Persistent Cell Populations Research Articles

Lactobacillus Persisters Formation and Resuscitation.

Lactobacillus is a commonly used probiotic, and many researchers have focused on its stress response to improve its functionality and survival. However, studies on persister cells, dormant cells that aid bacteria in surviving general stress, have focused on pathogenic bacteria that cause infection, not Lactobacillus. Thus, understanding Lactobacillus persister cells will provide essential clues for understanding how Lactobacillus survives and maintains its function under various environmental conditions. We treated Lactobacillus strains with various antibiotics to determine the conditions required for persister formation using kill curves and transmission electron microscopy. In addition, we observed the resuscitation patterns of persister cells using single-cell analysis. Our results show that Lactobacillus creates a small population of persister cells (0.0001-1% of the bacterial population) in response to beta-lactam antibiotics such as ampicillin and amoxicillin. Moreover, only around 0.5-1% of persister cells are heterogeneously resuscitated by adding fresh media; the characteristics are typical of persister cells. This study provides a method for forming and verifying the persistence of Lactobacillus and demonstrates that antibiotic-induced Lactobacillus persister cells show characteristics of dormancy, sensitivity of antibiotics, same as exponential cells, multi-drug tolerance, and resuscitation, which are characteristics of general persister cells. This study suggests that the mechanisms of formation and resuscitation may vary depending on the characteristics, such as the membrane structure of the bacterial species.

Salicylic acids and pathogenic bacteria: new perspectives on an old compound.

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid-immune modulation in response to microbial infection-was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines.

Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms.

Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

The recognition, control, and stress tolerance of bacterial persister cells

Bacterial persister cells are metabolically dormant cells in which cell activities such as protein synthesis and cell division are somehow drastically slowed down, so that they can survive under intensive antibiotic treatment that targets such cell activities. This review summarizes information about persister cells such as their history, definition, mechanism of formation, threats to clinical environments, and how to control them. In addition to such information, this review also introduces our recent studies on the physical stress tolerance of persister cells and their possible control in food environments. We demonstrated that some bacterial persister cells are indeed highly heat and acid tolerant compared to normal cells, although diversity was discovered in persister cell populations. We also found that indole and its derivatives or acetic acid are promising candidates for persister cell eradication in food environments. We believe that this review will aid in wider attention to and understanding of the importance of these quiet yet clever bacterial persister cells.

Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish.

ABSTRACTMelanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.

Abstract 1598: Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy characterized by robust, however transient, responses to frontline platinum-based therapy that are rapidly followed by refractory relapses. Treatment resistance is a significant concern in SCLC and a major factor regulating resistance is an emergence of unique cellular populations and an increase in intratumoral heterogeneity (ITH). We hypothesize that these emerging populations expressing various resistance-associated pathways, represent multiple, unique drug-resistant persister cell populations that underlie increased ITH and promote therapeutic resistance and, eventually, relapse, despite the initial response to therapy. Persister cells represent a unique population of cancer cells that are resistant to therapeutic pressure. While most persister cells remain arrested under treatment, a rare sub-population exists that can reverse state and re-enter the cell cycle. We performed single cell RNAseq of relapsed, extensive stage SCLC patient paired core biopsies collected 1) following relapse to standard of care (SOC) platinum chemotherapy plus immune checkpoint blockade (ICB) and 2) after six weeks of further therapy. Unlike what we found in platinum-sensitive disease, there is not a meaningful change in ITH score between cancer cells in the first and second biopsy. Presumably, maximum heterogeneity developed along with relapse to SOC treatment. Biopsies represent SCLC across subtypes (SCLC-A/N/P/I) with a modest loss of transcription factor expression following treatment (e.g., 76.8% to 57.9% NEUROD1). Cancer cells were classified as cycling or non-cycling to identify potential persister cell populations in paired patient core biopsies. Molecular subtype marker ASCL1 was reduced in non-cycling cells, but there was no change in NEUROD1 or POU2F3. Cycling cells demonstrate increased expression of NE genes (SYP, INSM1, UCHL1), biomarkers of response (SLFN11 and CDH1), but also genes associated with resistance (EZH2, NFIB). Non-cycling cells exhibit some common resistance mechanisms (e.g., EMT and MYC, AXL, ZFP36L1, and REST overexpression) and increased expression of inflammatory genes (e.g. HLA family) compared to cycling cells, reminiscent of the SCLC-Inflamed (SCLC-I) subtype. These data suggest that non-cycling persister cells may be more sensitive to ICB, AXL and/or AURK inhibition, while cycling cells may be more responsive to platinum chemotherapy, epigenetic modifiers or DNA repair targeted therapies. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors, while highlighting the transcriptional complexity underlying SCLC’s profound treatment resistance following SOC and address the major need to develop combination therapies to target these distinct cell populations. Citation Format: C. Allison Stewart, Yuanxin Xi, Runsheng Wang, Michael M. Frumovitz, Jing Wang, Lauren A. Byers, Carl M. Gay. Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1598.

Synergistic effect of antibiotic combinations on Staphylococcus aureus biofilms and their persister cell populations

Treatment of staphylococcal infections is difficult due to multidrug resistance with their persister forms posing an added threat of recalcitrant infections. Antibiotic combinations are widely studied as an alternative strategy to combat them; therefore, they merit further investigation into their effect on the number of persister cells. In the present study, the fractional inhibitory concentrations of antibiotic combinations ciprofloxacin-daptomycin, ciprofloxacin-vancomycin, daptomycin-tobramycin, and tobramycin-vancomycin (checkerboard assay) were determined against two previously studied clinical (S48 and J6) and one standard (NCIM 5021) isolate of Staphylococcus aureus. They showed synergistic effects with a 2 to 256-fold reduction in MICs. All combinations also resulted in inhibition and disruption of biofilms in a concentration-dependent manner. All antibiotic combinations, except ciprofloxacin-daptomycin, showed total biofilm inhibition at 100X MICs. Similarly, antibiotic combination at 100X MIC showed 77–97% disruption of preformed biofilms. Time-kill assays performed at a 100X MIC combination against stationary-phase cells showed a two to six log10 reduction in CFU followed by a plateau indicating the presence of persisters. Significant differences were observed in persister cell fraction remaining after treatment with antibiotic combinations compared to monotherapies (p < 0.05) and therefore merit further investigation in clinical use for treatment against persisters.

Leveraging Marine Natural Products as a Platform to Tackle Bacterial Resistance and Persistence.

Antimicrobial resistance to existing antibiotics represents one of the greatest threats to human health and is growing at an alarming rate. To further complicate treatment of bacterial infections, many chronic infections are the result of bacterial biofilms that are tolerant to treatment with antibiotics because of the presence of metabolically dormant persister cell populations. Together these threats are creating an increasing burden on the healthcare system, and a "preantibiotic" age is on the horizon if significant action is not taken by the scientific and medical communities. While the golden era of antibiotic discovery (1940s-1960s) produced most of the antibiotic classes in clinical use today, followed by several decades of limited development, there has been a resurgence in antibiotic drug discovery in recent years fueled by the academic and biotech sectors. Historically, great success has been achieved by developing next-generation variants of existing classes of antibiotics, but there remains a dire need for the identification of novel scaffolds and/or antimicrobial targets to drive future efforts to overcome resistance and tolerance. In this regard, there has been no more valuable source for the identification of antibiotics than natural products, with 69-77% of approved antibiotics either being such compounds or being derived from them.Our group has developed a program centered on the chemical synthesis and chemical microbiology of marine natural products with unusual structures and promising levels of activity against multidrug-resistant (MDR) bacterial pathogens. As we are motivated by preparing and studying the biological effects of these molecules, we are not initially pursuing a biological question but instead are allowing the observed phenotypes and activities to guide the ultimate project direction. In this Account, our recent efforts on the synoxazolidinone, lipoxazolidinone, and batzelladine natural products will be discussed and placed in the context of the field's greatest challenges and opportunities. Specifically, the synoxazolidinone family of 4-oxazolidinone-containing natural products has led to the development of several chemical methods to prepare antimicrobial scaffolds and has revealed compounds with potent activity as adjuvants to treat bacterial biofilms. Bearing the same 4-oxazolidinone core, the lipoxazolidinones have proven to be potent single-agent antibiotics. Finally, our synthetic efforts toward the batzelladines revealed analogues with activity against a number of MDR pathogens, highlighted by non-natural stereochemical isomers with superior activity and simplified synthetic access. Taken together, these studies provide several distinct platforms for the development of novel therapeutics that can add to our arsenal of scaffolds for preclinical development and can provide insight into the biochemical processes and pathways that can be targeted by small molecules in the fight against antimicrobial-resistant and -tolerant infections. We hope that this work will serve as inspiration for increased efforts by the scientific community to leverage synthetic chemistry and chemical microbiology toward novel antibiotics that can combat the growing crisis of MDR and tolerant bacterial infections.

A Robust Method for Generating, Quantifying, and Testing Large Numbers of Escherichia coli Persisters.

Bacteria can exhibit phenotypes that render them tolerant against antibiotics. However, often only a few cells of a bacterial population show the so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient shifts generate Escherichia coli populations that consist almost entirely of antibiotic-tolerant cells. These nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this chapter, we provide a detailed protocol for generating a large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and we provide a new way to assess antibiotic tolerance using flow cytometry. We envision that these methods will facilitate research into the important and exciting phenotype of bacterial persister cells.

Biofilm and persister cell fomation variability in clinical isolates of Klebsiella pneumoniae in Colombia

Klebsiella pneumoniae is an opportunistic pathogen associated with nosocomial infections. Persister cells are a fraction of a bacterial population that can escape antibiotic treatment and are associated with antibiotic therapy failure. In this work, we analyzed persistent cells in planktonic cultures and biofilms using10 K. pneumoniae clinical isolates and four different antibiotic types. The isolates had different antibiotic susceptibility profiles that did not correlate with their capacity to form biofilms. Persister cells were found under all conditions tested, although their population numbers varied depending on the antibiotic used. A larger number of persister cells were found in biofilms than in planktonic cultures. Antibiotic treatment with trimethoprim-sulfamethoxazole resulted in the largest persister cell sub-population compared with other antibiotics tested, while ciprofloxacin was the antibiotic that produced fewer persister cells. These results indicate that K. pneumoniae clinical isolates vary not only in their susceptibility to antibiotics but also in properties relevant to diseases, such as biofilm formation and persister cell populations.

Copper Kills Escherichia coli Persister Cells.

Due to their reduced metabolism, persister cells can survive most antimicrobial treatments, which usually rely on corrupting active biochemical pathways. Therefore, molecules that kill bacterial persisters should function in a metabolism-independent manner. Some anti-persister compounds have been found previously, such as the DNA-crosslinkers mitomycin C and cisplatin, but more effective and lower cost alternatives are needed. Copper alloys have been used since ancient times due to their antimicrobial properties, and they are still used in agriculture to control plant bacterial diseases. By stopping transcription with rifampicin and by treating with ampicillin to remove non-persister cells, we created a population that consists solely of Escherichia coli persister cells. Using this population of persister cells, we demonstrate that cupric compounds kill E. coli persister cells. Hence, copper ions may be used in controlling the spread of important bacterial strains that withstand treatment with conventional antimicrobials by forming persister cells.

Optimal Treatment Regimen of Using Chemo-Immunotherapy for Solid Tumors

Abstract Chemotherapeutic agents not only cause apoptosis and necrosis of cancer cells but also induce cellular senescence (TIS), resulting in a persistent cell population that re-enters the cell cycle with stemness characteristics via the senescence-associated secretory phenotype. These senescent cancer cells, therefore, seed disease relapses if they are not effectively eliminated. TIS cancer cells lack HLA expression and cannot be targeted by T cells. Herein, we show that chemotherapy-induced senescent B16F10 cells express the NK ligands Rae1e and Ulbp1, and are effectively killed by mouse NK cells in vitro. Docetaxel treatment also induced senescent B16F10 cells in vivo. We have previously shown that the TGF-β trap/IL-15 fusion protein, aka HCW9218, effectively activates NK cells in vivo. Therefore, we treated B16F10 tumor bearing mice with docetaxel and then a single dose of HCW9218 in combination with the ADCC antibody TA99. This treatment regimen exhibited potent antitumor activity compared to docetaxel alone. End point analysis showed that HCW9218-treated tumors have lower mitotic activity, reduced expression of senescence markers, and increased frequencies of infiltrating CD8 T cells. We further showed in this model that HCW9218 treatment followed by anti-PD-L1 therapy was more effective than anti-PD-L1 therapy followed by HCW9218. These results suggest that the optimal sequence of cancer treatment is: (1) standard-of-care chemotherapy to control tumor burden and induce cellular senescence, (2) immunotherapy for removal of senescent cells and enhanced tumor infiltration of CD8+ T cells and (3) checkpoint blockade to maintain infiltrated T cell effector function to enhance therapeutic responses and treatment durability.

Changes of cell permeability during Listeria monocytogenes persistence under nisin treatment

The study examined free radical production of Listeria monocytogenes under nisin stress by colorimetric cell proliferation assay, and used flow cytometry (FC) to investigate the persister population of L. monocytogenes since agar plating is limited in providing information on persister cells exposed to nisin. The stationary phase populations in spent medium or resuspended in fresh TSB medium were individually treated with 75 μg/ml nisin for 24 hr at 30°C. Intracellular and extracellular free radical production was detected in the resuspended group simultaneously, while free radicals were only detected within cells in the spent medium group during the nisin treatment, and FC indicated possible population differentiation from quadrants H1-1 (“dead region,”) H1-2 (“damaged region) and H1-3(“live region”). The change in cell permeability was different under the two conditions (spent medium vs. fresh medium) when facing exposure to nisin, indicating the effect of the environment on cell persistence under nisin stress. Practical applications The practical application of this work is an awareness of how the sensitivity of a food isolate of L. monocytogenes exposed to nisin as a food preservative, and survival of tested strains can be affected by the environment in generating a population of persistent cells. A nutrient rich environment appears to enhance the population of persister cells. This is an important consideration for the food industry trying to control L. monocytogenes in a nutrient rich environment such as food. In addition, FC can be used to rapidly assess the population of persister cells compared with dead cells and provide a useful tool in risk assessment of L. monocytogenes in a food where nisin is used as a preservative.

Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073

Urinary tract infections (UTIs) caused by Escherichia coli create a large burden on healthcare and frequently lead to recurrent infections. Part of the success of E. coli as an uropathogenic bacterium can be attributed to its ability to form quiescent intracellular reservoirs in bladder cells and its persistence after antibiotic treatment. Cranberry juice and related products have been used for the prevention of UTIs with varying degrees of success. In this study, a group of cranberry pectic oligosaccharides (cPOS) were found to both inhibit quiescence and reduce the population of persister cells formed by the uropathogenic strain, CFT073. This is the first report detailing constituents of cranberry with the ability to modulate these important physiological aspects of uropathogenic E. coli. Further studies investigating cranberry should be keen to include oligosaccharides as part of the ‘active’ cocktail of chemical compounds.

Interkingdom signal indole inhibits Pseudomonas aeruginosa persister cell waking.

Persister cells are stressed cells that have transient tolerance to antibiotics; these cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how persisters resuscitate, so we explored the waking of cells in the presence of the interkingdom signal indole. To generate a large population of persister cells, we induced the persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pretreating cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single cell observations, that proline is sufficient to wake the persister cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa persister cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa persister cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary-phase cultures, the indole produced by E. coli completely inhibits persister cell resuscitation of P. aeruginosa. Therefore, E. coli has devised a method to outcompete its competitors by preventing their resuscitation with indole. This work provides insight into why indole is produced by commensal bacteria.

Patient-Specific Tumor Growth Trajectories Determine Persistent and Resistant Cancer Cell Populations during Treatment with Targeted Therapies.

The importance of preexisting versus acquired drug resistance in patients with cancer treated with small-molecule tyrosine kinase inhibitors (TKI) remains controversial. The goal of this study is to provide a general estimate of the size and dynamics of a preexisting, drug-resistant tumor cell population versus a slow-growing persister population that is the precursor of acquired TKI resistance. We describe a general model of resistance development, including persister evolution and preexisting resistance, solely based on the macroscopic trajectory of tumor burden during treatment. We applied the model to 20 tumor volume trajectories of EGFR-mutant lung cancer patients treated with the TKI erlotinib. Under the assumption of only preexisting resistant cells or only persister evolution, it is not possible to explain the observed tumor trajectories with realistic parameter values. Assuming only persister evolution would require very high mutation induction rates, while only preexisting resistance would lead to very large preexisting populations of resistant cells at the initiation of treatment. However, combining preexisting resistance with persister populations can explain the observed tumor volume trajectories and yields an estimated preexisting resistant fraction varying from 10-4 to 10-1 at the time of treatment initiation for this study cohort. Our results also demonstrate that the growth rate of the resistant population is highly correlated to the time to tumor progression. These estimates of the size of the resistant and persistent tumor cell population during TKI treatment can inform combination treatment strategies such as multi-agent schedules or a combination of targeted agents and radiotherapy. SIGNIFICANCE: These findings quantify pre-existing resistance and persister cell populations, which are essential for the integration of targeted agents into the management of locally advanced disease and the timing of radiotherapy in metastatic patients.

The role of ATP pools in persister cell formation in (fluoro)quinolone-susceptible and -resistant strains of Salmonella enterica ser. Typhimurium

In this study, we investigated the reported dependence on the ATP pools for persister cell formation in fluoroquinolone-resistant variants of the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. We compared the generation of persister cell populations after ciprofloxacin challenge of wildtype and a nalidixic acid-resistant variant of S. Typhimurium with reduced ciprofloxacin-susceptibility, as well as strains containing a deletion of the atp operon or harbouring the cloned atp genes. A gyrA mutation (D87Y) was found to contribute to increased stationary phase formation of persister cells in S. Typhimurium. However, in contrast to expectations from prior studies, while treatment with the ATP synthase poison arsenate showed the expected increase in persister cells surviving ciprofloxacin treatment, a more direct approach using a strain of Salmonella deleted for the atp operon showed severe reductions in persister cell formation. Persister cell formation was recovered after introduction of the cloned atp operon which restored the reduced ATP levels. These results suggest either an alternative explanation for previous studies, or that persister cell formation in Salmonella is differently regulated.

Relating switching rates between normal and persister cells to substrate and antibiotic concentrations: a mathematical modelling approach supported by experiments.

SummaryWe developed and compared two mathematical models of variable phenotypic switching rates between normal and persister cells that depend on substrate concentration and antibiotic presence. They could be used to simulate the formation of persisters in environments with concentration gradients such as biofilms. Our models are extensions of a previous model of the dynamics of normal and persistent cell populations developed by Balaban et al. (2004, Science 305: 1622). We calibrated the models’ parameters with experimental killing curves obtained after ciprofloxacin treatment of samples regularly harvested from planktonic batch cultures of Klebsiella pneumoniae. Our switching models accurately reproduced the dynamics of normal and persistent populations in planktonic batch cultures and under antibiotic treatment. Results showed that the models are valid for a large range of substrate concentrations and for zero or high doses of antibiotics.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro.

Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

FGFR signaling maintains a drug persistent cell population following epithelial-mesenchymal transition.

An emerging characteristic of drug resistance in cancer is the induction of epithelial-mesenchymal transition (EMT). However, the mechanisms of EMT-mediated drug resistance remain poorly defined. Therefore, we conducted long-term treatments of human epidermal growth factor receptor-2 (Her2)-transformed breast cancer cells with either the EGFR/Her2 kinase inhibitor, Lapatinib or TGF-β, a known physiological inducer of EMT. Both of these treatment regimes resulted in robust EMT phenotypes, but upon withdrawal a subpopulation of TGF-β induced cells readily underwent mesenchymal-epithelial transition, where as Lapatinib-induced cells failed to reestablish an epithelial population. The mesenchymal population that remained following TGF-β stimulation and withdrawal was quickly selected for during subsequent Lapatinib treatment, manifesting in inherent drug resistance. The Nanostring cancer progression gene panel revealed a dramatic upregulation of fibroblast growth factor receptor 1 (FGFR1) and its cognate ligand FGF2 in both acquired and inherent resistance. Mechanistically, FGF:Erk1/2 signaling functions to stabilize the EMT transcription factor Twist and thus maintain the mesenchymal and drug resistant phenotype. Finally, Lapatinib resistant cells could be readily eliminated using recently characterized covalent inhibitors of FGFR. Overall our data demonstrate that next-generation targeting of FGFR can be used in combination with Her2-targeted therapies to overcome resistance in this breast cancer subtype.