Image-based dynamic phenotyping reveals genetic determinants of filamentation-mediated beta-lactam tolerance

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. beta-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to beta-lactam tolerance. Systematic discovery of genetic factors that affect beta-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the beta-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by beta-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after beta-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated beta-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains

Persisters show heritable phenotype and generate bacterial heterogeneity and noise in protein expression

Persisters are a small subpopulation of bacteria that survive a lethal concentration of antibiotic without antibiotic resistance genes. Isolation of persisters from normally dividing population is considered difficult due to their slow growth, low numbers and phenotypic shift i.e. when re-grown in antibiotic free medium, they revert to parent population. Inability to isolate persisters is a major hindrance in this field of research. Here we reject the ‘phenotypic shift’ phenomenon exhibited by persisters. Persisters, on the other hand, exhibit a heritable phenotype and can be easily isolated from a normally dividing population that allows their selective growth. Rather than a single subset, they comprise many distinct subgroups each exhibiting different growth rates, colony sizes, antibiotic tolerance and protein expression levels. Clearly, they are one of the sources of bacterial heterogeneity and noise in protein expression. Existence of persisters in normally dividing population can explain some of the unsolved puzzles like antibiotic tolerance, post-antibiotic effect and viable but non-culturable bacterial state. We hypothesize that persisters are aging bacteria

Persisters show heritable phenotype and generate bacterial heterogeneity and noise in protein expression

Persisters are a small subpopulation of bacteria that survive a lethal concentration of antibiotic without antibiotic resistance genes. Isolation of persisters from normally dividing population is considered difficult due to their slow growth, low numbers and phenotypic shift i.e. when re-grown in antibiotic free medium, they revert to parent population. Inability to isolate persisters is a major hindrance in this field of research. Here we reject the ‘phenotypic shift’ phenomenon exhibited by persisters. Persisters, on the other hand, exhibit a heritable phenotype and can be easily isolated from a normally dividing population that allows their selective growth. Rather than a single subset, they comprise many distinct subgroups each exhibiting different growth rates, colony sizes, antibiotic tolerance and protein expression levels. Clearly, they are one of the sources of bacterial heterogeneity and noise in protein expression. Existence of persisters in normally dividing population can explain some of the unsolved puzzles like antibiotic tolerance, post-antibiotic effect and viable but non-culturable bacterial state. We hypothesize that persisters are aging bacteria

Restriction of arginine induces antibiotic tolerance in <i>Staphylococcus aureus</i>

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections

pruR and PA0065 Genes Are Responsible for Decreasing Antibiotic Tolerance by Autoinducer Analog-1 (AIA-1) in Pseudomonas aeruginosa

Pseudomonas aeruginosa infection is considered a high-risk nosocomial infection and is very difficult to eradicate because of its tolerance to antibiotic treatment. A new compound, autoinducer analog-1 (AIA-1), has been demonstrated to reduce antibiotic tolerance, but its mechanisms remain unknown. This study aimed to investigate the mechanisms of AIA-1 in the antibiotic tolerance of P. aeruginosa. A transposon mutant library was constructed using miniTn5pro, and screening was performed to isolate high tolerant mutants upon exposure to biapenem and AIA-1. We constructed a deletion mutant and complementation strain of the genes detected in transposon insertion site determination, pruR and PA0066-65-64, and performed killing assays with antibiotics and AIA-1. Gene expression upon exposure to biapenem and AIA-1 and their relationship to stress response genes were analyzed. High antibiotic tolerance was observed in Tn5-pruR and Tn5-PA0065 transposon mutants and their deletion mutants, ΔpruR and ΔPA0066-65-64. Complemented strains of pruR and PA0066-65-64 with their respective deletion mutants exhibited suppressed antibiotic tolerance. It was determined that deletion of PA0066-65-64 increased rpoS expression, and PA0066-65-64 affects antibiotic tolerance via the rpoS pathway. Additionally, antibiotics and AIA-1 were found to inhibit pruR and PA0066-65-64. This study proposed that pruR and PA0066-65-64 are members of the antibiotic tolerance suppressors

Compounding Effects of Climate Warming and Antibiotic Resistance.

Bacteria have evolved diverse mechanisms to survive environments with antibiotics. Temperature is both a key factor that affects the survival of bacteria in the presence of antibiotics and an environmental trait that is drastically increasing due to climate change. Therefore, it is timely and important to understand links between temperature changes and selection of antibiotic resistance. This review examines these links by synthesizing results from laboratories, hospitals, and environmental studies. First, we describe the transient physiological responses to temperature that alter cellular behavior and lead to antibiotic tolerance and persistence. Second, we focus on the link between thermal stress and the evolution and maintenance of antibiotic resistance mutations. Finally, we explore how local and global changes in temperature are associated with increases in antibiotic resistance and its spread. We suggest that a multidisciplinary, multiscale approach is critical to fully understand how temperature changes are contributing to the antibiotic crisis

Robustness analysis of culturing perturbations on Escherichia coli colony biofilm beta-lactam and aminoglycoside antibiotic tolerance

<p>Abstract</p> <p>Background</p> <p>Biofilms are ubiquitous. For instance, the majority of medical infections are thought to involve biofilms. However even after decades of investigation, the <it>in vivo </it>efficacy of many antimicrobial strategies is still debated suggesting there is a need for better understanding of biofilm antimicrobial tolerances. The current study's goal is to characterize the robustness of biofilm antibiotic tolerance to medically and industrially relevant culturing perturbations. By definition, robust systems will return similar, predictable responses when perturbed while non-robust systems will return very different and potentially unpredictable responses. The predictability of an antibiotic tolerance response is essential to developing, testing, and employing antimicrobial strategies.</p> <p>Results</p> <p>The antibiotic tolerance of <it>Escherichia coli </it>colony biofilms was tested against beta-lactam and aminoglycoside class antibiotics. Control scenario tolerances were compared to tolerances under culturing perturbations including 1) different nutritional environments 2) different temperatures 3) interruption of cellular quorum sensing and 4) different biofilm culture ages. Here, antibiotic tolerance was defined in terms of culturable biofilm cells recovered after a twenty four hour antibiotic treatment.</p> <p>Colony biofilm antibiotic tolerances were not robust to perturbations. Altering basic culturing parameters like nutritional environment or temperature resulted in very different, non-intuitive antibiotic tolerance responses. Some minor perturbations like increasing the glucose concentration from 0.1 to 1 g/L caused a ten million fold difference in culturable cells over a twenty four hour antibiotic treatment.</p> <p>Conclusions</p> <p>The current study presents a basis for robustness analysis of biofilm antibiotic tolerance. Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.</p

Exploring Heterogeneous Phenotypes in Response to Stress

This work combines traditional microbiology with bioinformatic and synthetic biology approaches to study antibiotic tolerance. Antibiotic tolerance is a widespread phenomenon that facilitates antibiotic resistance and decreases the effectiveness of antibiotic treatment. Tolerance is distinct from antibiotic resistance, because tolerance is short term survival and typically results from phenotypic variations rather than genetic variation. The molecular mechanisms underlying tolerance are varied and debated in the literature. I have explored two intracellular processes related to tolerance, toxin-antitoxin (TA) systems (Chapter 2) and proteases (Chapter 4). Specifically, I focus on the ratio of antitoxin-to-toxin in type II TA systems, because type II TA systems must be regulated in such a way that antitoxins are more prevalent than their toxins. Our analysis of RNA-sequencing and ribosome profiling data demonstrates that most type II TA systems in E. coli are regulated at the translational level, while others rely on various combinations of transcriptional and post-transcriptional regulation. Before publishing this article, researchers often cited transcriptional regulation as the primary method of regulating TA systems. Studying antibiotic tolerance and other subpopulations necessitates the ability to study single-cell dynamics in the context of the whole population. To facilitate single-cell analysis, we have developed single-cell tracking software that leverages machine learning to identify cells. The software then tracks the cell based on this classification and returns data on cell size, location, division and fluorescence. The software provides the means of quantifying cell behavior before and after antibiotic treatment. One such system we would like to apply this software to is our work on proteolytic queueing and antibiotic tolerance. Proteases are responsible for protein degradation and, as such, regulate many cellular functions. To better identify the role proteases play in persistence, we used proteolytic queueing to interfere with proteolytic activity. We found that interfering with degradation at the protease ClpXP increases antibiotic tolerance ~80 and ~60 fold in an E. coli population treated with ampicillin and ciprofloxacin, respectively. I used stochastic modeling to support our results, and we have experimentally determined that altering the expression of the synthetic system affects the level of tolerance in the population. I am currently using next-generation sequencing to identify the systems being affected by the queue

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems

Fitness Landscape of Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, we used genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1 in biofilm and planktonic growth conditions with and without tobramycin to systematically quantify the contribution of each locus to antibiotic tolerance under these two states. We identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only, offering global insights into the differences and similarities between biofilm and planktonic antibiotic tolerance. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections