Phage infection reinstates antibiotic sensitivity in MDR Pseudomonas aeruginosa: A study on phage and bacterial evolution

The emergence of antibiotic resistance among bacterial pathogens is a significant public health threat affecting humans worldwide. In Europe, Pseudomonas aeruginosa contributes to almost 9% of overall multi-drug-resistant (MDR) infections. Alternative methods for controlling MDR pathogens have been explored for several decades. Bacteriophage therapy is one of the oldest and most efficient alternative solutions. The study described in this thesis began with the isolation and characterization of 25 MDR P. aeruginosa clinical strains and eigth novel lytic phages. The investigation disclosed the infection with two phage isolates, PIAS and PAPSZ1, led to the sequential appearance of phage-resistant colonies with two phenotypes (green and brown). We examined the evolutionary basis for the two types of mutants and uncovered phage mutants capable of infecting green mutants. Simultaneously we also learned that PIAS phage infected the host via the OrpM-MexXY system involved in drug efflux. Thus, the PIAS-resistant mutants decreased the minimum inhibitory concentrations (MIC) for several non-effective antibiotics. After this new insight into the evolutionary arms race between hosts and phages, we decided to use this window to comprehensively eradicate mutants by treating MDR strain with previously resistant antibiotics combined with PIAS phage. The in vitro study with PIAS phage-antibiotic combination completely prevented the formation and growth of mutants. We tested the same strategy in an in vivo rescue experiment in the mouse lung infection model, when combined with PIAS phage and fosfomycin. The combination therapy saved 75% of the animals. Later, we used PAPSZ1 phage to investigate whether phage mutants can suppress bacterial resistance. We isolated multiple PAPSZ1 mutants after a continuous infection cycle, which can block or suppress bacterial resistance and mutant formation and broaden the host specificity of the phages. Phages like PIAS and PAPSZ1 offer a unique window that can exploit to eradicate MDR bacteria. This study highlights the importance of preliminary and detailed examinations of phage-host bacterium interactions preceding the application of a given phage. The experimental data in this thesis shows that studying phage-host bacterium interactions and coevolution will help to utilize phage therapy’s full potential when treating MDR infections

Send more data: a systematic review of mathematical models of antimicrobial resistance

Abstract Background Antimicrobial resistance is a global health problem that demands all possible means to control it. Mathematical modelling is a valuable tool for understanding the mechanisms of AMR development and spread, and can help us to investigate and propose novel control strategies. However, it is of vital importance that mathematical models have a broad utility, which can be assured if good modelling practice is followed. Objective The objective of this study was to provide a comprehensive systematic review of published models of AMR development and spread. Furthermore, the study aimed to identify gaps in the knowledge required to develop useful models. Methods The review comprised a comprehensive literature search with 38 selected studies. Information was extracted from the selected papers using an adaptation of previously published frameworks, and was evaluated using the TRACE good modelling practice guidelines. Results None of the selected papers fulfilled the TRACE guidelines. We recommend that future mathematical models should: a) model the biological processes mechanistically, b) incorporate uncertainty and variability in the system using stochastic modelling, c) include a sensitivity analysis and model external and internal validation. Conclusion Many mathematical models of AMR development and spread exist. There is still a lack of knowledge about antimicrobial resistance, which restricts the development of useful mathematical models

Can biowarfare agents be defeated with light?

Biological warfare and bioterrorism is an unpleasant fact of 21st century life. Highly infectious and profoundly virulent diseases may be caused in combat personnel or in civilian populations by the appropriate dissemination of viruses, bacteria, spores, fungi, or toxins. Dissemination may be airborne, waterborne, or by contamination of food or surfaces. Countermeasures may be directed toward destroying or neutralizing the agents outside the body before infection has taken place, by destroying the agents once they have entered the body before the disease has fully developed, or by immunizing susceptible populations against the effects. A range of light-based technologies may have a role to play in biodefense countermeasures. Germicidal UV (UVC) is exceptionally active in destroying a wide range of viruses and microbial cells, and recent data suggests that UVC has high selectivity over host mammalian cells and tissues. Two UVA mediated approaches may also have roles to play; one where UVA is combined with titanium dioxide nanoparticles in a process called photocatalysis, and a second where UVA is combined with psoralens (PUVA) to produce “killed but metabolically active” microbial cells that may be particularly suitable for vaccines. Many microbial cells are surprisingly sensitive to blue light alone, and blue light can effectively destroy bacteria, fungi, and Bacillus spores and can treat wound infections. The combination of photosensitizing dyes such as porphyrins or phenothiaziniums and red light is called photodynamic therapy (PDT) or photoinactivation, and this approach cannot only kill bacteria, spores, and fungi, but also inactivate viruses and toxins. Many reports have highlighted the ability of PDT to treat infections and stimulate the host immune system. Finally pulsed (femtosecond) high power lasers have been used to inactivate pathogens with some degree of selectivity. We have pointed to some of the ways light-based technology may be used to defeat biological warfare in the future

Epidemiology Insights

This book represents an overview on the diverse threads of epidemiological research, brings together the expertise and enthusiasm of an international panel of leading researchers to provide a state-of-the art overview of the field. Topics include the epidemiology of dermatomycoses and Candida spp. infections, the epidemiology molecular of methicillin-resistant Staphylococcus aureus (MRSA) isolated from humans and animals, the epidemiology of varied manifestations neuro-psychiatric, virology and epidemiology, epidemiology of wildlife tuberculosis, epidemiologic approaches to the study of microbial quality of milk and milk products, Cox proportional hazards model, epidemiology of lymphoid malignancy, epidemiology of primary immunodeficiency diseases and genetic epidemiology family-based. Written by experts from around the globe, this book is reading for clinicians, researchers and students, who intend to address these issues

Phytotherapy and oral health

Phytotherapy is the use of extracts from natural origin as medicines or healthpromoting agents. The use of medicinal herbs in the treatment of a variety of ailments is beneficial and efficient (Jaikaria et al., 2016). Many different herbal plants can be used to extract phytotherapeutic chemicals, which are thought to offer a wide range of therapeutic effects and fewer adverse effects than synthetic medications. A significant contribution to pharmacotherapy has historically been made by natural compounds and their structural analogues. The World Health Organization (WHO) estimates that around 80% of people worldwide utilise traditional medicine, mostly plant extracts, for their healthcare. Due to cultural familiarity, accessibility, and price, traditional medicine has seen a resurgence in interest considering that it has been used to treat illnesses for thousands of years (Schuhladen et al., 2019)

Killing of organisms responsible for wound infections using a light-activated antimicrobial agent

Infected wounds are a major cause of hospital-acquired infections and these are difficult to treat due to the emergence of antibiotic-resistant bacteria. This project is concerned with evaluating a novel antimicrobial approach involving the photosensitizer indocyanine green (ICG) which generates reactive oxygen species when irradiated with near-infrared (NIR) light which enables good tissue penetration. The photo-susceptibility of common wound-infecting organisms to ICG coupled with NIR-light was investigated. All species were susceptible to killing. ICG at a concentration of 25 μg/mL enabled the killing of the Gram-positive species (Staphylococcus aureus and Streptococcus pyogenes), higher concentrations (100-200μg/mL) were necessary to achieve substantial kills of the Gram-negative species (Pseudomonas aeruginosa and Escherichia coli). Both high and low fluences were able to kill 99.999% of the Gram-positive bacteria. High fluence irradiation was necessary to kill 99.99% of the Gram-negative bacteria. The pulsed-mode of irradiation was as effective as the continuous-mode for killing the Gram-positive species. Yet only the continuous-mode of irradiation was able to kill P. aeruginosa. Biofilms of Staph. aureus and P. aeruginosa were susceptible to disruption and killing by ICG-photosensitization. A significant enhancement of lethal photosensitization of Staph. aureus was achievable using gold-nanoparticles and antioxidants. Significant kills (>99%) were achieved in the presence of serum and 100 μg/mL ICG. A low oxygen concentration reduced the kills to 96.77% and 71.62% for Staph. aureus and Strep. pyogenes respectively. Mechanistic studies revealed that killing was mediated mainly by reactive-oxygen species. In vivo studies in mice showed that ICG and continuous-NIR light could achieve kills of 96%, 93% and 78-91% for P. aeruginosa, Strep. pyogenes and Staph. aureus respectively. The results of these in vitro and in vivo studies imply that ICG-PDT could be an effective means of decreasing the microbial burden in wounds

Host Resistance, Genomics and Population Dynamics in a Salmonella Enteritidis and Phage System

Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella enterica serovar Enteritidis and φSan23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of Salmonella under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of Salmonella under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy

Part – I: Development of a Two-step Regiospecific Synthetic Route for Multigram Scale Synthesis of Β-carboline Analogs for Studies in Primates as Anti-alcohol Agents,part – II: Design and Synthesis of Novel Antimicrobials for the Treatment of Drug Resistant Bacterial Infections Part – Iii: A Novel Synthetic Method for the Synthesis of the Key Quinine Metabolite (3S)-3-Hydroxyquinine

PART – I Development of a Two-Step Regiospecifc Synthetic Route for Multigram-Scale Synthesis of β-Carboline Analogs for Studies in Primates as Anti-Alcohol Agents β-Carboline and their derivatives are important structural motifs in synthetic organic and medicinal chemistry because of their novel biological activity, especially in regard to the reduction of alcohol self-administration [binge drinking (BD)], a major problem increasing day by day in modern society. This anti-alcohol effect is proposed to be due to the activity of ligands at the benzodiazepine site of the GABAA receptor in the central nervous system acting as antagonists at the α1 subunit. The past evidence by June, Gondre-Lewis, and Weerts et al. of the biological importance of β-carbolines for the treatment of alcohol abuse has prompted the design and synthesis of a new series of analogs to improve the in vitro and in vivo pharmacological properties. Initial SAR studies on these β-carbolines revealed that βCCt (3) and the more water soluble analog, 3-PBC·HCl (1·HCl) were lead ligands for they had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats by June et al. with little or no effect on sucrose self-administration and no anhedonia nor depression. With this important activity, further studies were designed in higher animal models such as non-human primates (Weerts). However, the availability of these ligands for biological studies was the limiting step because of the long synthetic route and overall low yields. Consequently, a novel short two-step palladium catalyzed protocol was developed which consisted of a combined regioselective Buchwald-Hartwig amination and an intramolecular Heck-type cyclization to gain regiospecific access to 3,6-disubstituted β-carbolines. This regiospecific two-step synthetic protocol reduced the number of steps from 6 to 2 and permitted execution in excellent yields on a large scale (50 - 80 grams). To obtain ligands with anti-alcohol effects that were more water soluble than the active anti-alcohol compound βCCt (3) by using 3-PBC·HCl (1·HCl) as the guide, 3-ISOPBC·HCl (2·HCl) was synthesized which showed more potent activity in the reduction of alcohol self-administration than 1·HCl in a maternally deprived (MD) rat model for binge drinking. Later pre-clinical studies were conducted in non-human primate models such as baboons which required 80-100 grams of 3-ISOPBC·HCl. This can now be accomplished with ease using the new Pd chemistry. The pronounced activity of 2·HCl in non-human primates does imply it is a potential ligand to treat human alcoholics without the side effects of diazepam (one of the drugs employed now). These results led to the synthesis of 3-cycloPBC·HCl (20·HCl) which was active, to date, in MD rats without effecting the sucrose responding. The 3-cycloPBC·HCl was not cytotoxic at all when compared to βCCt, 3-PBC·HCl, 3-ISOPBC·HCl; the latter 3 ligands of which did exhibit some toxicity but only at very high concentrations. The microsomal stability studies on human and mouse liver microsomes of 20·HCl revealed it was longer lived in vitro than 3-PBC·HCl, and 3-ISOPBC·HCl. Further studies will need to be carried out in primate models to see if 20·HCl is a potential novel therapeutic agent to combat alcohol drinking and substance use disorders. PART – II Design and Synthesis of Novel Antimicrobials for the Treatment of Drug Resistant Bacterial Infections The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of tuberculosis (TB) and methicillin-resistant Staphylococcus aureus (MRSA) has emerged and is responsible for several million deaths per year. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes, and evaluated as potential agents against a panel of gram positive, negative, mycobacterial, and clinically significant resistant strains including M. tuberculosis (Mtb) for minimum inhibitory concentrations (MIC). In depth structure activity relationship (SAR) studies of acrylic acid ethyl ester derivatives revealed that the ethyl esters 59 and 63 were found to be very potent (MIC = 0.72 and 0.69 µg/mL) against actively replicating Mtb. Importantly, scaffolds 59 and 63 exhibited six and four fold greater inhibition, respectively, against nonreplicating persistent (dormant) phenotypes under low oxygen conditions than isoniazid; this is essential to decrease the duration of tuberculosis treatment from many months to less time. Further evaluation of these selected analogs 59 and 63 against a panel of single-drug resistant Mtb strains indicated a similar level of activity as against wild type Mtb. This encouraging safety profile is key with a selective index greater than 10. Gratifyingly, the ethyl ester 59 retained excellent inhibition with MIC values of 0.25-4.0 µg/mL against a wide variety of virulent antibiotic-resistant clinical isolates (MRSA, MDR MRSA, VISA MRSA, and VRE). This exciting activity provided a path to determine the molecular target for this novel class of compounds with the copper catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) Click reaction because of the availability of alkyne functionality in 59. By treating Staphylococcus aureus lysates with Alexa fluor 647 picolyl azide (AF647), one identified S. aureus proteins that had been covalently modified by propargyl ligand 59. SDS-PAGE analysis of the fluorescently labeled proteins showed that only two proteins were labeled. Encouraged by the results, AF647 was replaced with biotin azide to isolate the target proteins using streptavidin beads. Later, the purified protein fractions were subjected to peptide mass fingerprinting for protein identification. Data analysis of these samples using MaxQuant 1.4.1.2 against the Uniport database for E. coli and S. aureus identified three enzymes as potential targets: enolase (Uniprot ID: P64079), dihydrolipoyllysine-residue acetyltransferase (Uniprot ID: Q8NX76), and glyceraldehyde-3-phosphate dehydrogenase (Uniprot ID: P0A037). These enzymes are well-known to be involved in glycolysis and act as virulence factors responsible for the pathogenicity of S. aureus. Thus far, attempts to validate the structure of S. aureus enolase by X-ray diffraction analysis have been unsuccessful, since one has been unable to obtain diffraction-quality crystals of this protein; however, protein docking experiments with S. aureus enolase have been successful. Further work on these potent antimicrobial agents would benefit from the knowledge of the binding site as well as interactions between the ligand and the proteins; the mode of inhibition. The identification of the bimolecular interaction between the ligand 59 and target proteins would potentially result in new drugs to treat drug resistant infections from bacteria, including MRSA, MDR VISA, and VRE. The investigation of ADMET medicinal chemistry properties of select agents including 59 and 63 is ongoing in our laboratories. Part – III A Novel Synthetic Method for the Synthesis of the Key Quinine Metabolite (3S)-3-Hydroxyquinine The Cinchona alkaloid quinine (1) remains unique among the thousands of natural products isolated and characterized to date because it still remains the drug of choice for the treatment of severe and complicated malaria in most parts of the world. Apart from biological activity, Cinchona alkaloids play a vital role in organic chemistry from racemate resolutions to promote enantioselective transformations in both homogeneous and heterogeneous catalysis. The synthesis of the major metabolite of quinine (1), 3(S)-3-hydroxyquinine (7) has been accomplished by a shorter route, devoid of the previously employed toxic reagent (HBr gas) and separated from its epimeric mixture [4(S):1(R)] at C-3 by conversion into the 9-aceto analogue followed by flash column chromatography. The molecular structure of the major acetate diastereomer 9 was further confirmed by X-ray crystallographic analysis, and this unambiguously confirms the absolute configuration of 3(S)-3-hydroxyquinine (7). The new synthetic protocol increased the overall yield from 16% to 53% and makes essential metabolite 7 more readily available now for scientists and doctors to study drug-drug interactions when using quinine with another agent to treat, malaria combined with HIV (ritonavir) or other comorbid situations. For instance, a doctor in Nigeria, using 7 found that in healthy volunteers, to treat patients with HIV and malaria one needed a ratio of ~5:1 ritonavir and quinine, not 1:1, as used previously

HISTOLOGICAL STUDIES OF BREWERY SPENT GRAINS IN DIETARY PROTEIN FORMULATION IN DONRYU RATS

The increasing production of large tonnage of products in brewing industries continually generates lots of solid waste which includes spent grains, surplus yeast, malt sprout and cullet. The disposal of spent grains is often a problem and poses major health and environmental challenges, thereby making it imminently necessary to explore alternatives for its management. This paper focuses on investigating the effects of Brewery Spent Grain formulated diet on haematological, biochemical, histological and growth performance of Donryu rats. The rats were allocated into six dietary treatment groups and fed on a short-term study with diet containing graded levels of spent grains from 0, 3, 6, 9, 12 and 100% weight/weight. The outcome demonstrated that formulated diet had a positive effect on the growth performance of the rats up to levels of 6% inclusions, while the haematological and biochemical evaluation revealed that threshold limit should not exceed 9% of the grain. However, the histological study on the liver indicated a limit of 3% inclusion in feed without serious adverse effect. Thus invariably showing that blend between ranges 1-3% is appropriate for the utilization of the waste in human food without adverse effect on the liver organ. The economic advantage accruing from this waste conversion process not only solves problem of waste disposal but also handle issues of malnutrition in feeding ration