Deletion ofliaRReverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background

ABSTRACTWe have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain ofEnterococcus faecalisreversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved inEnterococcus faecium, we investigated the role of LiaR in a variety of clinicalE. faeciumstrains representing the most common DAP-R genetic backgrounds. Deletion ofliaRin DAP-RE. faeciumR446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, theliaRdeletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR inE. faeciuminvolves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion ofliaRnot only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.

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Environment Shapes the Accessible Daptomycin Resistance Mechanisms in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00790-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 5

Author(s):

Amy G. Prater ◽

Heer H. Mehta ◽

Abigael J. Kosgei ◽

William R. Miller ◽

Truc T. Tran ◽

...

Keyword(s):

Enterococcus Faecium ◽

Cell Envelope ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Clinical Strain ◽

Content Type ◽

Component Cell ◽

Evolutionary Trajectories ◽

High Level ◽

Cell Envelopes

ABSTRACT Daptomycin binds to bacterial cell membranes and disrupts essential cell envelope processes, leading to cell death. Bacteria respond to daptomycin by altering their cell envelopes to either decrease antibiotic binding to the membrane or by diverting binding away from septal targets. In Enterococcus faecalis, daptomycin resistance is typically coordinated by the three-component cell envelope stress response system, LiaFSR. Here, studying a clinical strain of multidrug-resistant Enterococcus faecium containing alleles associated with activation of the LiaFSR signaling pathway, we found that specific environments selected for different evolutionary trajectories, leading to high-level daptomycin resistance. Planktonic environments favored pathways that increased cell surface charge via yvcRS upregulation of dltABCD and mprF, causing a reduction in daptomycin binding. Alternatively, environments favoring complex structured communities, including biofilms, evolved both diversion and repulsion strategies via divIVA and oatA mutations, respectively. Both environments subsequently converged on cardiolipin synthase (cls) mutations, suggesting the importance of membrane modification across strategies. Our findings indicate that E. faecium can evolve diverse evolutionary trajectories to daptomycin resistance that are shaped by the environment to produce a combination of resistance strategies. The accessibility of multiple and different biochemical pathways simultaneously suggests that the outcome of daptomycin exposure results in a polymorphic population of resistant phenotypes, making E. faecium a recalcitrant nosocomial pathogen.

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MgrB Inactivation Is Responsible for Acquired Resistance to Colistin in Enterobacter hormaechei subsp. steigerwaltii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00128-20 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 2

Author(s):

Amel Mhaya ◽

Dominique Bégu ◽

Slim Tounsi ◽

Corinne Arpin

Keyword(s):

Acquired Resistance ◽

Regulatory System ◽

Multidrug Resistant ◽

Negative Regulator ◽

Clinical Strain ◽

Sequencing Analysis ◽

Content Type ◽

Health Care Associated Infections ◽

Enterobacter Hormaechei

ABSTRACT Multidrug-resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV, and C-VIII, have increasingly emerged as a leading cause of health care-associated infections, with colistin used as one of the last lines of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of the PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hormaechei subsp. steigerwaltii. An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. The wild-type mgrB gene from Eh22 and that of a clinical strain of Klebsiella pneumoniae used as controls were cloned, and the corresponding recombinant plasmids were used for complementation assays. The results showed a fully restored susceptibility to colistin and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains.

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Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids

mBio ◽

10.1128/mbio.00281-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 96

Author(s):

Truc T. Tran ◽

Diana Panesso ◽

Nagendra N. Mishra ◽

Eugenia Mileykovskaya ◽

Ziqianq Guan ◽

...

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Antimicrobial Peptides ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Multidrug Resistant ◽

Content Type ◽

Vancomycin Resistant Enterococci ◽

Vancomycin Resistant ◽

Cardiolipin Synthase

ABSTRACT Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides. IMPORTANCE The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

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The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02122-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 19

Author(s):

Ana M. Guzmán Prieto ◽

Jessica Wijngaarden ◽

Johanna C. Braat ◽

Malbert R. C. Rogers ◽

Eline Majoor ◽

...

Keyword(s):

Enterococcus Faecium ◽

Response Regulator ◽

Normal Growth ◽

Multidrug Resistant ◽

Cross Tolerance ◽

Two Component System ◽

Component System ◽

Content Type ◽

A Genome ◽

Two Component

ABSTRACT Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium. Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR. Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans. Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

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Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

Journal of Bacteriology ◽

10.1128/jb.02491-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 861-871 ◽

Cited By ~ 4

Author(s):

Kumiko Kurabayashi ◽

Yuko Hirakawa ◽

Koichi Tanimoto ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Phosphate Uptake ◽

Response Regulator ◽

Anaerobic Respiration ◽

Regulatory System ◽

Carbon Substrate ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component ◽

Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

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Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains

mBio ◽

10.1128/mbio.00534-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 204

Author(s):

François Lebreton ◽

Willem van Schaik ◽

Abigail Manson McGuire ◽

Paul Godfrey ◽

Allison Griggs ◽

...

Keyword(s):

Enterococcus Faecium ◽

Sequence Data ◽

Mobile Element ◽

Multidrug Resistant ◽

Human Infection ◽

Animal Origin ◽

Loss Of Function ◽

Content Type ◽

Hospital Acquired Infection ◽

Hospital Acquired

ABSTRACTEnterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand howE. faeciumemerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.IMPORTANCEEnterococci, in particular vancomycin-resistantEnterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistantE. faeciuminfections.

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Gene Cluster Responsible for Secretion of and Immunity to Multiple Bacteriocins, the NKR-5-3 Enterocins

Applied and Environmental Microbiology ◽

10.1128/aem.02312-14 ◽

2014 ◽

Vol 80 (21) ◽

pp. 6647-6655 ◽

Cited By ~ 14

Author(s):

Naoki Ishibashi ◽

Kohei Himeno ◽

Yoshimitsu Masuda ◽

Rodney Honrada Perez ◽

Shun Iwatani ◽

...

Keyword(s):

Gene Cluster ◽

Abc Transporter ◽

Response Regulator ◽

Regulatory System ◽

Content Type ◽

Expression Studies ◽

A Genome ◽

Wide Range ◽

Close Proximity ◽

Immunity Genes

ABSTRACTEnterococcus faeciumNKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA[ent53A],enkC[ent53C],enkD[ent53D], andenkZ[ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIazandenkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies ofenkTand ΔenkTmutant strains showed thatenkTis responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRKmutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.

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The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

mBio ◽

10.1128/mbio.01821-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 6

Author(s):

Karine Loth ◽

Agnès Vergnes ◽

Cairé Barreto ◽

Sébastien N. Voisin ◽

Hervé Meudal ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Activity ◽

Bactericidal Activity ◽

Multidrug Resistant ◽

High Salt ◽

Clinical Isolates ◽

Host Defense Peptides ◽

Hydrophobic Domain ◽

Content Type ◽

Terminal Domain

ABSTRACT Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward β-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus. We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the β-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate β-defensins. IMPORTANCE β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus. Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations.

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Evaluation of the Bactericidal Activity of Plazomicin and Comparators against Multidrug-Resistant Enterobacteriaceae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00236-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 7

Author(s):

M. Thwaites ◽

D. Hall ◽

D. Shinabarger ◽

A. W. Serio ◽

K. M. Krause ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Bactericidal Activity ◽

Minimum Bactericidal Concentration ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Next Generation ◽

Content Type ◽

Time Kill

ABSTRACT The next-generation aminoglycoside plazomicin, in development for infections due to multidrug-resistant (MDR) Enterobacteriaceae, was evaluated alongside comparators for bactericidal activity in minimum bactericidal concentration (MBC) and time-kill (TK) assays against MDR Enterobacteriaceae isolates with characterized aminoglycoside and β-lactam resistance mechanisms. Overall, plazomicin and colistin were the most potent, with plazomicin demonstrating an MBC50/90 of 0.5/4 μg/ml and sustained 3-log10 kill against MDR Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp.

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Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Infection and Immunity ◽

10.1128/iai.02700-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1199-1209 ◽

Cited By ~ 38

Author(s):

Kivanc Bilecen ◽

Jiunn C. N. Fong ◽

Andrew Cheng ◽

Christopher J. Jones ◽

David Zamorano-Sánchez ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Lipid A ◽

Response Regulator ◽

Regulatory Region ◽

Regulatory System ◽

Polymyxin B ◽

Content Type ◽

Two Component ◽

Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

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