scholarly journals Multiple Roles for Enterococcus faecalis Glycosyltransferases in Biofilm-Associated Antibiotic Resistance, Cell Envelope Integrity, and Conjugative Transfer

2015 ◽  
Vol 59 (7) ◽  
pp. 4094-4105 ◽  
Author(s):  
Jennifer L. Dale ◽  
Julian Cagnazzo ◽  
Chi Q. Phan ◽  
Aaron M. T. Barnes ◽  
Gary M. Dunny
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Conjugative Transfer ◽  
Content Type ◽  
Resistance Determinants

ABSTRACTThe emergence of multidrug-resistant bacteria and the limited availability of new antibiotics are of increasing clinical concern. A compounding factor is the ability of microorganisms to form biofilms (communities of cells encased in a protective extracellular matrix) that are intrinsically resistant to antibiotics.Enterococcus faecalisis an opportunistic pathogen that readily forms biofilms and also has the propensity to acquire resistance determinants via horizontal gene transfer. There is intense interest in the genetic basis for intrinsic and acquired antibiotic resistance inE. faecalis, since clinical isolates exhibiting resistance to multiple antibiotics are not uncommon. We performed a genetic screen using a library of transposon (Tn) mutants to identifyE. faecalisbiofilm-associated antibiotic resistance determinants. Five Tn mutants formed wild-type biofilms in the absence of antibiotics but produced decreased biofilm biomass in the presence of antibiotic concentrations that were subinhibitory to the parent strain. Genetic determinants responsible for biofilm-associated antibiotic resistance include components of the quorum-sensing system (fsrA,fsrC, andgelE) and two glycosyltransferase (GTF) genes (epaIandepaOX). We also found that the GTFs play additional roles inE. faecalisresistance to detergent and bile salts, maintenance of cell envelope integrity, determination of cell shape, polysaccharide composition, and conjugative transfer of the pheromone-inducible plasmid pCF10. TheepaOXgene is located in a variable extended region of the enterococcal polysaccharide antigen (epa) locus. These data illustrate the importance of GTFs inE. faecalisadaptation to diverse growth conditions and suggest new targets for antimicrobial design.

scholarly journals Environmental Spread of New Delhi Metallo-β-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Mohammad Aminul Islam ◽  
Moydul Islam ◽  
Rashedul Hasan ◽  
M. Iqbal Hossain ◽  
Ashikun Nabi ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Tap Water ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
New Delhi ◽  
Multidrug Resistant Bacteria ◽  
Content Type ◽  
Wastewater Samples

ABSTRACT Resistance to carbapenem antibiotics through the production of New Delhi metallo-β-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the bla NDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among bla NDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including bla CTX-M-1 (80%), bla CTX-M-15 (63%), bla TEM (76%), bla SHV (33%), bla CMY-2 (16%), bla OXA-48-like (2%), bla OXA-1 (53%), and bla OXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying bla NDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community. IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the bla NDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

scholarly journals CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Valerie J. Price ◽  
Wenwen Huo ◽  
Ardalan Sharifi ◽  
Kelli L. Palmer
Keyword(s):  
Antibiotic Resistance ◽  
High Risk ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Treatment Options ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Content Type ◽  
New Genes ◽  
Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

scholarly journals DNA Microarray for Genotyping Antibiotic Resistance Determinants in Acinetobacter baumannii Clinical Isolates

2013 ◽  
Vol 57 (10) ◽  
pp. 4761-4768 ◽  
Author(s):  
Simon Dally ◽  
Karin Lemuth ◽  
Martin Kaase ◽  
Steffen Rupp ◽  
Cornelius Knabbe ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Predictive Value ◽  
Handling Time ◽  
Multidrug Resistant ◽  
Testing System ◽  
Content Type ◽  
Resistance Determinants ◽  
Epidemiologic Surveillance ◽  
National Reference Center

ABSTRACTIn recent decades,Acinetobacter baumanniihas emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants inA. baumanniiin terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains ofA. baumanniiin a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants ofA. baumanniiin parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance.

scholarly journals Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing

2015 ◽  
Vol 197 (11) ◽  
pp. 1939-1951 ◽  
Author(s):  
Wenwen Huo ◽  
Hannah M. Adams ◽  
Michael Q. Zhang ◽  
Kelli L. Palmer
Keyword(s):  
Antibiotic Resistance ◽  
Horizontal Gene Transfer ◽  
Enterococcus Faecalis ◽  
Single Molecule ◽  
Bisulfite Sequencing ◽  
Treatment Options ◽  
Conjugative Transfer ◽  
Content Type ◽  
Genome Modification ◽  
Life Threatening

ABSTRACTEnterococcus faecalisis a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR)E. faecalisstrains have emerged, reducing treatment options for these infections. MDRE. faecalisstrains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDRE. faecalis. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can be distinguished from self DNA. A cognate restriction endonuclease digests improperly modified nonself DNA. Little is known about R-M inE. faecalis. Here, we use genome resequencing to identify DNA modifications occurring in the oral isolate OG1RF. OG1RF has one of the smallestE. faecalisgenomes sequenced to date and possesses few MGEs. Single-molecule real-time (SMRT) and bisulfite sequencing revealed that OG1RF has global 5-methylcytosine (m5C) methylation at 5′-GCWGC-3′ motifs. A type II R-M system confers the m5C modification, and disruption of this system impacts OG1RF electrotransformability and conjugative transfer of an antibiotic resistance plasmid. A second DNA MTase was poorly expressed under laboratory conditions but conferred globalN4-methylcytosine (m4C) methylation at 5′-CCGG-3′ motifs when expressed inEscherichia coli. Based on our results, we conclude that R-M can act as a barrier to MGE acquisition and likely influences antibiotic resistance gene dissemination in theE. faecalisspecies.IMPORTANCEThe horizontal transfer of antibiotic resistance genes among bacteria is a critical public health concern.Enterococcus faecalisis an opportunistic pathogen that causes life-threatening infections in humans. Multidrug resistance acquired by horizontal gene transfer limits treatment options for these infections. In this study, we used innovative DNA sequencing methodologies to investigate how a model strain ofE. faecalisdiscriminates its own DNA from foreign DNA, i.e., self versus nonself discrimination. We also assess the role of anE. faecalisgenome modification system in modulating conjugative transfer of an antibiotic resistance plasmid. These results are significant because they demonstrate that differential genome modification impacts horizontal gene transfer frequencies inE. faecalis.

scholarly journals Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

2019 ◽  
Author(s):  
Marinelle Rodrigues ◽  
Sara W. McBride ◽  
Karthik Hullahalli ◽  
Kelli L. Palmer ◽  
Breck A. Duerkop
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Bacterial Infections ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Conjugative Plasmid ◽  
Antibiotic Resistant ◽  
Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

scholarly journals Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis

2013 ◽  
Vol 81 (8) ◽  
pp. 2662-2668 ◽  
Author(s):  
Nosheen Fatima Rana ◽  
Nicolas Sauvageot ◽  
Jean-Marie Laplace ◽  
YinYin Bao ◽  
Ingolf Nes ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Enterococcus Faecalis ◽  
Drug Target ◽  
Systemic Infection ◽  
Opportunistic Pathogen ◽  
Redox Balance ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Hostile Environments

ABSTRACTEnterococcus faecalisis a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability ofE. faecalisto maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published forStaphylococcus aureus(A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672–1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.

scholarly journals Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
William Brewer ◽  
Johnathan Harrison ◽  
Holly E. Saito ◽  
Elizabeth M. Fozo
Keyword(s):  
Fatty Acids ◽  
Antibiotic Resistance ◽  
Fatty Acid ◽  
Stearic Acid ◽  
Enterococcus Faecalis ◽  
Cell Envelope ◽  
Negative Effects ◽  
Gram Positive ◽  
Bacterial Populations ◽  
Content Type

ABSTRACT Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid. IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

scholarly journals Comparative Biofilm Assays Using Enterococcus faecalis OG1RF Identify New Determinants of Biofilm Formation

mBio ◽  
2021 ◽  
Author(s):  
Julia L. E. Willett ◽  
Jennifer L. Dale ◽  
Lucy M. Kwiatkowski ◽  
Jennifer L. Powers ◽  
Michelle L. Korir ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Complete Understanding ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Hospital Acquired

E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients.

scholarly journals Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Anthony O. Gaca ◽  
Jessica K. Kajfasz ◽  
James H. Miller ◽  
Kuanqing Liu ◽  
Jue D. Wang ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Bacterial Infections ◽  
Stringent Response ◽  
Growth Conditions ◽  
Multidrug Resistant ◽  
Care Providers ◽  
Content Type ◽  
Bacterial Physiology ◽  
Balanced Growth ◽  
Significant Public Health

ABSTRACTThe stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. InEnterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for “Rel SpoT homologue”; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization ofE. faecalisstrains lackingrsh,relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays ofE. faecalisstrains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the ∆rsh∆relQ[(p)ppGpp0] strain, suggesting that a lack of basal (p)ppGpp places the cell in a “transcriptionally relaxed” state. Alterations in the fermentation profile and increased production of H2O2in the (p)ppGpp0strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen inBacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.IMPORTANCEDrug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogenEnterococcus faecalisas a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.

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

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
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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