"Killing them softly" … challenges in the Bacillus subtilis spore inactivation by plasma sterilization

The elimination of bacterial endospores is absolutely essential in numerous fields, ranging from hospital hygiene, the food processing industry, all the way to the space industry. A major goal of space exploration is the search for signatures of life forms and biomolecules on other planetary bodies and moons in our solar system. The transfer of microorganisms or biomolecules of terrestrial origin to critical areas of exploration is of particular risk to impact the development and integrity of life-detection missions.1 Plasma sterilization is a promising alternative to conventional sterilization methods for spaceflight purposes. Due to their extraordinary resistance properties, spores of the Gram-positive bacterium Bacillus subtilis are used as biological indicators for decontamination studies to identify the relevant mechanism that leads to the rapid bacterial inactivation.1,3 Here, we present novel insights into the key factors involved in spore inactivation by low pressure plasma sterilization using a double inductively-coupled plasma reactor. (2,4) In order to standardize the assessment of inactivation efficiencies by plasma discharges, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of B. subtilis spore monolayers. We demonstrate that plasma discharges caused significant physical damage to spore surface structures as visualized by atomic force microscopy. A systematic analysis of B. subtilis spores lacking individual coat and crust layers - the first barrier to environmental influences – revealed the coat to be one of the contributing factors in the spore resistance to plasma sterilization. (2-4) Furthermore, we identified spore-specific and general protection mechanisms and DNA repair pathways during spore germination and outgrowth after plasma treatment, leading to a better understanding of the complex molecular mechanisms involved in the inactivation by plasma sterilization processes

Irreversible depletion of intestinal CD4+ T cells is associated with T cell activation during chronic HIV infection

HIV infection in the human gastrointestinal (GI) tract is thought to be central to HIV progression, but knowledge of this interaction is primarily limited to cohorts within Westernized countries. Here, we present a large cohort recruited from high HIV endemic areas in South Africa and found that people living with HIV (PLWH) presented at a younger age for investigation in the GI clinic. We identified severe CD4(+) T cell depletion in the GI tract, which was greater in the small intestine than in the large intestine and not correlated with years on antiretroviral treatment (ART) or plasma viremia. HIV-p24 staining showed persistent viral expression, particularly in the colon, despite full suppression of plasma viremia. Quantification of mucosal antiretroviral (ARV) drugs revealed no differences in drug penetration between the duodenum and colon. Plasma markers of gut barrier breakdown and immune activation were elevated irrespective of HIV, but peripheral T cell activation was inversely correlated with loss of gut CD4(+) T cells in PLWH alone. T cell activation is a strong predictor of HIV progression and independent of plasma viral load, implying that the irreversible loss of GI CD4(+) T cells is a key event in the HIV pathogenesis of PLWH in South Africa, yet the underlying mechanisms remain unknown

Obstructive jaundice: Studies on predictors of biliary infection and microbiological analysis in an HIV setting

Background. Early diagnosis of biliary infection is critical for timely antimicrobial therapy and biliary drainage. HIV infection may influence the spectrum and severity of biliary infection in an environment with a high HIV prevalence. Charcot’s triad has low sensitivity and higher specificity for biliary infection, and more sensitive markers are required.Objectives. To investigate possible predictors of biliary infection (bacteriobilia) and identify the microbiological spectrum in patients presenting with biliary obstruction to a tertiary institute in an environment with a high prevalence of HIV.Methods. Bile was assessed for infection at endoscopic retrograde cholangiopancreatography, percutaneous transhepatic cholangiography and surgery, and the roles of clinical/haematological factors, C-reactive protein (CRP) and procalcitonin (PCT) in determining biliary infection were evaluated. Results: One hundred and six patients with obstructive jaundice had a mean age of 52 years (range 21 - 58); most were female (74%), and 36 (34%) were infected with HIV, with a mean CD4 count of 495 cells/µL. Choledocholithiasis (53%), biliary strictures (21%) and head of pancreas tumour (8%) were the main aetiopathologies. Bile was obtained for microbial culture from 104 patients (98%), and 56 (54%) were infected. Gram-negative bacteria were most frequent (58%), and 2 HIV-infected patients had fungal infections (Candida albicans and Aspergillus fumigatus). Screening for endoscopy-associated infections revealed Pseudomonas aeruginosa. PCT was a poor predictor of bacterial infection, whereas CRP was a fair predictor.Conclusions. The majority of bacteria cultured were sensitive to ciprofloxacin or amoxicillin-clavulanate. Duodenoscopes were a potential source of Pseudomonas infection

Improved Plaque Assay Identifies a Novel Anti-Chlamydia Ceramide Derivative with Altered Intracellular Localization

Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C(16) as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C(16) acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C(16) represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections

Dual pro-drugs of 2 '-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus

We have previously reported the power of combining a 5′-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2′-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism

Explanatory case studies: Implications and applications for clinical research

Explanatory case study methodology has been used to research complex systems in the fields of business, public policy and urban planning, to name a few. While it has been suggested by some that this might be a useful way to progress complex research issues in health science research, to date, there has been little evidence of this happening. The purpose of this paper is to review the explanatory case study method and present a framework for understanding how it differs from other case study methods. A review of the literature is undertaken to ascertain to what extent this method is currently being used in health science research. Finally, a model for guiding the implementation of the explanatory case study method is proposed in order to promote quality, rigour and applicability of this approach within clinical settings

HIV infection drives interferon signaling within intestinal SARS-CoV-2 target cells

SARS-CoV-2 infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analyzed single-cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV-infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest coexpression of SARS-CoV-2 receptors ACE2, TMPRSS2, and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a substantial reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2-infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV-coinfected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV-infected individuals does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity, and vaccine responses remains unclear and requires further investigation

Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-HCV agents

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2′-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 μM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases