Convergence of PASTA Kinase and Two-Component Signaling in Response to Cell Wall Stress inEnterococcus faecalis

ABSTRACTTwo common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryote-like Ser/Thr kinases and phosphatases (eSTK/Ps).Enterococcus faecalisis a Gram-positive bacterium and a serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and a response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to a modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance toward cell wall-targeting antibiotics, we hypothesized that these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS, which revealed the IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through the phosphorylation of CroS to promote antimicrobial resistance inE. faecalis.IMPORTANCETwo-component signaling systems (TCSs) and eukaryote-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogenEnterococcus faecalisrelies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing the convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance inE. faecalis. This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance inE. faecalis.

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Intrinsic Antimicrobial Resistance Determinants in the Superbug Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.01603-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 47

Author(s):

Justine L. Murray ◽

Taejoon Kwon ◽

Edward M. Marcotte ◽

Marvin Whiteley

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Good Predictor ◽

Molecular Mechanisms ◽

Financial Burden ◽

Resistant Bacteria ◽

Intrinsic Resistance ◽

Content Type ◽

Resistance Determinants

ABSTRACT Antimicrobial-resistant bacteria pose a serious threat in the clinic. This is particularly true for opportunistic pathogens that possess high intrinsic resistance. Though many studies have focused on understanding the acquisition of bacterial resistance upon exposure to antimicrobials, the mechanisms controlling intrinsic resistance are not well understood. In this study, we subjected the model opportunistic superbug Pseudomonas aeruginosa to 14 antimicrobials under highly controlled conditions and assessed its response using expression- and fitness-based genomic approaches. Our results reveal that gene expression changes and mutant fitness in response to sub-MIC antimicrobials do not correlate on a genomewide scale, indicating that gene expression is not a good predictor of fitness determinants. In general, fewer fitness determinants were identified for antiseptics and disinfectants than for antibiotics. Analysis of gene expression and fitness data together allowed the prediction of antagonistic interactions between antimicrobials and insight into the molecular mechanisms controlling these interactions. IMPORTANCE Infections involving multidrug-resistant pathogens are difficult to treat because the therapeutic options are limited. These infections impose a significant financial burden on infected patients and on health care systems. Despite years of antimicrobial resistance research, we lack a comprehensive understanding of the intrinsic mechanisms controlling antimicrobial resistance. This work uses two fine-scale genomic approaches to identify genetic loci important for antimicrobial resistance of the opportunistic pathogen Pseudomonas aeruginosa. Our results reveal that antibiotics have more resistance determinants than antiseptics/disinfectants and that gene expression upon exposure to antimicrobials is not a good predictor of these resistance determinants. In addition, we show that when used together, genomewide gene expression and fitness profiling can provide mechanistic insights into multidrug resistance mechanisms.

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Oxidative Stress Enhances Cephalosporin Resistance of Enterococcus faecalis through Activation of a Two-Component Signaling System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03984-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 159-169 ◽

Cited By ~ 28

Author(s):

Dušanka Djorić ◽

Christopher J. Kristich

Keyword(s):

Oxidative Stress ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Signaling System ◽

Intrinsic Resistance ◽

Content Type ◽

Prior Therapy ◽

Two Component ◽

Cephalosporin Resistance ◽

Hospital Acquired

ABSTRACTEnterococcus faecalisis a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance inE. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H2O2, activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance inE. faecalis. We find that CroR-CroS is required for adaptation to H2O2stress and that H2O2potentiates the activities of cephalosporins againstE. faecaliswhen the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H2O2, our data suggest that the CroR-CroS system responds to cell envelope damage caused by H2O2exposure in order to promote cell envelope repair and enhanced cephalosporin resistance.

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Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00995-15 ◽

2016 ◽

Vol 198 (8) ◽

pp. 1326-1336 ◽

Cited By ~ 16

Author(s):

Stephanie L. Kellogg ◽

Christopher J. Kristich

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Histidine Kinase ◽

Response Regulator ◽

Wall Stress ◽

Opportunistic Pathogen ◽

Content Type ◽

Cell Wall Stress ◽

Cephalosporin Resistance ◽

Sense And Respond

ABSTRACTBacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogenEnterococcus faecalisutilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants ofE. faecaliscarrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylationin vivo. Deletion ofcroSin various lineages ofE. faecalisrevealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded byOG1RF_12162; here calledcisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress.IMPORTANCETCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogenEnterococcus faecalisutilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance inE. faecaliscells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance inE. faecalis.

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Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00392-17 ◽

2017 ◽

Vol 199 (23) ◽

Cited By ~ 3

Author(s):

Dušanka Djorić ◽

Christopher J. Kristich

Keyword(s):

Enterococcus Faecalis ◽

Signal Sequence ◽

Cell Envelope ◽

Intrinsic Resistance ◽

Content Type ◽

Genetic Lineages ◽

Two Component ◽

Cephalosporin Resistance ◽

Biochemical Mechanisms ◽

And Function

ABSTRACT Enterococci are major causes of hospital-acquired infections. Intrinsic resistance to cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic cephalosporin resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for cephalosporin resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent cephalosporin resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic cephalosporin resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote cephalosporin resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis. IMPORTANCE Resistance to cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. Disabling cephalosporin resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of cephalosporin resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes cephalosporin resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal cephalosporin resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote cephalosporin resistance.

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Enterococcal Physiology and Antimicrobial Resistance: The Streetlight Just Got a Little Brighter

mBio ◽

10.1128/mbio.03511-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Louis B. Rice

Keyword(s):

Antimicrobial Resistance ◽

Enterococcus Faecalis ◽

Defense Mechanisms ◽

Antimicrobial Agents ◽

Acquired Resistance ◽

Complex Phenomenon ◽

Human Pathogens ◽

Intrinsic Resistance ◽

Content Type ◽

Resistance Determinants

ABSTRACT Enterococcus faecalis differs from many other common human pathogens in its physiology and in its susceptibility to antimicrobial agents. Multiresistant E. faecalis strains owe their phenotypes to a combination of intrinsic and acquired antimicrobial resistance determinants. Acquired resistance is due to E. faecalis frequenting multicultural environments, its capacity to mate with different species, and the nullification of its own defense mechanisms in some lineages. Intrinsic resistance is a complex phenomenon that is intimately tied to the physiology of the species. In their recent study in mBio, Gilmore and colleagues (M. S. Gilmore, R. Salamzade, E. Selleck, N. Bryan, et al., mBio 11:e02962-20, 2020, https://doi.org/10.1128/mBio.02962-20) use functional genomics to explore the genetic underpinnings of E. faecalis physiology and antimicrobial resistance. While they do not come up with many definitive answers, their work points the way toward new and fruitful areas of investigation.

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Nutritional Regulation of the Sae Two-Component System by CodY inStaphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00012-18 ◽

2018 ◽

Vol 200 (8) ◽

Cited By ~ 14

Author(s):

Kevin D. Mlynek ◽

William E. Sause ◽

Derek E. Moormeier ◽

Marat R. Sadykov ◽

Kurt R. Hill ◽

...

Keyword(s):

Gene Expression ◽

Virulence Factors ◽

Virulence Gene ◽

Nutrient Depletion ◽

Global Regulator ◽

Two Component System ◽

Component System ◽

Content Type ◽

Virulence Gene Expression ◽

Two Component

ABSTRACTStaphylococcus aureussubverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via theS. aureusexoprotein expression (SaeR/SaeS [SaeR/S]) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S, but the mechanism remained unknown. Here, we identified two CodY binding motifs upstream of thesaeP1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and that alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY repressessaeexpression by blocking SaeR binding. Epistasis experiments support a model that CodY also controlssaeindirectly through Agr and Rot-mediated repression of thesaeP1 promoter. We also demonstrate that CodY repression ofsaerestrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.IMPORTANCEBacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle are among the biggest unanswered questions in infectious disease research. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues and that pathogens likeS. aureusproduce virulence factors in order to gain access to nutrients in the host. Here, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by whichS. aureusdelays the production of immune evasion and immune-cell-killing proteins until key nutrients are depleted.

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Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽

10.1128/msystems.00980-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Kumari Sonal Choudhary ◽

Julia A. Kleinmanns ◽

Katherine Decker ◽

Anand V. Sastry ◽

Ye Gao ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Target Gene ◽

Target Genes ◽

Rna Seq ◽

Content Type ◽

E Coli ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

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Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression

Journal of Bacteriology ◽

10.1128/jb.00703-18 ◽

2019 ◽

Vol 201 (10) ◽

Cited By ~ 2

Author(s):

Karan Gautam Kaval ◽

Margo Gebbie ◽

Jonathan R. Goodson ◽

Melissa R. Cruz ◽

Wade C. Winkler ◽

...

Keyword(s):

Gene Expression ◽

Enterococcus Faecalis ◽

Small Rna ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Bioinformatics Analysis ◽

Maximum Efficiency ◽

Gi Tract ◽

Content Type ◽

Regulatory Factors

ABSTRACT Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR). IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella. Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

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Increased Resistance to Multiple Antimicrobials and Altered Resistance Gene Expression in CMY-2-Positive Salmonella enterica following a Simulated Patient Treatment with Ceftriaxone

Applied and Environmental Microbiology ◽

10.1128/aem.02077-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 8062-8066 ◽

Cited By ~ 7

Author(s):

Russell D. Hamilton ◽

Holly J. Hulsebus ◽

Samina Akbar ◽

Jeffrey T. Gray

Keyword(s):

Gene Expression ◽

Antimicrobial Resistance ◽

Resistance Gene ◽

Resistance Genes ◽

Salmonella Enterica ◽

The United States ◽

Simulated Patient ◽

Patient Treatment ◽

Content Type

ABSTRACTSalmonellosis is one of the most common causes of food-borne disease in the United States. Increasing antimicrobial resistance and corresponding increases in virulence present serious challenges. Currently, empirical therapy for invasiveSalmonella entericainfection includes either ceftriaxone or ciprofloxacin (E. L. Hohmann, Clin. Infect. Dis. 32:263–269, 2001). TheblaCMY-2gene confers resistance to ceftriaxone, the antimicrobial of choice for pediatric patients with invasiveSalmonella entericainfections, making these infections especially dangerous (J. M. Whichard et al., Emerg. Infect. Dis. 11:1464–1466, 2005). We hypothesized thatblaCMY-2-positiveSalmonella entericawould exhibit increased MICs to multiple antimicrobial agents and increased resistance gene expression following exposure to ceftriaxone using a protocol that simulated a patient treatmentin vitro. SevenSalmonella entericastrains survived a simulated patient treatmentin vitroand, following treatment, exhibited a significantly increased ceftriaxone MIC. Not only would these isolates be less responsive to further ceftriaxone treatment, but because theblaCMY-2genes are commonly located on large, multidrug-resistant plasmids, increased expression of theblaCMY-2gene may be associated with increased expression of other drug resistance genes located on the plasmid (N. D. Hanson and C. C. Sanders, Curr. Pharm. Des. 5:881–894, 1999). The results of this study demonstrate that a simulated patient treatment with ceftriaxone can alter the expression of antimicrobial resistance genes, includingblaCMY-2andfloRinS. entericaserovar Typhimurium andS. entericaserovar Newport. Additionally, we have shown increased MICs following a simulated patient treatment with ceftriaxone for tetracycline, amikacin, ceftriaxone, and cefepime, all of which have resistance genes commonly located on CMY-2 plasmids. The increases in resistance observed are significant and may have a negative impact on both public health and antimicrobial resistance ofSalmonella enterica.

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Evolution of Daptomycin Resistance in Coagulase-Negative Staphylococci Involves Mutations of the Essential Two-Component Regulator WalKR

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01926-18 ◽

2019 ◽

Vol 63 (3) ◽

Cited By ~ 7

Author(s):

Jhih-Hang Jiang ◽

Carina Dexter ◽

David R. Cameron ◽

Ian R. Monk ◽

Sarah L. Baines ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Cell Surface ◽

Surface Charge ◽

Sequencing Analysis ◽

Care Hospital ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Staphylococcus Capitis ◽

Cell Surface Charge ◽

Two Component

ABSTRACTCoagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably,Staphylococcus capitisaccounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains ofS. capitisandStaphylococcus epidermidisunderwentin vitrodaptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen withStaphylococcus aureus, alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) inwalKR, the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing ofwalKandwalRfrom 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of thewalKSNP inS. epidermidis, which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed thatwalKRis shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that inS. aureus.

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