The role of population dynamics in driving bacterial persistence

Bacterial persister cells can survive a broad range of antibiotics despite being genetically susceptible. This trait is clinically relevant, yet evolutionary explanations for persistence remain mysterious. Significant focus has been given to single cell studies with the aim of identifying genes responsible for persistence. However, other studies have indicated that persistence may not be genetically-driven at all but instead simply a by-product of other cellular processes. Moving away from the single cell approach, more needs to be done to consider persistence at the population level – strikingly, the proportion of persisters is maintained at a remarkably low level across populations and even species, between 0.0001-1%. Uncovering the conditions that promote persistence could tell us more about the regulatory mechanisms behind this trait. In my thesis, I explore the population-wide dynamics behind elevated levels of bacterial persistence in Pseudomonas aeruginosa. I first look at how changing bacterial population dynamics (growth rate and cell density) affect persistence by varying the culture inoculum density to detect the effect of growth, while also controlling for age of culture. This reveals inoculum density dependent effects where persistence peaks are not always associated with stationary phase as is commonly assumed in the literature, but perhaps with the number of divisions of individual cells. To expand this work, I replicate the study with, Escherichia coli, the most widely studied species in the persister research field. I find that E. coli displays different persistence patterns to P. aeruginosa, with limited inoculation density-dependent responses. While persistence patterns varied between species, extracellular ATP, which can be an indicator of cell lysis, was consistent with persistence patterns across both species, presenting intriguing avenues to explore in future. Lastly, I look at an unusual phenomenon I came across in my early work where I saw antibiotic-induced growth occurring in P. aeruginosa which I term a ‘stress divide’. Here, I look at potential links with another antibiotic survival mechanism: heteroresistance, a less understood survival trait whereby a bacterial subpopulation is heterogeneously resistant to the antibiotic treatment. This introduces the concept of looking at how heterogenous phenotypic traits such as persistence and heteroresistance could work synergistically to promote population survival. In summary, by testing a broad range of growth conditions, I have found that persistence is not always greatest in stationary phase, is species-specific, and can work in tandem with traits like heteroresistance to enhance survival. My thesis highlights the significance of examining the impact of population dynamics on persistence, considering the mechanisms driving persistence on a species-by-species basis

Prevalence of ticks and tick-borne pathogens:<i>Babesia </i>and <i>Borrelia </i>species in ticks infesting cats of Great Britain

In a study of tick and tick-borne pathogen prevalence, between May and October 2016, 278 veterinary practices in Great Britain examined 1855 cats. Six-hundred and one cats were found to have attached ticks. The most frequently recorded tick species was Ixodes ricinus (57.1%), followed by Ixodes hexagonus (41.4%) and Ixodes trianguliceps (1.5%). Male cats, 4–6 years of age living in rural areas were most likely to be carrying a tick; hair length and tick treatment history had no significant association with attachment. For cats that were parasitized by ticks in large urban areas, I. hexagonus was the most frequent species recorded. Molecular analysis was possible for 541 individual tick samples, others were too damaged for analysis; Babesia spp., and Borrelia burgdorferi sensu lato were identified in 1.1% (n\ua0=\ua06) and 1.8% (n\ua0=\ua010) of these, respectively. Babesia spp. included Babesia vulpes sp. nov./Babesia microti-like (n\ua0=\ua04) in I. hexagonus and Babesia venatorum (n\ua0=\ua02) in I. ricinus. Borrelia burgdorferi s.l. species included Borrelia garinii (n\ua0=\ua06) and Borrelia afzelii (n\ua0=\ua04). The majority of B. burgorferi s.l. cases were found in I. ricinus, with B. afzelii in one I. hexagonus nymph. No Borrelia or Babesia spp. were present in I. trianguliceps. To determine a true prevalence for ticks on cats, practices that only submitted questionnaires from cats with ticks and practices that submitted fewer than 5 returns per week were removed; amongst those considered to have adhered strictly to the collection protocol, feline tick prevalence amongst cats that had access to the outdoors was 6.6%. These results show that ticks can be found on cats throughout Great Britain, which harbour a range of species of Babesia and B. burgdorferi s.l. and that cats, particularly in green spaces within urban areas, may form an important host for I. hexagonus, a known vector of pathogens

Acute panuveitis with hypopyon in Crohn's disease secondary to medical therapy: a case report

<p>Abstract</p> <p>Background</p> <p>A case report to highlight the association between rifabutin and hypopyon</p> <p>Methods</p> <p>A 56 year old male presented with a one day history of blurred vision in his right eye. He had an established diagnosis of Crohn's disease which was in remission following treatment with rifabutin and clarithromycin. A brisk anterior uveitis with hypopyon and a mild vitritis was detected in the right eye. The acute inflammatory episode resolved following treatment with topical corticosteroids and withdrawal of rifabutin.</p> <p>Results</p> <p>The presence of hypopyon is atypical in uveitis associated with inflammatory bowel disease. The association between rifabutin treatment and hypopyon uveitis is well recognised in Mycobacterium avium paratuberculosis. However, use of rifabutin in the management of Crohn's disease is controversial and not widely known to an ophthalmic readership.</p> <p>Conclusion</p> <p>This report highlights the importance of keeping abreast of novel therapeutic developments in systemic conditions likely to be encountered in ophthalmology.</p

<i>Anaplasma phagocytophilum</i>, <i>Bartonella</i> spp., haemoplasma species and <i>Hepatozoon</i> spp. in ticks infesting cats:A large-scale survey

Background: Ticks derived from cats have rarely been evaluated for the presence of pathogens. The aim of this study was to determine the prevalence of Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks collected from cats in the UK. Methods: Five hundred and forty DNA samples extracted from 540 ticks collected from cats presenting to veterinarians in UK practices were used. Samples underwent a conventional generic PCR assay for detection of Hepatozoon spp. and real-time quantitative PCR assays for detection of Anaplasma phagocytophilum and three feline haemoplasma species and a generic qPCR for detection of Bartonella spp. Feline 28S rDNA served as an endogenous internal PCR control and was assessed within the haemoplasma qPCR assays. Samples positive on the conventional and quantitative generic PCRs were submitted for DNA sequencing for species identification. Results: Feline 28S rDNA was amplified from 475 of the 540 (88.0%) ticks. No evidence of PCR inhibition was found using an internal amplification control. Of 540 ticks, 19 (3.5%) contained DNA from one of the tick-borne pathogens evaluated. Pathogens detected were: A. phagocytophilum (n = 5; 0.9%), Bartonella spp. (n = 7; 1.3%) [including Bartonella henselae (n = 3; 0.6%) and Bartonella clarridgeiae (n = 1; 0.2%)], haemoplasma species (n = 5; 0.9%), "Candidatus Mycoplasma haemominutum" (n = 3; 0.6%), Mycoplasma haemofelis (n = 1; 0.2%), "Candidatus Mycoplasma turicensis" (n = 1; 0.2%), Hepatozoon spp. (n = 2; 0.4%), Hepatozoon felis (n = 1; 0.2%) and Hepatozoon silvestris (n = 1; 0.2%). Conclusion: These data provide important information on the prevalence of tick-borne pathogens in ticks infesting cats, with the identification of haemoplasma species, A. phagocytophilum, H. felis and Bartonella spp. (including B. henselae and B. clarridgeiae). This study also documents the first report of H. silvestris in ticks collected from domestic cats

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo

This is the final published versionAvailable from Ivyspring International Publisher via the DOI in this recordInvasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy and bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant 1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.This work was supported by the European Union Seventh Framework Programme FP7/2007-2013 under Grant 602820, the Deutsche Forschungsgemeinschaft (Grant WI3777/1-2 to SW), and the Werner Siemens Foundation. We thank Sven Krappman for use of the A. fumigatustdTomato strain, and acknowledge the Imaging Centre Essen (IMCES) for assistance with optical imaging of lungs

Minimum energy conformations of DNA dimeric subunits: Potential energy calculations for dGpdC, dApdA, dCpdC, dGpdG, and dTpdT

Minimum energy conformations have been calculated for the deoxydinucleoside phosphates dGpdC, dApdA, dCpdC, dGpdG, and dTpdT. In these potential energy calculations the eight diheldral angles and the sugar pucker were flexible parameters. A substantial survey of conformation space was made in which all staggred combination of the dihedral angles ω′,ω, and ψ, in conjunction with C(2′)-endo pucker, were used as starting conformers for the energy minimization. The most important conformations in the C(3′)-endo-puckering domain have ψ = g+; ω′,ω = g−,g−(A-form),g+, g+, and g−,t. With C(2′)-endo-type pucker the most important conformations have ψ = g+; ω′,ω =g-,g-(B-form) and g+,t; and ψ =t; ω′,ω =g-,t(Watson-Crick from) and t,g+ (skewed). Stacked bases are a persistent feature of the low-energy conformations, the g+ conformer being an exception. Freeing the suger pucker allowed this conformation to become low energy, with C(3′)-exo pucker. It also caused other low-energy forms, such and the Waston-Crick conformation, to become more favourable. Conformation flexibility in the sugar pucker and in ψ, as well as the ω′,ω angle pair, is indicated for the dimeric subunits of DNA

Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study

Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study

An atmospheric radiation model for Cerro Paranal. I. The optical spectral range

The Earth's atmosphere affects ground-based astronomical observations. Scattering, absorption, and radiation processes deteriorate the signal-to-noise ratio of the data received. For scheduling astronomical observations it is, therefore, important to accurately estimate the wavelength-dependent effect of the Earth's atmosphere on the observed flux. In order to increase the accuracy of the exposure time calculator of the European Southern Observatory's (ESO) Very Large Telescope (VLT) at Cerro Paranal, an atmospheric model was developed as part of the Austrian ESO In-Kind contribution. It includes all relevant components, such as scattered moonlight, scattered starlight, zodiacal light, atmospheric thermal radiation and absorption, and non-thermal airglow emission. This paper focuses on atmospheric scattering processes that mostly affect the blue (< 0.55 mum) wavelength regime, and airglow emission lines and continuum that dominate the red (> 0.55 mum) wavelength regime. While the former is mainly investigated by means of radiative transfer models, the intensity and variability of the latter is studied with a sample of 1186 VLT FORS1 spectra. For a set of parameters such as the object altitude angle, Moon-object angular distance, ecliptic latitude, bimonthly period, and solar radio flux, our model predicts atmospheric radiation and transmission at a requested resolution. A comparison of our model with the FORS1 spectra and photometric data for the night-sky brightness from the literature, suggest a model accuracy of about 20%. This is a significant improvement with respect to existing predictive atmospheric models for astronomical exposure time calculators.Comment: 23 pages, 21 figures, accepted for publication in A&

Spectrophotometric analysis of lipid used to examine the phenology of the tick <i>Ixodes ricinus</i>

Ticks store lipid as an energy souce, which depletes progressively between blood meals. The amount of lipid and rate of lipid depletion can be used as a good indicator of the feeding history and assist in explaining the phenology of tick populations. However, existing gravimetric approaches to lipid measurement are relatively imprecise. To improve our ability to accurately measure lipid accumulation and metabolism in individual ticks, a microquantity colorimetric sulfophosphovanillan method of lipid estimation was standardised and used to explore the seasonal variations in the lipid content of I. ricinus nymphs.Lipid values for field-derived questing ticks, collected by blanket dragging, varied between 5-45 μg and clear patterns of lipid depletion were demonstrated under controlled laboratory conditions. For field populations collected monthly over two years, the results indicate that two different cohorts of nymphs enter the questing tick population in autumn and spring, with very few nymphs joining the population in summer.The data illustrate the seasonal change in lipid content of nymphal ticks, reflecting their feeding history and highlight the utility of the spectrophotometric technique for analysis of lipid in ticks in helping to improve our understanding of seasonal activity patterns

Ground Water Chemistry Changes before Major Earthquakes and Possible Effects on Animals

Prior to major earthquakes many changes in the environment have been documented. Though often subtle and fleeting, these changes are noticeable at the land surface, in water, in the air, and in the ionosphere. Key to understanding these diverse pre-earthquake phenomena has been the discovery that, when tectonic stresses build up in the Earth’s crust, highly mobile electronic charge carriers are activated. These charge carriers are defect electrons on the oxygen anion sublattice of silicate minerals, known as positive holes, chemically equivalent to O− in a matrix of O2−. They are remarkable inasmuch as they can flow out of the stressed rock volume and spread into the surrounding unstressed rocks. Travelling fast and far the positive holes cause a range of follow-on reactions when they arrive at the Earth’s surface, where they cause air ionization, injecting massive amounts of primarily positive air ions into the lower atmosphere. When they arrive at the rock-water interface, they act as •O radicals, oxidizing water to hydrogen peroxide. Other reactions at the rock-water interface include the oxidation or partial oxidation of dissolved organic compounds, leading to changes of their fluorescence spectra. Some compounds thus formed may be irritants or toxins to certain species of animals. Common toads, Bufo bufo, were observed to exhibit a highly unusual behavior prior to a M6.3 earthquake that hit L’Aquila, Italy, on April 06, 2009: a few days before the seismic event the toads suddenly disappeared from their breeding site in a small lake about 75 km from the epicenter and did not return until after the aftershock series. In this paper we discuss potential changes in groundwater chemistry prior to seismic events and their possible effects on animals