Salmonella Persistence During Infection: Trade-off Between Mitigating DNA Damage and Ability to Relapse

Sophie Helaine • Harvard Medical School

My lab studies the molecular mechanisms of bacterial persistence during infection. Bacterial persistence, characterized by chronic and relapsing infections, is a major threat to human health as these infections cause considerable morbidity and frequently require multiple courses of antibiotics. Such long-lasting infections are caused by a variety of bacterial pathogens including Mycobacterium tuberculosis, Salmonella, Pseudomonas and pathogenic Escherichia coli. We developed single cell reporters to track Salmonella growth history during macrophage and murine infections. It revealed the presence of non-growing bacteria in infected host cells, which had been hypothesized for decades but had remained elusive. Interaction between Salmonella and host macrophages has then proven to be a powerful and relevant model to study persister biology since we showed that the bacteria specifically respond to engulfment by the host defence cells by forming high proportions of persisters. I will present our characterization of how persisters survive antibiotics in this challenging environment.

Cryptic Phenotypic Resistance: “Differentially Detectable” Bacteria

Carl F. Nathan • Weill Cornell Medicine

Lysis of bacteria is the one certain way to define their death. The gold standard surrogate is inability to form a colony. Mycobacterium tuberculosis (Mtb), the leading pre-COVID cause of death from infection, poses the clinical challenge of phenotypic resistance to antibiotics on the part of viable Mtb that are not colony-forming units and are therefore misconstrued as being absent or dead. Their viability is revealed in vivo by their ability to cause disease and in vitro by alternative methods of culture, such as limiting dilution, making them “differentially detectable” (DD). DD Mtb have undergone a degree of oxidative damage intermediate between what does not interfere with colony growth and what precludes recovery during replicative delay. DD Mtb are not “dormant”, but they are profoundly resistant to antibiotics selected for their ability to kill replicating bacteria. We need antibiotics that can kill bacteria in DD states.

The Impact of the Disrupted Cellular State on Drug Response

Nathalie Balaban • The Hebrew University of Jerusalem

The evolution of antibiotic resistance in microorganisms is a major health issue. Understanding the factors affecting evolutionary trajectories from susceptibility to resistance is crucial. Quantitative experiments and mathematical modelling can shed light on the processes that speed up or delay the evolution of resistance. We previously showed that tolerance, a form of survival under antibiotics that is distinct from resistance, plays a major role in promoting the evolution of resistance in vitro. In order to determine the relevance of the in vitro experimental evolution results for the clinic, we followed the course of infection in patients with life threatening bloodstream infection. Further dissection of the response of the clinical strains to combinations of antibiotics reveals a new way by which resistance evolution is strongly promoted by the tolerance phenotype. Finally, we develop a global understanding of how stresses can push bacteria into a disrupted state which drives subsequent antibiotic tolerance.

Antibiotic Resistance is the Tip of the Iceberg of Ecological Change

Martin J. Blaser • Rutgers University

Like that of all mammals, the human microbiome is ancient, diverse, numerous, niche-specific, and is comprised of both conserved and host-specific features. In early life, the microbiome develops in a choreographed manner, and is a part of the development of host metabolism, immunity and cognition. Antibiotic treatments, designed to suppress or eliminate pathogens, have substantial collateral effects on the microbiome, especially in early life. If the ill-effects of antibiotics on human biology were drawn as an iceberg, the tip would be the well-recognized selection for antibiotic resistance. But the body would represent the transient or long-term antibiotic-induced changes that affect developmental, situational, senescent, and generational phenomena, which themselves have metabolic, immunologic, neoplastic, or degenerative effects. There now is extensive evidence that correlates early life antibiotic exposures and subsequent disease risks. We have conducted experimental studies in animal models to determine the effects of such exposures. Multiple studies provide consistent evidence of causal roles, including those crossing host generations. The widespread use of antibiotics across several human generations now presents serious and increasing problems that require directed solutions. These include more restricted clinical use of antibiotics, narrow spectrum approaches to anti-bacterial activities, and strategies for the restoration of missing microbes and their host-signaling modalities and pathways.

Non-anthropogenic Selective Pressures on Resistance Evolution

Michael Baym • Harvard Medical School 

While the human use of antibiotics does contribute to the evolution of resistance, the epidemiology of resistance we observe is inconsistent with it being the sole, or potentially even the primary, driver of resistance evolution. So what else is selecting on resistance? I will propose some possibilities, show how to discover phages that select against both resistance and the horizontal transmission of resistance, and show how evolutionary insight can teach us how to build better diagnostics.

Using Fitness Seascapes and Counterdiabatic Driving to Enable Control of Evolving Populations of Microbes

Jacob Scott • Cleveland Clinic 

Antibiotic resistance represents a growing health crisis necessitating the immediate discovery of novel treatment strategies. One treatment strategy involves probabilistically directing the evolution of bacterial populations toward increased drug-sensitive states. This strategy however requires multiple rounds of prolonged drug exposure which select for resistant cells in a stochastic manner. We will present results highlighting the stochasticity of these methods, in particular the possibility of evolutionary escape to genotypes which could access high fitness. We then present a solution to these limitations involving a quantum-inspired method to control the trajectory of evolving bacterial populations. This approach allows one to calculate a time-dependent drug dosage that will steer a bacterial population, in a finite time, toward a target genotype distribution. Finally, we will present novel data in the form of fitness seascapes for commonly used antibiotics — a sine qua non for these methods — and present a path toward validation of this control method.

Short-Range Bacterial Communication and Its Implications

Avigdor Eldar • Tel Aviv University 

Bacterial quorum sensing allows for coordinated cellular response through the secretion and detection of diffusible molecules. While many quorum-sensing bacteria grow in spatially structured and genetically heterogeneous communities, the principles that govern quorum-sensing under these conditions are largely unknown. Combining microfluidic experiments with mathematical modeling, we quantified signal propagation in synthetic bacterial communities at a single-cell resolution. We identify two generic quorum-sensing designs that profoundly differ in the spatial scale of their effect: one design allows for global communication among cell clusters in the community, whereas the other design allows localized communication with signaling length-scale of about 10 microns. We further show that phages and conjugative elements, employ short-range quorum to sense their frequency in a highly localized neighborhood and initiate horizontal gene transfer when this fraction is small. Finally, we constructed a synthetic gene network based on short range signaling which leads to pattern formation at the cellular scale.

Understanding Metabolic Preferences of Bacterial Pathogens as Novel Metabolotherapies to Infection

Roi Avraham • Weizmann Institute of Science 

Management of many bacterial infections is becoming increasingly difficult due to new, rapidly evolving pathogens with increased virulence and drug resistance. Promising alternatives to targeting pathogens, novel anti-infective approaches harness the host’s own response to infection or target virulent processes of the pathogen. To realize the promise of these alternative therapeutic approaches, a comprehensive, systematic understanding of the complex dynamics between host and pathogen is required. Specifically, we believe that the early stages of infection, when bacterial numbers are relatively small, play a particularly critical role in determining infection outcome, and offers a unique opportunity to eradicate infection. Despite the huge medical importance and global health burden, our ability to assess the impact of host-pathogen interactions on infection outcome is limited, especially in the context of human infection. For this, one of the least-understood periods in the course of infectious disease lies between the initial inoculation with a dose of pathogens and the appearance of the first symptoms of disease, or the lack of appearance thereof. In my talk, I will present recent findings in our lab indicating that this can now be done by modelling the dynamics of metabolism, phenotype, and function of immune cells so as to identify cell type-specific states that represent their activation process during infection. I will present tools and approaches to study early infection dynamics and define two novel concepts of infection biology: 1) Immune cell dynamic states (immDS) – the range of metabolic and immunological changes of cells in response to pathogens, without losing their identity. 2) Host-Patho-Cells, cellular states determined by the immuno-metabolic- virulence crosstalk between infected host cells and intracellular bacteria. I will demonstrate how the landscape of immDS and Host-Patho-Cells at critical stages of early human infection can define the trajectory of infection outcome, and provide bona-fide targets to better treat infection.

Phage Therapy to Combat Infections by Antibiotic-Resistant Bacterial Pathogens

Paul Turner • Yale University 

One possible strategy to combat the antibiotic resistance crisis is a renewed approach to ‘phage therapy,’ where these administered viruses not only kill the target bacteria, but also predictably select for phage resistance that reduces virulence and/or increases antibiotic sensitivity (evolutionary trade-offs). By utilizing virulence factors as receptor binding sites, the phages exert selection for bacteria to evolve phage resistance by modifying (or losing) the virulence factor, potentially reducing bacterial pathogenicity. We present examples of naturallyisolated phages that kill target bacteria while selecting for phage resistance that coincides with useful clinical traits, and compare in vitro data to phenotypic, genetic and metagenomics analyses of microbes isolated longitudinally from patient samples before, during and after emergency phage therapy treatments.