Convergent phenotypic evolution towards fosfomycin collateral sensitivity of Pseudomonas aeruginosa antibiotic-resistant mutants

The rise of antibiotic resistance and the reduced amount of novel antibiotics support the need of developing novel strategies to fight infections, based on improving the use of the antibiotics we already have. Collateral sensitivity is an evolutionary trade-off associated with the acquisition of antibiotic resistance that can be exploited to tackle this relevant health problem. However, different works have shown that patterns of collateral sensitivity are not always conserved, thus precluding the exploitation of this evolutionary trade-off to fight infections. In this work, we identify a robust pattern of collateral sensitivity to fosfomycin in Pseudomonas aeruginosa antibiotic-resistant mutants, selected by antibiotics belonging to different structural families. We characterize the underlying mechanism of the collateral sensitivity observed, which is a reduced expression of the genes encoding the peptidoglycan-recycling pathway, which preserves the peptidoglycan synthesis in situations where its de novo synthesis is blocked, and a reduced expression of fosA, encoding a fosfomycin-inactivating enzyme. We propose that the identification of robust collateral sensitivity patterns, as well as the understanding of the molecular mechanisms behind these phenotypes, would provide valuable information to design evolution-based strategies to treat bacterial infections.     

Novel antimicrobial strategies to treat multi-drug resistant Staphylococcus aureus infections

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.     

A combination therapy of Phages and Antibiotics: Two is better than one

Emergence of antibiotic resistance presents a major setback to global health, and shortage of antibiotic pipelines has created an urgent need for development of alternative therapeutic strategies. Bacteriophage (phage) therapy is considered as a potential approach for treatment of the increasing number of antibiotic-resistant pathogens. Phage-antibiotic synergy (PAS) refers to sublethal concentrations of certain antibiotics that enhance release of progeny phages from bacterial cells. A combination of phages and antibiotics is a promising strategy to reduce the dose of antibiotics and the development of antibiotic resistance during treatment. In this review, we highlight the state-of-the-art advancements of PAS studies, including the analysis of bacterial-killing enhancement, bacterial resistance reduction, and anti-biofilm effect, at both in vitro and in vivo levels. A comprehensive review of the genetic and molecular mechanisms of phage antibiotic synergy is provided, and synthetic biology approaches used to engineer phages, and design novel therapies and diagnostic tools are discussed. In addition, the role of engineered phages in reducing pathogenicity of bacteria is explored.     

Dismantling the bacterial virulence program

In the face of rising antimicrobial resistance, there is an urgent need for the development of efficient and effective anti-infective compounds. Adaptive resistance, a reversible bacterial phenotype characterized by the ability to surmount antibiotic challenge without mutation, is triggered to cope in situ with several stressors and is very common clinically. Thus, it is important to target stress-response effectors that contribute to in vivo adaptations and associated lifestyles such as biofilm formation. Interfering with these proteins should provide a means of dismantling bacterial virulence for treating infectious diseases, in combination with conventional antibiotics.     

Tetracycline and Chloramphenicol Efficiency Against Selected Biofilm Forming Bacteria

Despite the constantly increasing need for new antimicrobial agents, antibiotic drug discovery and development seem to have greatly decelerated in recent years. Presented with the significant problem of advancing antimicrobial resistance, the global scientific community has attempted to find alternative solutions; one of the most promising ones is the evaluation and use of old antibiotic compounds. A number of old antibiotic compounds, such as aminoglycosides, chloramphenicol, and tetracycline, are re-emerging as valuable alternatives for the treatment of difficult-to-treat infections. This study examined the in vitro potency for biofilm formation of five isolates (Klebsiella sp., Pseudomonas aeruginosa, Achromobacter sp., Klebsiella pneumoniae, and Bacillus pumilis) and the effects of antibiotics on these biofilms. Furthermore the quantitative analysis of planktonic, loosely attached cells, and their susceptibility to antibiotics was also determined. Twitching motility was observed to determine any effect in the biofilm forming capability of the isolates. All the isolates tested were efficient biofilm-forming strains in the polypropylene and borosilicate test tubes. Standard bacterial enumeration technique and CV staining produced equivalent results both in biofilm and planktonic assays. The biofilm formation of all the strains was affected in the presence of tetracycline or chloramphenicol. Highly significant decrease (P < 0.01) in biofilm formation was observed by treatment with chloramphenicol compared to tetracycline. In addition, the two antibiotics also affected adversely the planktonic and loosely attached cells of all isolates. Thus, testing the effects of older antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis.     

Queuing up the ribosome: nutrition and the microbiome control protein synthesis

The health of an organism is intricately linked to its gut microbiome. However, the mechanisms by which the microbiome affect the host gene regulation are still not well established. A new study by Tuorto et al ( 2018 ) shows that queuine, a nitrogenous base obtained from the gut microbiota, is used to modify tRNAs and affects cellular behavior. Dietary queuine is required for proper protein synthesis, and its depletion activates cellular stress responses in vitro and in vivo.     

Systems for in vivo hypermutation: a quest for scale and depth in directed evolution

Traditional approaches to the directed evolution of genes of interest (GOIs) place constraints on the scale of experimentation and depth of evolutionary search reasonably achieved. Engineered genetic systems that dramatically elevate the mutation of target GOIs in vivo relieve these constraints by enabling continuous evolution, affording new strategies in the exploration of sequence space and fitness landscapes for GOIs. We describe various in vivo hypermutation systems for continuous evolution, discuss how different architectures for in vivo hypermutation facilitate evolutionary search scale and depth in their application to problems in protein evolution and engineering, and outline future opportunities for the field.     

Mining for novel antibiotics in the age of antimicrobial resistance

The misuse of antimicrobials is causing an alarming increase in the appearance of antibiotic-resistant microorganisms. In this context, the identification of novel antibiotics against new targets and with low rates of resistance development is a major global challenge. In this article, we highlight a number of recent articles that exploit a variety of in vitro, in vivo and in silico state-of-the-art approaches to identify and develop new antimicrobials. Rapid progress in this research field will be crucial to combating a global health problem, antimicrobial resistance, that is expected to be the leading cause of death by 2050.     

Effects of Piper cubeba L. essential oil on methicillin‐resistant Staphylococcus aureus: an AFM and TEM study

The increasing prevalence of antibiotic‐resistant bacteria is creating a real challenge for health care systems worldwide, making the development of novel antibiotics a necessity. In addition to the development of new antibiotics, there is an urgent need for in‐depth characterization of the mechanisms of bacterial resistance toward new drugs. Here, we used essential oils extracted in our laboratory from Piper cubeba against methicillin‐resistant Staphylococcus aureus ATCC 43300, one of the most prominent antibiotic‐resistant bacteria. Effects of the essential oils extracted from P cubeba on bacteria were mainly evaluated using 2 powerful microscopy techniques: atomic force microscopy and transmission electron microscopy. High‐resolution atomic force microscopy images of the cells were obtained close to their native environment by immobilizing the cells on porous Polyether sulfone membranes, which were prepared in our laboratory with a wide range and distribution of pore sizes and depth. Inhibition zones (mm) and minimum inhibitory concentrations were determined. Two different concentrations of the oil were used to treat the cells: 50 μg/mL minimum inhibitory concentration and 25 μg/mL. The 50 μg/mL oil solution caused severe damage to the bacterial cells at microscopic levels while the 25 μg/mL solution showed no effects compared to the control. However, at nanoscopic levels, the 25 μg/mL oil solution caused significant changes in the cell wall, which could potentially impair bacterial activities. These results were also confirmed by transmission electron microscopy micrographs. Our results indicate that the extract has a good biological activity against methicillin‐ and oxacillin‐resistant S aureus and that it acts on the cell wall and plasma (cytoplasmic) membrane.     

Decipher enzymes from human microbiota for drug discovery and development

The human microbiota plays an important role in human health and contributes to the metabolism of therapeutic drugs affecting their potency. However, the current knowledge on human gut bacterial metabolism is limited and lacks an understanding of the underlying mechanisms of observed drug biotransformations. Despite the complexity of the gut microbial community, genomic and metagenomic sequencing provides insights into the diversity of chemical reactions that can be carried out by the microbiota and poses new challenges to functionally annotate thousands of bacterial enzymes. Here, we outline methods to systematically address the structural and functional space of the human microbiome, highlighting a combination of in silico and in vitro approaches. Systematic knowledge about microbial enzymes could eventually be applied for personalized therapy, the development of prodrugs and modulators of unwanted bacterial activity, and the further discovery of new antibiotics.     

Zoonosis: An Emerging Link to Antibiotic Resistance Under “One Health Approach”

Current scenario in communicable diseases has generated new era that identifies the “One health” approach to understand the sharing and management of etiological agents with its impact on ecosystem. Under this context the relevance of zoonotic diseases generates major concern. The indiscriminate and higher use of antibiotics in animal husbandry creates substantial pressure on the gut microbiome for development of resistance due to shorter generation time and high density. Thus, gut works as a bioreactor for the breeding of ARBs in this scenario and are continuously released in different niches. These ARBs transfer resistance genes among native flora through horizontal gene transfer events, vectors and quorum sensing. About 60% of infectious diseases in human are caused by zoonotic pathogens have potential to carry ARGs which could be transmitted to humans. The well documented zoonotic diseases are anthrax cause by Bacillus anthracis, bovine tuberculosis by Mycobacterium tuberculosis, brucellosis by Brucella abortus, and hemorrhagic colitis by Escherichia coli. Similarly, most of the antibiotics are not completely metabolized and released in unmetabolized forms which enters the food chain and affect various ecological niches through bioaccumulation. The persistence period of antibiotics ranges from?<?1 to 3466 days in environment. The consequences of misusing the antibiotic in livestock and their fate in various ecological niches have been discussed in this review. Further the light sheds on antibiotics persistence and it biodegradation through different abiotic and biotic approaches in environment. The knowledge on personnel hygiene and strong surveillance system for zoonotic disease including ARBs transmission, prevention and control measures should be established to regulate the spread of AMR in the environment and subsequently to the human being through a food web.

     

Cholesterol glucosylation–based survival strategy in Helicobacter pylori

Helicobacter pylori is a major chronic health problem, infecting more than half of the population worldwide. H. pylori infection is linked with various clinical complications ranging from gastritis to gastric cancer. The resolution of gastritis and peptic ulcer appears to be linked with the eradication of H. pylori. However, resistance to antibiotics and eradication failure rates are reaching alarmingly high levels. This calls for urgent action in finding alternate methods for H. pylori eradication. Here, we discuss the recently identified mechanism of H. pylori known as cholesterol glucosylation, mediated by the enzyme cholesterol-α-glucosyltransferase, encoded by the gene cgt. Cholesterol glucosylation serves several functions that include promoting immune evasion, enhancing antibiotic resistance, maintaining the native helical morphology, and supporting functions of prominent virulence factors such as CagA and VacA. Consequently, strategies aiming at inhibition of the cholesterol glucosylation process have the potential to attenuate the potency of H. pylori infection and abrogate H. pylori immune evasion capabilities. Knockout of H. pylori cgt results in unsuccessful colonization and elimination by the host immune responses. Moreover, blocking cholesterol glucosylation can reverse antibiotic susceptibility in H. pylori. In this work, we review the main roles of cholesterol glucosylation in H. pylori and evaluate whether this mechanism can be targeted for the development of alternate methods for eradication of H. pylori infection.     

Thermal proteome profiling in bacteria: probing protein state in vivo

Increasing antibiotic resistance urges for new technologies for studying microbes and antimicrobial mechanism of action. We adapted thermal proteome profiling (TPP) to probe the thermostability of Escherichia coli proteins in vivo. E. coli had a more thermostable proteome than human cells, with protein thermostability depending on subcellular location—forming a high‐to‐low gradient from the cell surface to the cytoplasm. While subunits of protein complexes residing in one compartment melted similarly, protein complexes spanning compartments often had their subunits melting in a location‐wise manner. Monitoring the E. coli meltome and proteome at different growth phases captured changes in metabolism. Cells lacking TolC, a component of multiple efflux pumps, exhibited major physiological changes, including differential thermostability and levels of its interaction partners, signaling cascades, and periplasmic quality control. Finally, we combined in vitro and in vivo TPP to identify targets of known antimicrobial drugs and to map their downstream effects. In conclusion, we demonstrate that TPP can be used in bacteria to probe protein complex architecture, metabolic pathways, and intracellular drug target engagement.     

Lactobacillus plantarum Lp62 exerts probiotic effects against Gardnerella vaginalis ATCC 49154 in bacterial vaginosis

The severe side-effects elicited by conventional antibiotic therapy and the recurrence of Bacterial vaginosis-associated bacteria and bacterial resistance have led to the development of novel alternative therapies, among which genital probiotics are widely used. In this study, we aimed to evaluate the antimicrobial activities of Lactobacillus plantarum Lp62 and its supernatant against Gardnerella vaginalis, using both in vitro and in vivo approaches. In vitro assays were used to evaluate the viability of the strain and the antimicrobial activities of the supernatant in different pH ranges. An in vivo assay was performed on female BALB/c mice, wherein the animals were divided into eight groups: four control groups and four treated groups (for curative and preventive therapies). After infecting and treating the mice, the animals were killed to quantify the bacterial load using qPCR, evaluate leucocyte cellular response, determine vaginal cytokine levels and perform cytokine tissue gene expression. Our analyses revealed significant activity of the strain and its supernatant against G. vaginalis. Preliminary in vitro tests showed that the strain grew with equal efficiency in different pH ranges. Meanwhile, the presence of halo and inhibition of pathogen growth established the significant activity of the supernatant against G. vaginalis. We observed that both micro-organisms are resident bacteria of mouse microbiota and that the lactobacilli population growth was affected by G. vaginalis and vice versa. We also observed that the treated groups, with their low bacterial load, absence of leucocyte recruitment, reduced cytokine levels in the vaginal lavage and normalized cytokine gene expression, successfully controlled the infection.     

Preconditioning influences mesenchymal stem cell properties in vitro and in vivo

Various diseases and toxic factors easily impair cellular and organic functions in mammals. Organ transplantation is used to rescue organ function, but is limited by scarce resources. Mesenchymal stem cell (MSC)‐based therapy carries promising potential in regenerative medicine because of the self‐renewal and multilineage potency of MSCs; however, MSCs may lose biological functions after isolation and cultivation for a long time in vitro. Moreover, after they are injected in vivo and migrate into the damaged tissues or organs, they encounter a harsh environment coupled with death signals due to the inadequate tensegrity structure between the cells and matrix. Preconditioning, genetic modification and optimization of MSC culture conditions are key strategies to improve MSC functions in vitro and in vivo, and all of these procedures will contribute to improving MSC transplantation efficacy in tissue engineering and regenerative medicine. Preconditioning with various physical, chemical and biological factors is possible to preserve the stemness of MSCs for further application in studies and clinical tests. In this review, we mainly focus on preconditioning and the corresponding mechanisms for improving MSC activities in vitro and in vivo; we provide a glimpse into the promotion of MSC‐based cell therapy development for regenerative medicine. As a promising consequence, MSC transplantation can be applied for the treatment of some terminal diseases and can prolong the survival time of patients in the near future.