the gut must be considered. <I>Bacteroides</I> species outnumber the enterobacteria such as <I>E.coli</I> in the gut by approximately 1000-fold. Although <I>Bacteroides</I> LPS is less endotoxic, by simple arithmetic there must be as much biological potential from the LPS of <I>Bacteroides</I> as from <I>E.coli</I>. This thesis re-examines the biological activity of <I>Bacteroides</I> LPS and its possible role in the development of SIRS. LPSs were extracted from seven <I>Bacteroides</I> species by three different techniques: the phenol-water (PW), the phenol-chloroform-petroleum (PCP) and Triton-Mg<SUP>2+</SUP>. The biological activity of these <I>Bacteroides</I> LPSs was compared to that of an <I>E.coli</I> O18K<SUP>-</SUP> LPS control. In general, <I>Bacteroides</I> LPSs prepared by the PW method were found to have a significantly higher activity in a mouse lethality model, LAL assay, TNF and IL-8 induction assays, than LPS extracted by the PCP or Triton methods. <I>Bacteroides</I> LPS extracted by the PCP method had consistently low activity in all assays. LPS from <I>B.fragilis</I> NCTC 9343 and <I>B.caccae</I> had a consistently higher activity than LPS from <I>B.vulgatus</I> and <I>B.thetaiotaomicron</I> in most assays. Differences in activity between <I>B.fragilis</I> NCTC 9343 LPS grown in different media was seen. The PW method selected for greater amounts of carbohydrate and KDO and the PCP the least. Further information from sub-population studies, Percoll profiles, chemotype on PAGE and chemical analysis failed to account for differences in biological activity between extraction methods and <I>Bacteroides</I> species.

This project has focused on the interaction of <I>B. cepacia</I> with neutrophils, the predominant immune cell in CF. As <I>B. cepacia</I> is resistant to neutrophil non-oxidative killing mechanisms, respiratory burst induction by <I>B. cepacia</I> was investigated using another CF pathogen, <I>Pseudomonas aeruginosa,</I> as a comparison. In general, non-opsonised CF isolates of <I>B. cepacia</I> induced little respiratory burst activity. By contrast, <I>P. aeruginosa</I> strains induced a greater range of responses, with a subset of strains inducing considerable activity. Opsonisation with specific immune sera increased neutrophil responses to both <I>B. cepacia</I> and <I>P. aeruginosa</I>. However, in view of the cleavage of opsonins and opsonin receptors within the CF lung, opsonisation may have little impact on host defences to <I>B. cepacia</I> within the respiratory tract. Lipopolysaccharide (LPS) extracted from <I>B. cepacia</I> was shown to upregulate neutrophil expression of complement receptor 3 (CR3) and to prime respiratory burst responses to fMet-Leu-Phe and to whole bacteria. Significantly LPS from the major epidemic strain of <I>B. cepacia, </I>ET12, increased neutrophil respiratory burst responses to <I>P. aeruginosa</I> but not to the ET12 strain itself. Thus it was speculated that <I>B. cepacia</I> may establish a foothold in the <I>P. aeruginosa</I>-infected lung by selectively upregulating immune responses to <I>P. aeruginosa</I>. In view of the development of environmental <I>B. cepacia</I> as biocontrol agents, environmental and clinical isolates were compared for potential virulence determinants. Few obvious differences were found between CF, non-CF clinical and environmental strains. Significantly LPS from two environmental strains of <I>B. cepacia </I>primed neutrophil responses to a similar degree as LPS from CF strains of <I>B. cepacia</I>. A surprising result was that an environmental <I>B. cepacia</I> strain was less effectively cleared in both CF and non-CF mice than the ET12 strain. This project has provided evidence to support the hypothesis that <I>B. cepacia</I> colonisation is associated with a marked and damaging immune response in CF patients.

Plasmodium falciparum var genes encode the PfEMPI family of variant antigens expressed on the surface of infected erythrocytes. PfEMPlmediates the adhesion of the parasitized erythrocyte to the venular endothelium and to uninfected erythrocytes. PfEMP 1 variants use a range of different host molecules as receptors in these adhesive interactions. The expression of different PfEMPI variants may directly affect the clinical course of malaria infection by defining the distribution and intensity of parasite adhesion in the microvasculature of various organs within the host. PfEMPI is a major focus of the host immune response, and the slow onset of clinical immunity in endemic areas may be explained by the gradual accumulation of effective responses to a wide range of PfEMPI variants present in the local parasite population. It has been hypothesised that the immune response to PfEMPI may act to stratify the parasite population into co-circulating 'strains' defined by discrete, non-overlapping repertoires of var genes. Here, the DBL1 region of 56 var gene variants from 6 genetically distinct cocirculating Sudanese parasites have been cloned and sequenced. Sequence comparisons suggest that recombination and gene duplication are important mechanisms in the generation of new var variants. A model of the basic structural framework of DBL1 sequences is described and "sequence subtypes" identified within variable regions of sequence. Phylogenetic analysis of the Sudanese and other var sequences from GenBank fail to support the 'strain' model for Sudanese P. falciparum and suggests that the global pool of var genes is finite. 40 Sudanese variants have been expressed as GST-fusion proteins with yields of varying quantity and degree of degradation. 10 of the recombinant proteins have been tested against a cohort of sera in a pilot ELISA, and the role of anti-PfEMPI immune responses in the development of clinical immunity is discussed.

No description available.

This study investigated systemic and local cell mediated immunity to HPV in patients with cutaneous warts in order to understand the basis for their persistence or regression. The aim of the first part of the project was to examine HPV-specific lymphoproliferative responses from the peripheral blood of patients with cutaneous HPV infection. Proliferative T cell responses towards purified HPV <I>in vitro</I> were demonstrated in some individuals but frequently responses were weak or non existent. Patients whose warts were regressing (or had fully resolved) did not show increased lymphoproliferative responses towards purified HPV <I>in vitro</I> compared with patients whose warts showed no change. HPV-specific T cell clones were not obtained either directly from peripheral blood mononuclear cells stimulated with purified HPV, or after expansion of these cells with anti-CD3 and anti-CD28 antibodies. Any HPV-specific modulation of immunity may be more apparent locally in the lesion, than in systemic circulation. Therefore three studies were conducted in order to investigate local immunity in cutaneous warts. In the first, an immunohistochemical approach was taken. The density of Langerhans' cells was reduced in the epidermis of warts compared with normal skin. Epidermal keratinocytes did not express intercellular adhesion molecule-1 (ICAM-1) in cutaneous warts, but the vascular expression of ICAM-1 and E-selectin was increased in the dermis of warts compared with normal skin. This may account in part for the observed increase in numbers of T cells in the dermis of warts compared with normal skin. However, few T cells were found in the epidermis of warts, compared with an inflammatory skin disease such as psoriasis.