Persister control by leveraging dormancy associated reduction of antibiotic efflux

Persistent bacterial infections do not respond to current antibiotic treatment and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 μg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 μg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles’ heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.

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Zinc Acetate Potentiates the Action of Tosufloxacin against Escherichia coli Biofilm Persisters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00069-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 2

Author(s):

Masaru Usui ◽

Hayato Yokoo ◽

Yutaka Tamura ◽

Chie Nakajima ◽

Yasuhiko Suzuki ◽

...

Keyword(s):

Escherichia Coli ◽

Zinc Acetate ◽

Bacterial Infections ◽

Sos Response ◽

Persister Cells ◽

Major Health ◽

Content Type ◽

E Coli ◽

Potential Strategy ◽

Multiple Antibiotics

ABSTRACT Formation of bacterial biofilms is a major health threat due to their high levels of tolerance to multiple antibiotics and the presence of persisters responsible for infection relapses. We previously showed that a combination of starvation and induction of SOS response in biofilm led to increased levels of persisters and biofilm tolerance to fluoroquinolones. In this study, we hypothesized that inhibition of the SOS response may be an effective strategy to target biofilms and fluoroquinolone persister cells. We tested the survival of Escherichia coli biofilms to different classes of antibiotics in starved and nonstarved conditions and in the presence of zinc acetate, a SOS response inhibitor. We showed that zinc acetate potentiates, albeit moderately, the activity of fluoroquinolones against E. coli persisters in starved biofilms. The efficacy of zinc acetate to increase fluoroquinolone activity, particularly that of tosufloxacin, suggests that such a combination may be a potential strategy for treating biofilm-related bacterial infections.

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Control of Bacterial Persister Cells by Trp/Arg-Containing Antimicrobial Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.02440-10 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4878-4885 ◽

Cited By ~ 53

Author(s):

Xi Chen ◽

Mi Zhang ◽

Chunhui Zhou ◽

Neville R. Kallenbach ◽

Dacheng Ren

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Future Development ◽

Bacterial Population ◽

Lower Threshold ◽

Chronic Infections ◽

Persister Cells ◽

Normal Cells ◽

Content Type ◽

High Concentrations

ABSTRACTPersister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells ofEscherichia coliHM22, a hyper-persister producer. The activities of three linear peptides [(RW)n-NH2, wherenis 2, 3, or 4] and a dendrimeric peptide, (RW)4D, in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)4Drequires a lower threshold to kill planktonic persisters, octameric peptide (RW)4-NH2is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 μM (RW)4-NH2for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)4-NH2and (RW)4D. These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)4-NH2was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections.

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Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01481-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6867-6871 ◽

Cited By ~ 28

Author(s):

Peng Cui ◽

Hongxia Niu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Hao Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Enhancement Effect ◽

Bacterial Cells ◽

Sulfa Drugs ◽

Content Type ◽

Bacterial Membranes ◽

Highly Active ◽

High Concentrations

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

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Garlic Extract (Allium sativum L.) Effectively Inhibits Staphylococcus aureus and Escherichia coli by Invitro Test

Tropical Health and Medical Research ◽

10.35916/thmr.v0i0.22 ◽

2020 ◽

Vol 2 (2) ◽

pp. 61-68

Author(s):

Agnina Listya Anggraini ◽

Ratih Dewi Dwiyanti ◽

Anny Thuraidah

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Bacterial Infections ◽

Allium Sativum ◽

Antibacterial Properties ◽

Herbal Medicines ◽

Garlic Extract ◽

Dilution Method ◽

Initial Stage

Infection is a disease caused by the presence of pathogenic microbes, including Staphylococcus aureus and Escherichia coli. Garlic (Allium sativum L.) has chemical contents such as allicin, alkaloids, flavonoids, saponins, tannins, and steroids, which can function as an antibacterial against Staphylococcus aureus and Escherichia coli. This study aims to determine the antibacterial properties of garlic extract powder against Staphylococcus aureus and Escherichia coli. This research is the initial stage of the development of herbal medicines to treat Staphylococcus aureus and Escherichia coli infections. The antibacterial activity test was carried out by the liquid dilution method. The concentrations used were 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL and 70 mg/mL. The results showed that the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus and Escherichia coli was 40 mg/mL and 50 mg / mL. Minimum Bactericidal Concentration (MBC) results for Staphylococcus aureus and Escherichia coli are 50 mg/mL and 70 mg/mL. Based on the Simple Linear Regression test, the R2 value of Staphylococcus aureus and Escherichia coli is 0.545 and 0.785, so it can be concluded that there is an effect of garlic extract powder on the growth of Staphylococcus aureus and Escherichia coli by 54.5% and 78.5%. Garlic (Allium sativum L.) extract powder has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

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The Potential of Human Peptide LL-37 as an Antimicrobial and Anti-Biofilm Agent

Antibiotics ◽

10.3390/antibiotics10060650 ◽

2021 ◽

Vol 10 (6) ◽

pp. 650

Author(s):

Kylen E. Ridyard ◽

Joerg Overhage

Keyword(s):

Bacterial Infections ◽

Antimicrobial Properties ◽

Cross Resistance ◽

Resistant Bacteria ◽

Peptide Antibiotic ◽

Adaptive Responses ◽

Antibiotic Resistant ◽

Structural Modifications ◽

Immobilization Techniques ◽

Conventional Antibiotics

The rise in antimicrobial resistant bacteria threatens the current methods utilized to treat bacterial infections. The development of novel therapeutic agents is crucial in avoiding a post-antibiotic era and the associated deaths from antibiotic resistant pathogens. The human antimicrobial peptide LL-37 has been considered as a potential alternative to conventional antibiotics as it displays broad spectrum antibacterial and anti-biofilm activities as well as immunomodulatory functions. While LL-37 has shown promising results, it has yet to receive regulatory approval as a peptide antibiotic. Despite the strong antimicrobial properties, LL-37 has several limitations including high cost, lower activity in physiological environments, susceptibility to proteolytic degradation, and high toxicity to human cells. This review will discuss the challenges associated with making LL-37 into a viable antibiotic treatment option, with a focus on antimicrobial resistance and cross-resistance as well as adaptive responses to sub-inhibitory concentrations of the peptide. The possible methods to overcome these challenges, including immobilization techniques, LL-37 delivery systems, the development of LL-37 derivatives, and synergistic combinations will also be considered. Herein, we describe how combination therapy and structural modifications to the sequence, helicity, hydrophobicity, charge, and configuration of LL-37 could optimize the antimicrobial and anti-biofilm activities of LL-37 for future clinical use.

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How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile

Membranes ◽

10.3390/membranes11010048 ◽

2021 ◽

Vol 11 (1) ◽

pp. 48

Author(s):

Ana Rita Ferreira ◽

Cátia Teixeira ◽

Carla F. Sousa ◽

Lucinda J. Bessa ◽

Paula Gomes ◽

...

Keyword(s):

Electrostatic Interactions ◽

Bacterial Infections ◽

Electron System ◽

Bacterial Membranes ◽

Highly Active ◽

Large Unilamellar Vesicles ◽

Biophysical Profile ◽

N Terminus ◽

Lysine Residues ◽

Preferential Location

In the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π–electron system of tryptophan, resulting in negatively charged clouds, that participate in cation–π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.

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Three-Year Trend in Escherichia coli Antimicrobial Resistance among Children’s Urine Cultures in an Italian Metropolitan Area

Children ◽

10.3390/children8070597 ◽

2021 ◽

Vol 8 (7) ◽

pp. 597

Author(s):

Luca Pierantoni ◽

Laura Andreozzi ◽

Simone Ambretti ◽

Arianna Dondi ◽

Carlotta Biagi ◽

...

Keyword(s):

Escherichia Coli ◽

Metropolitan Area ◽

Bacterial Infections ◽

Asymptomatic Bacteriuria ◽

Multidrug Resistant ◽

Resistance Rate ◽

First Line ◽

E Coli ◽

First Line Therapy ◽

Tract Infections

Urinary tract infections (UTIs) are among the most common bacterial infections in children, and Escherichia coli is the main pathogen responsible. Several guidelines, including the recently updated Italian guidelines, recommend amoxicillin-clavulanic acid (AMC) as a first-line antibiotic therapy in children with febrile UTIs. Given the current increasing rates of antibiotic resistance worldwide, this study aimed to investigate the three-year trend in the resistance rate of E. coli isolated from pediatric urine cultures (UCs) in a metropolitan area of northern Italy. We conducted a retrospective review of E. coli-positive, non-repetitive UCs collected in children aged from 1 month to 14 years, regardless of a diagnosis of UTI, catheter colonization, urine contamination, or asymptomatic bacteriuria. During the study period, the rate of resistance to AMC significantly increased from 17.6% to 40.2% (p < 0.001). Ciprofloxacin doubled its resistance rate from 9.1% to 16.3% (p = 0.007). The prevalence of multidrug-resistant E. coli rose from 3.9% to 9.2% (p = 0.015). The rate of resistance to other considered antibiotics remained stable, as did the prevalence of extended spectrum beta-lactamases and extensively resistant E. coli among isolates. These findings call into question the use of AMC as a first-line therapy for pediatric UTIs in our population, despite the indications of recent Italian guidelines.

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Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

Communications Biology ◽

10.1038/s42003-021-02074-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Amit Gaurav ◽

Varsha Gupta ◽

Sandeep K. Shrivastava ◽

Ranjana Pathania

Keyword(s):

Escherichia Coli ◽

Acinetobacter Baumannii ◽

Nalidixic Acid ◽

Bacterial Infections ◽

Clinical Isolates ◽

Hospital Environment ◽

Infection Model ◽

Drug Resistant ◽

E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

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Genomic Characterization of New Variant of Hydrogen Sulfide (H2S)-Producing Escherichia coli with Multidrug Resistance Properties Carrying the mcr-1 Gene in China

Antibiotics ◽

10.3390/antibiotics9020080 ◽

2020 ◽

Vol 9 (2) ◽

pp. 80 ◽

Cited By ~ 7

Author(s):

Silpak Biswas ◽

Mohammed Elbediwi ◽

Guimin Gu ◽

Min Yue

Keyword(s):

Escherichia Coli ◽

Hydrogen Sulfide ◽

Multidrug Resistance ◽

Bacterial Infections ◽

Multidrug Resistant ◽

Health Concern ◽

Colistin Resistance ◽

New Variant ◽

Genomic Characterization ◽

Human Infections

Colistin is considered to be a ‘last-resort’ antimicrobial for the treatment of multidrug-resistant Gram-negative bacterial infections. Identification of Enterobacteriaceae, carrying the transferable colistin resistance gene mcr-1, has recently provoked a global health concern. This report presents the first detection of a hydrogen sulfide (H2S)-producing Escherichia coli variant isolated from a human in China, with multidrug resistance (MDR) properties, including colistin resistance by the mcr-1 gene, which could have great implications for the treatment of human infections.

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Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013587 ◽

2020 ◽

Vol 295 (38) ◽

pp. 13314-13325

Author(s):

Yanyu Zhu ◽

James C. Weisshaar ◽

Mainak Mustafi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Binding Site ◽

Elongation Factor ◽

Microscopy Study ◽

Single Step ◽

Long Term Effects ◽

Bacterial Membranes

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

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