Biology subjects

Gil-Gil, T.

Publications and source records attributed to Gil-Gil, T..

11 recordsLinked to original sources

bioRxiv2026

Bacteriophage Heteroresistance as a Cause of Treatment Failure in Urinary Tract Infections

With the rise of antimicrobial resistance, urinary tract infections (UTIs) have become increasingly more difficult to treat, prompting renewed interest in bacteriophage (phage) therapy as an alternative or adjunct to antibiotics. UTIs are an attractive target for phage therapy because they generate a high density of actively replicating bacteria that supports phage propagation, and because the urinary tract is readily accessible for administration and monitoring. Yet studies of phage therapy for UTIs report mixed outcomes, including failures to meet clinical and microbiological endpoints. Here we follow the population dynamics of a clinical Escherichia coli UTI strain and two phages, HP3 and ES19, to which the strain appears susceptible by standard testing. Despite this apparent susceptibilty, both phages fail to suppress the strain, with resistance emerging almost immediately. Using the measured mutation rate, our mathematical model shows that traditional resistance cannot account for these dynamics. We instead demonstrate, including by a phage-specific population analysis profile assay we developed, that heteroresistance drives this rapid failure, offering a plausible explanation for treatment failures in UTI phage therapy.

evolutionary biology↗

bioRxiv2026

The Joint Action of Bacteriophage and Antibiotics in the Treatment of Staphylococcus aureus infections in Galleria mellonella

Given the global rise of antibiotic resistance, there has been a resurgence of interest in bacteriophage therapy, typically administered concomitantly with antibiotics and currently used as a last resort treatment. In this study, we use the Galleria mellonella model to investigate the treatment outcomes and dynamics of joint therapy against a toxigenic and pathogenic strain of Staphylococcus aureus. While our previous research demonstrated that single-agent therapy, whether using bactericidal or bacteriostatic antibiotics or lytic phage, could suppress infections below a critical threshold, it rarely achieved complete bacterial eradication. Here, we show that the coadministration of a phage PYOSa with antibiotics generally enhances clearance, regardless of the antibiotic class. Joint therapy with daptomycin resulted in the complete clearance of infecting bacteria in the majority of larvae. Notably, even when combined with ampicillin, to which the bacteria are highly resistant, approximately half of the larvae achieved infection clearance. Taken together, these results demonstrate that joint therapy with phage and antibiotics enhances clearance beyond what either agent achieves alone, while underscoring that treatment timing and drug-specific pharmacodynamics remain critical determinants of therapeutic outcome. Significance StatementThe global rise of antibiotic resistance has renewed interest in bacteriophage therapy, which is almost universally administered concomitantly with antibiotics in clinical practice. Using Galleria mellonella (the wax moth larvae), which possess an innate immune response functionally similar to that of mammals, we demonstrate that coadministration of bacteriophages and antibiotics significantly enhances infection clearance compared with single-agent therapies. Critically, this joint action of antibiotics and phage can achieve bacterial eradication even when employing antibiotics to which the bacteria are resistant. We also find that therapeutic efficacy is sensitive to treatment timing and the specific pharmacodynamics of each drug. These factors are not captured by standard in vitro assessments of antimicrobial activity. Together, these results motivate further quantitative study of clinically relevant dosing regimens to determine the impact of host-pathogen-drug interactions on treatment outcome.

microbiology↗

bioRxiv2026

The role of the innate immune system in shaping the dynamics of antimicrobial treatment

Contemporary antibiotic treatment almost solely considers the Minimum Inhibitory Concentration (MIC) when deciding to employ an antibiotic, often overlooking the critical role of host immunity. Using Galleria mellonella and a virulent strain of Staphylococcus aureus, we investigate the infection dynamics and treatment outcomes of antibiotics of different classes--including antibiotics to which the bacteria are resistant--and a lytic bacteriophage. Surprisingly, we find that the hosts ability to control bacterial density and survive infection does not depend on the specific type of antimicrobial agent nor the bacterias susceptibility to it. Our results demonstrate that the innate immune system is the primary factor in therapeutic success, capable of clearing even highly resistant infections, such as those involving beta-lactamase-producing or ribosomal mutant-resistant strains. These findings challenge the traditional antibiotic-centric view of infection outcomes and emphasize the need to account for host-pathogen-drug interactions beyond simple MIC measurements when designing clinical treatment regimens.

microbiology↗

bioRxiv2025

The joint action of antibiotics, bacteriophage, and the innate immune response in the treatment of bacterial infections

Studies of antimicrobial therapeutics have traditionally neglected the contribution of the host in determining the course of treatment and its outcome. One critical host element, which shapes the dynamics of treatment is the innate immune system. Studies of chemotherapeutics and complementary therapies such as bacteriophage (phage), are commonly performed with mice that purposely have an ablated innate immune system. Here, we generate a mathematical and computer-simulation model of the joint action of antibiotics, phage, and phagocytes. Our analysis of this model highlights the need for future studies to consider the role of the hosts innate immune system in determining treatment outcomes. Critically, our model predicts that the conditions under which resistance to the treatment agent(s) will emerge are much narrower than commonly anticipate. We also generate a second model to predict the dynamics of treatment when multiple phages are used. This model provides support for the application of cocktails to treat infections rather than individual phages. Overall, this study provides hypotheses that can readily be tested experimentally with both in vitro and in vivo experiments.

microbiology↗

bioRxiv2024

Antibiotic killing of drug-induced bacteriostatic cells

SynopsisO_ST_ABSBackgroundC_ST_ABSThere is a long-standing belief that bacteriostatic drugs are inherently antagonistic to the action of bactericidal antibiotics. This belief is primarily due to the fact that the action of most bactericidal antibiotics requires the target bacteria to be growing. Since bacteriostatic drugs stop the growth of treated bacteria, these drugs would necessarily work against one another. We have recently shown that bacteria treated with high concentrations of bacteriostatic drugs retain some metabolic activity, dividing on average once per day. ObjectivesWe seek to determine if this low level of growth is sufficient to allow for bactericidal antibiotics of different classes to still kill after bacteria are treated with bacteriostatic drugs. MethodsWe first treated Escherichia coli and Staphylococcus aureus with two different bacteriostatic drugs, followed by one of three bactericidal drugs of three different classes. The density of these bacteria was tracked over six days to determine the amount of killing that occurred. ResultsOur results question this long-standing belief by demonstrating conditions where sequential treatment with a bacteriostatic then bactericidal antibiotic is as or more effective than treatment with a bactericidal drug alone. ConclusionsThese results raise the need to investigate the pharmacodynamics of the joint action of bacteriostatic and bactericidal antibiotics in vitro and in vivo.

microbiology↗

bioRxiv2024

The dynamics of Staphylococcal infection and their treatment with antibiotics and bacteriophage in the Galleria mellonella model system

Critical to our understanding of infections and their treatment is the role the innate immune system plays in controlling bacterial pathogens. Nevertheless, many in vivo systems are made or modified such that they do not have an innate immune response. Use of these systems denies the opportunity to examine the synergy between the immune system and antimicrobial agents. In this study we demonstrate that the larva of Galleria mellonella is an effective in vivo model for the study of the population and evolutionary biology of bacterial infections and their treatment. To do this we test three hypotheses concerning the role of the innate immune system during infection. We show: i) sufficiently high densities of bacteria are capable of saturating the innate immune system, ii) bacteriostatic drugs and bacteriophages are as effective as bactericidal antibiotics in preventing mortality and controlling bacterial densities, and iii) minority populations of bacteria resistant to a treating antibiotic will not ascend. Using a highly virulent strain of Staphylococcus aureus and a mathematical computer-simulation model, we further explore how the dynamics of the infection within the short term determine the ultimate infection outcome. We find that immune activation in response to high densities of bacteria leads to a strong but short-lived immune response which ultimately results in a high degree of mortality. Overall, our findings illustrate the utility of the G. mellonella model system in conjunction with established in vivo models in studying infectious disease progression and treatment. Significance statementCentral to our understanding of the course of bacterial infections and their treatment is the contribution of the innate immune system. We use the larvae of Galleria mellonella to test hypothesis about the role of the innate immune system on Staphylococcus aureus infections. We demonstrate that the innate immune system of these larvae can control the infection and be saturated by high bacterial densities. As a consequence of this innate immune system, bacteriostatic drugs and phages are as effective as bactericidal drugs, and minority populations of bacteria resistant to antibiotics do not ascend. Our findings illustrate the utility of G. mellonella as a model for studying infections dynamics and therapeutic strategies.

microbiology↗

bioRxiv2024

The Tradeoffs Between Persistence and Mutation Rates at Sub-Inhibitory Antibiotic Concentrations in Staphylococcus aureus

The rational design of the antibiotic treatment of bacterial infections employs these drugs to reach concentrations that exceed the minimum needed to prevent the replication of the target bacteria. However, within a treated patient, spatial and physiological heterogeneity promotes antibiotic gradients such that the concentration of antibiotics at specific sites is below the minimum needed to inhibit bacterial growth. Here, we investigate the effects of sub-inhibitory antibiotic concentrations on three parameters central to bacterial infection and the success of antibiotic treatment, using in vitro experiments with Staphylococcus aureus and mathematical-computer simulation models. Our results, using drugs of six different classes, demonstrate that exposure to sub-inhibitory antibiotic concentrations not only alters the dynamics of bacterial growth but also increases the mutation rate to antibiotic resistance and decreases the rate of production of persister cells thereby reducing the persistence level. Understanding this trade-off between mutation rates and persistence levels resulting from sub-inhibitory antibiotic exposure is crucial for optimizing, and mitigating the failure of, antibiotic therapy.

microbiology↗

bioRxiv2023

The Evolution of Heteroresistance via Small Colony Variants in Escherichia coli Following Long Term Exposure to Bacteriostatic Antibiotics

Traditionally, bacteriostatic antibiotics are agents able to arrest bacterial growth. Despite being traditionally viewed as unable to kill bacterial cells, when they are used clinically the outcome of these drugs is frequently as effective as when a bactericidal drug is used. We explore the dynamics of Escherichia coli after exposure to two ribosome-targeting bacteriostatic antibiotics, chloramphenicol and azithromycin, for thirty days. The results of our experiments provide evidence that bacteria exposed to these drugs replicate, evolve, and generate a sub-population of small colony variants (SCVs) which are resistant to multiple drugs. These SCVs contribute to the evolution of heteroresistance and rapidly revert to a susceptible state once the antibiotic is removed. Stated another way, exposure to bacteriostatic drugs selects for the evolution of heteroresistance in populations previously lacking this trait. More generally, our results question the definition of bacteriostasis as populations exposed to bacteriostatic drugs are replicating despite the lack of net growth.

microbiology↗

bioRxiv2023

Theoretical Considerations and Empirical Predictions of the Pharmaco- and Population Dynamics of Heteroresistance

Antibiotics are considered one of the most important contributions to clinical medicine in the last 100 years. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulations models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend the definition of heteroresistance should include a consideration of the rate of return to susceptibility. Significance StatementThis mathematical modeling and computer-simulation study quantitatively explores two broadly different, previously undescribed, classes of heteroresistance. In our analysis of the properties of these models, we consider the response of heteroresistant populations to antibiotic exposure, focusing on the conditions where heteroresistance could lead to clinical treatment failure. We also provide novel consideration to the rate of reversion from a resistant to sensitive state. Our analysis illustrates the need to include the reversion rate from resistant to sensitive in the definition of heteroresistance and questions the sufficiency of the method currently used to identify heteroresistance.

microbiology↗

bioRxiv2022

What's the matter with MICs: The contribution of nutrients and limiting resources to the pharmacodynamics of antibiotics and bacteria

The minimum inhibitory concentration (MIC) of an antibiotic required to prevent replication is used both as a measure of the susceptibility/resistance of bacteria to that drug and as the single pharmacodynamic parameter for the rational design of antibiotic treatment regimes. MICs are estimated in vitro under conditions optimal for the action of the antibiotic. However, bacteria rarely grow in these optimal conditions. Using a mathematical model of the pharmacodynamics of antibiotics, we make predictions about the nutrient dependency of bacterial growth in the presence of antibiotics. We test these predictions with experiments in a rich media and a glucose-limited minimal media with Escherichia coli and eight different antibiotics. Our experiments uncover properties that question the sufficiency of using MICs and simple pharmacodynamic functions as measures of the pharmacodynamics of antibiotics under the nutritional conditions of infected tissues. To an extent that varies among drugs: (i) The estimated MICs obtained in rich media are greater than those estimated in minimal media. (ii) Exposure to these drugs increases the time before logarithmic growth starts, their lag. (iii) The stationary phase density of E. coli populations declines with greater sub-MIC antibiotic concentrations. We postulate a mechanism to account for the relationship between the sub-MIC concentration of antibiotics and the stationary phase density of bacteria and provide evidence in support of this hypothesis. We discuss the implications of these results to our understanding of the MIC as the unique pharmacodynamic parameter used to design protocols for antibiotic treatment.

microbiology↗

bioRxiv2019

The inactivation of enzymes belonging to the central carbon metabolism, a novel mechanism of developing antibiotic resistance

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate (PEP) that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from PEP to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly used in the last years, mainly for treating infections caused by Gram-negative multidrug resistant bacteria as Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to antibiotics of common use. The mechanisms of mutational resistance to fosfomycin in S. maltophilia were studied in the current work. None of the mechanisms so far described for other organisms, which include the production of fosfomycin inactivating enzymes, target modification, induction of alternative peptidoglycan biosynthesis pathway and the impaired entrance of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Rather the unique cause of resistance in the studied mutants is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants showing that neither the inactivation nor the transport of the antibiotic were involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes neither any related enzyme. Finally, the mutants did not present an increased PEP concentration that might compete with fosfomycin for its binding to MurA. Based on these results, we describe a completely novel mechanism of antibiotic resistance based on the remodeling of S. maltophilia metabolism.\n\nSignificanceAntibiotic resistance (AR) has been largely considered as a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated in mechanisms that affect the antibiotics (mutations in transporters, the activity of efflux pumps and antibiotic modifying enzymes) or their targets (i.e.: target mutations, protection or bypass). Usually, AR-associated metabolic changes were considered to be a consequence (fitness costs) and not a cause of AR. Herein, we show that strong alterations in the bacterial metabolism can also be the cause of AR. In the study here presented, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that AR can be under metabolic control, our results provide evidence that AR is intertwined with the bacterial metabolism.

microbiology↗