Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to β-Lactams

ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia coli(p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purifiedE. colicomponents, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

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Discovery of a Novel Class of Boron-Based Antibacterials with Activity against Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02058-12 ◽

2013 ◽

Vol 57 (3) ◽

pp. 1394-1403 ◽

Cited By ~ 102

Author(s):

Vincent Hernandez ◽

Thibaut Crépin ◽

Andrés Palencia ◽

Stephen Cusack ◽

Tsutomu Akama ◽

...

Keyword(s):

Resistance Mechanisms ◽

Trna Synthetase ◽

Gram Negative Bacteria ◽

Aminoacyl Trna Synthetase ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Drug Classes ◽

Medical Need ◽

Infection Models

ABSTRACTGram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms inEscherichia coliandPseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, includingEnterobacteriaceaebearing NDM-1 and KPC carbapenemases, as well asP. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious againstE. coliandP. aeruginosain murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

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Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02552-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 2089-2097 ◽

Cited By ~ 47

Author(s):

Lee W. Goneau ◽

Nigel S. Yeoh ◽

Kyle W. MacDonald ◽

Peter A. Cadieux ◽

Jeremy P. Burton ◽

...

Keyword(s):

Differential Susceptibility ◽

Type I ◽

Antibiotic Tolerance ◽

Persister Cells ◽

Content Type ◽

E Coli ◽

Cellular Processes ◽

Multidrug Tolerance ◽

External Stimuli ◽

Susceptibility Patterns

ABSTRACTPersister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed betweenStaphylococcus saprophyticusandEscherichia coliuropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancyper secannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenicE. coliisolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

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Activity of Fosfomycin and Amikacin against Fosfomycin-Heteroresistant Escherichia coli Strains in a Hollow-Fiber Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02213-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

I. Portillo-Calderón ◽

M. Ortiz-Padilla ◽

B. de Gregorio-Iaria ◽

V. Merino-Bohorquez ◽

J. Blázquez ◽

...

Keyword(s):

Escherichia Coli ◽

Hollow Fiber ◽

Infection Model ◽

Bacterial Burden ◽

Content Type ◽

E Coli ◽

Bacterial Cultures ◽

Agar Dilution ◽

Time Kill ◽

Risk Of Treatment

ABSTRACT We evaluated human-like the efficacy of intravenous doses of fosfomycin of 8 g every 8 h (8 g/Q8h) and of amikacin (15 mg/kg/Q24h) in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM). Six fosfomycin-heteroresistant E. coli isolates (four with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100 mg/liter), fosfomycin (50 and 200 mg/liter), and amikacin (32 mg/liter) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip assay (GSA), and broth microdilution (BMD). Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. The efficacies of fosfomycin (8 g/Q8h) and amikacin (15 mg/kg/Q24h) alone and in combination were assessed using an HFIM. Five isolates were determined to be resistant to fosfomycin by AD and BMD, but all were determined to be susceptible by GSA. All isolates were determined to be susceptible to amikacin. Antibiotic combinations were synergistic in two isolates, and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64 mg/liter, although at 307 mg/liter only the normomutators and two hypermutators survived. Four isolates survived under 16 mg/liter amikacin, and none survived at 45 mg/liter. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilize bacterial cultures; however, the combination of fosfomycin and amikacin yielded a rapid eradication. There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the amikacin-fosfomycin combination can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains.

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Enzymatic Synthesis of Bioinformatically Predicted Microcin C-Like Compounds Encoded by Diverse Bacteria

mBio ◽

10.1128/mbio.01059-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 16

Author(s):

Olga Bantysh ◽

Marina Serebryakova ◽

Kira S. Makarova ◽

Svetlana Dubiley ◽

Kirill A. Datsenko ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Streptococcus Thermophilus ◽

Trna Synthetase ◽

Open Reading Frames ◽

Peptide Substrates ◽

Content Type ◽

E Coli ◽

Gram Positive Bacteria ◽

Synechococcus Sp

ABSTRACT The Trojan horse Escherichia coli antibiotic microcin C (McC) consists of a heptapeptide attached to adenosine through a phosphoramidate linkage. McC is synthesized by the MccB enzyme, which terminally adenylates the ribosomally synthesized heptapeptide precursor MccA. The peptide part is responsible for McC uptake; it is degraded inside the cell to release a toxic nonhydrolyzable aspartyl-adenylate. Bionformatic analysis reveals that diverse bacterial genomes encoding mccB homologues also contain adjacent short open reading frames that may encode MccA-like adenylation substrates. Using chemically synthesized predicted peptide substrates and recombinant cognate MccB protein homologs, adenylated products were obtained in vitro for predicted MccA peptide-MccB enzyme pairs from Helicobacter pylori, Streptococcus thermophilus, Lactococcus johnsonii, Bartonella washoensis, Yersinia pseudotuberculosis, and Synechococcus sp. Some adenylated products were shown to inhibit the growth of E. coli by targeting aspartyl-tRNA synthetase, the target of McC. IMPORTANCE Our results prove that McC-like adenylated peptides are widespread and are encoded by both Gram-negative and Gram-positive bacteria and by cyanobacteria, opening ways for analyses of physiological functions of these compounds and for creation of microcin C-like antibiotics targeting various bacteria.

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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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Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02393-19 ◽

2020 ◽

Vol 64 (5) ◽

Author(s):

Prashant Karki ◽

Sayed Golam Mohiuddin ◽

Pouria Kavousi ◽

Mehmet A. Orman

Keyword(s):

Stress Factors ◽

General Notion ◽

Late Stationary Phase ◽

Complete Understanding ◽

Persister Cells ◽

Content Type ◽

E Coli ◽

Phenotype Microarrays ◽

Ph Conditions ◽

Slow Growing

ABSTRACT Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

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Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

Applied and Environmental Microbiology ◽

10.1128/aem.04285-13 ◽

2014 ◽

Vol 80 (12) ◽

pp. 3597-3603 ◽

Cited By ~ 7

Author(s):

Vicki Adams ◽

Radhika Bantwal ◽

Lauren Stevenson ◽

Jackie K. Cheung ◽

Milena M. Awad ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Genetic Manipulation ◽

Antibiotic Resistance Genes ◽

Dna Fragments ◽

Site Specific ◽

Content Type ◽

E Coli ◽

Marker Recycling ◽

A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

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Nitrogen Starvation Induces Persister Cell Formation inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00622-18 ◽

2018 ◽

Vol 201 (3) ◽

Cited By ~ 4

Author(s):

Daniel R. Brown

Keyword(s):

Stress Responses ◽

Nitrogen Starvation ◽

Cell Formation ◽

Nitrogen Regulation ◽

Adaptive Responses ◽

Persister Cells ◽

Content Type ◽

E Coli ◽

Secondary Messenger ◽

Regulatory Cascade

ABSTRACTTo cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such asEscherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of therelAgene, the major (p)ppGpp synthetase inE. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starvedE. colisynthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCEBacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

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Ribosome•RelA structures reveal the mechanism of stringent response activation

eLife ◽

10.7554/elife.17029 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 81

Author(s):

Anna B Loveland ◽

Eugene Bah ◽

Rohini Madireddy ◽

Ying Zhang ◽

Axel F Brilot ◽

...

Keyword(s):

Antibiotic Resistance ◽

Stringent Response ◽

Amino Acid Starvation ◽

High Fidelity ◽

E Coli ◽

30S Subunit ◽

Transcriptional Modulators ◽

70S Ribosome ◽

A Site ◽

Response Activation

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.

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Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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