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ABSTRACT Many antibiotics that are used in healthcare, farming, and aquaculture end up in environments with different spatial structures that might promote heterogeneity in the emergence of antibiotic resistance. However, the experimental evolution of microbes at sub-inhibitory concentrations of antibiotics has been mainly carried out at the population level which does not allow capturing single-cell responses to antibiotics. Here, we investigate and compare the emergence of resistance to ciprofloxacin in Escherichia coli in well-mixed and structured environments using experimental evolution, genomics, and microfluidics-based time-lapse microscopy. We discover that resistance to ciprofloxacin and cross-resistance to other antibiotics is stronger in the well-mixed environment due to the emergence of target mutations, whereas efflux regulator mutations emerge in the structured environment. The latter mutants also harbor sub-populations of persisters that survive high concentrations of ciprofloxacin that inhibit bacterial growth at the population level. In contrast, genetically resistant bacteria that display target mutations also survive high concentrations of ciprofloxacin that inhibit their growth via population-level antibiotic tolerance. These resistant and tolerant bacteria keep doubling while shrinking in size in the presence of ciprofloxacin and regain their original size after antibiotic removal, which constitutes a newly discovered phenotypic response. This new knowledge sheds light on the diversity of strategies employed by bacteria to survive antibiotics and poses a stepping stone for understanding the link between mutations at the population level and phenotypic single-cell responses.IMPORTANCEThe evolution of antimicrobial resistance poses a pressing challenge to global health with an estimated 5 million deaths associated with antimicrobial resistance every year globally. Here, we investigate the diversity of strategies employed by bacteria to survive antibiotics. We discovered that bacteria evolve genetic resistance to antibiotics while simultaneously displaying tolerance to very high doses of antibiotics by doubling while shrinking in size.

Irritable bowel syndrome is a chronic gastrointestinal disease whose symptoms affect patients' daily lives. Exacerbation of symptoms can worsen patients' quality of life and lead to depressive-anxiety and somatoform disorders. The associated costs burden not only the patients, but also those around them. The etiology of IBS is multifactorial and not fully understood. The brain-gut axis remains a subject of research, which may involve the development of new, effective therapies for the treatment of IBS in the future. Pharmacological and non-pharmacological methods are used to treat IBS. In order to provide the patient with the best possible treatment results in the absence of significant side effects, research is being conducted on the possibilities of non-pharmacological patient management. Currently, there are numerous studies on the effect of physical activity on the severity of symptoms in IBS patients. The results show that the introduction of moderate-intensity physical activity has positive effects in terms of alleviating gastrointestinal symptoms, reducing the severity of psychological and somatoform symptoms, and improving patients' well-being as well as quality of life. Appropriate physical activity is acceptable to patients and rated by them as positive and having a beneficial effect on IBS symptoms. The lack of patient-reported significant side effects is important. Given the results of the study, it seems beneficial to recommend that IBS patients perform moderate physical activity to alleviate and control symptoms. It is necessary to educate patients and individualize physical activity recommendations to match the type of activity to patients' abilities and needs.

A fatigue fracture, also known as stress fracture or overuse fracture, is caused by the summation of micro-injuries resulting from a chronic state of bone overload. It affects 1.4 %- 4.4 % of professional athletes. The most common site of fatigue fractures are the lower limbs. This is related to the heavy load caused by body weight and performing dynamic activities like jumping, running. Typical fractures are fractures of the fifth metatarsal bone, fibula and tibia, and less common fractures of the femur. Fractures involving the bones of the upper limbs and thorax are less commonly reported in the literature. It affect athletes in strength sports and athletes who perform dynamic and repetitive movements with their upper limbs, most often boxers, weightlifters, wrestlers, judokas, swimmers, golfers, rowers. Fractures in the thorax most often involve the first rib. Fractures of ribs II through XII are extremely rarely described. We present an unusual case of a second rib fracture in a professional athlete. A 26-year-old athlete training in racewalking reported increasing pain in the left scapula area 7 days before competing in the European Championships. Immediately after the competition, the pain changed location to the anterior thorax area, making movement of the upper limb and breathing much more difficult. A chest tomography was performed and it revealed a fatigue fracture of the second rib on the left side. The case report presents an atypical fracture in a female racewalking athlete and presents the uncharacteristic symptoms accompanying this fracture. Incorrect initial diagnosis and application of physiotherapy procedures without performing basic diagnostic tests, delay the diagnosis and initiation of proper treatment, and thus the athlete's recovery.

The utility of cannabinoids as agents used to treat diseases is a popular topic of discussion these days. The approval of medical marijuana continues to stir up controversy. As far back as ancient times, hemp was used for medicinal purposes. These plants have the ability to produce phytochemicals and other compounds such as flavonoids and terpenes. Respective species of hemp differ in the content of particular chemical compounds and therefore have different possible uses. Among the best-known phytocannabinoids found in hemp are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). They have partially opposing effects and interact with the corresponding receptors of the endocannabinoid system in the body's tissues. We also distinguish between cannabinoids of endogenous origin - endocannabinoids (EC) and synthetic cannabinoids (SC). The effects of THC and CBD on the human body are still being studied. Information about them that appears in the media, is often reported inaccurately or incompletely. There is now a growing body of research into the use of cannabinoids to treat and alleviate the symptoms of many diseases. Due to the neuroprotective effects of THC and CBD, researchers' work has focused on using them to treat neurodegenerative diseases, which include Parkinson's disease (PD).

Alcohol withdrawal syndrome (AWS) is a complex set of symptoms that occur in alcohol-dependent individuals after sudden withdrawal or a significant reduction in alcohol consumption. AWS symptoms occur in about 50% of alcohol abusers. These symptoms may include restlessness, tremor, nausea, nervousness, tachycardia, elevated blood pressure, hyperhidrosis, insomnia, hyperactivity, and hallucinations. In some cases, seizures may occur and delirium tremens may develop, which is life-threatening and an absolute indication for hospitalization of the patient. Effective treatment of AWS is the key to prevent complications and to reduce the risk of death. Treatment of alcohol withdrawal syndrome requires a complex approach that combines properly performed diagnosis, careful monitoring of the patient's condition, pharmacotherapy, equalization of electrolyte disorders, adequate hydration of the patient, supplementation of thiamine deficiencies and, in the case of symptoms of alcoholic delirium, intensive medical care. Pharmacological treatment plays a key role, with the first line of treatment being benzodiazepines, which reduce the risk of epileptic seizures and delirium tremens, and reduce mortality in the course of AWS. Individualized therapy adjustment and patient monitoring are crucial to ensure effective and safe treatment.

The interactions between a virus and its host vary in space and time and are affected by the presence of molecules that alter the physiology of either the host or the virus. Determining the molecular mechanisms at the basis of these interactions is paramount for predicting the fate of bacterial and phage populations and for designing rational phage-antibiotic therapies. We study the interactions between stationary phase Burkholderia thailandensis and the phage ΦBp-AMP1. Although heterogeneous genetic resistance to phage rapidly emerges in B. thailandensis, the presence of phage enhances the efficacy of three major antibiotic classes, the quinolones, the beta-lactams and the tetracyclines, but antagonizes tetrahydrofolate synthesis inhibitors. We discovered that enhanced antibiotic efficacy is facilitated by reduced antibiotic efflux in the presence of phage. This new phage-antibiotic therapy allows for eradication of stationary phase bacteria, whilst requiring reduced antibiotic concentrations, which is crucial for treating infections in sites where it is difficult to achieve high antibiotic concentrations.

Abstract Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide−alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.

The combined nutritional and salinity limitation is shown to increase glucose uptake and degradation rates in individual E. coli bacteria.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

New virions of Ff bacteriophages are extruded from the host cell via the channel built from phage protein pIV, homologous to bacterial secretins. Here, the authors report the structure of this channel from the f1 filamentous bacteriophage and propose its use as an adjuvant to increase the uptake and efficacy of antibiotics.

Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

Bacteriophages represent an avenue to overcome the current antibiotic resistance crisis, but evolution of genetic resistance to phages remains a concern. In vitro, bacteria evolve genetic resistance, preventing phage adsorption or degrading phage DNA. In natural environments, evolved resistance is lower possibly because the spatial heterogeneity within biofilms, microcolonies, or wall populations favours phenotypic survival to lytic phages. However, it is also possible that the persistence of genetically sensitive bacteria is due to less efficient phage amplification in natural environments, the existence of refuges where bacteria can hide, and a reduced spread of resistant genotypes. Here, we monitor the interactions between individual planktonic bacteria in isolation in ephemeral refuges and bacteriophage by tracking the survival of individual cells. We find that in these transient spatial refuges, phenotypic resistance due to reduced expression of the phage receptor is a key determinant of bacterial survival. This survival strategy is in contrast with the emergence of genetic resistance in the absence of ephemeral refuges in well-mixed environments. Predictions generated via a mathematical modelling framework to track bacterial response to phages reveal that the presence of spatial refuges leads to fundamentally different population dynamics that should be considered in order to predict and manipulate the evolutionary and ecological dynamics of bacteria-phage interactions in naturally structured environments.