The pathogenicity and virulence of the opportunistic pathogen Staphylococcus epidermidis.

The pervasive presence of Staphylococcus epidermidis and other coagulase-negative staphylococci on the skin and mucous membranes has long underpinned a casual disregard for the infection risk that these organisms pose to vulnerable patients in healthcare settings. Prior to the recognition of biofilm as an important virulence determinant in S. epidermidis, isolation of this microorganism in diagnostic specimens was often overlooked as clinically insignificant with potential delays in diagnosis and onset of appropriate treatment, contributing to the establishment of chronic infection and increased morbidity or mortality. While impressive progress has been made in our understanding of biofilm mechanisms in this important opportunistic pathogen, research into other virulence determinants has lagged S. aureus. In this review, the broader virulence potential of S. epidermidis including biofilm, toxins, proteases, immune evasion strategies and antibiotic resistance mechanisms is surveyed, together with current and future approaches for improved therapeutic interventions.

mSphere of Influence: Targeting bacterial signaling and metabolism to overcome antimicrobial resistance.

Dr Merve Suzan Zeden works in the field of molecular bacteriology and antibiotic resistance. In this mSphere of Influence article, she reflects on how three papers, entitled "c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation," "Amino acid catabolism in Staphylococcus aureus and the function of carbon catabolite repression," and "Evolving MRSA: high-level ß-lactam resistance in Staphylococcus aureus is associated with RNA polymerase alterations and fine tuning of gene expression," made an impact on her work on bacterial metabolism and antimicrobial resistance and how it shaped her research in understanding the link in between.

Metabolic reprogramming and altered cell envelope characteristics in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.

Small-Scale Illumina Library Preparation Using the Illumina Nextera XT DNA Library Preparation Kit.

Here, we describe a protocol for a scaled-down version of a genomic DNA (gDNA)-fragmentation and tagmentation reaction using the Illumina Nextera XT DNA Library Preparation Kit. Using Staphylococcus aureus as an example, which has a genome size of ∼3 Mb, we show how 24 different samples can be pooled for a typical paired-end Illumina high-throughput sequencing run using the MiSeq Reagent V2 300-cycle kit, with which it is possible to sequence 5.1 Gb of DNA. As part of the protocol, a DNA size-selection method using a standard DNA agarose gel-extraction procedure and a final sample quality-control step using a Bioanalyzer are described.

Allelic-Exchange Procedure in Staphylococcus aureus.

This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.

Preparation of Electrocompetent Staphylococcus aureus Cells and Plasmid Transformation.

This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.

Bacterial Whole-Genome-Resequencing Analysis: Basic Steps Using the CLC Genomics Workbench Software.

In this protocol, we describe the basic steps for bacterial genome resequencing analysis using the QIAGEN CLC Genomics Workbench software. More specifically, we present how a reference genome sequence can be generated from Illumina reads of a wild-type reference bacterial strain and how this reference genome sequence can then be used to identify genomic alterations in mutant strains. As specific examples, Illumina reads from the Staphylococcus aureus RN4220 strain will be used to generate a consensus reference genome based on the publicly available S. aureus NCTC8325 genome sequence. The generated RN4220 consensus reference genome will subsequently be used to identify genomic mutations in an RN4220 mutant strain with increased oxacillin resistance (OxaR strain).

Selection of Antibiotic-Resistant Bacterial Strains and Identification of Genomic Alterations by Whole-Genome Sequencing: Using Staphylococcus aureus and Oxacillin Resistance as an Example.

Here, we discuss methods for the selection of antibiotic-resistant bacteria and the use of high-throughput whole-genome sequencing for the identification of the underlying mutations. We comment on sample requirements and the choice of specific DNA preparation methods depending on the strain used and briefly introduce a workflow we use for the selection of Staphylococcus aureus strains with increased oxacillin resistance and identification of genomic alterations.

Staphylococcus aureus Colony Polymerase Chain Reaction.

Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for Staphylococcus aureus The methodology involves the preparation of small S. aureus lysates by using the enzyme lysostaphin to degrade the peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.

Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps.

In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus using the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such as S. aureus are harder to lyse than Gram-negative bacteria. Hence, the first step in the procedure for isolating gDNA from Gram-positive bacteria consists of a mechanical lysis step (e.g., using a bead beating grinder or homogenizer) or an enzymatic lysis step. For the method described here, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, the gDNA can be purified using procedures similar to those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the gDNA is subsequently used for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.

Preparation of Staphylococcus aureus Genomic DNA Using a Chloroform Extraction and Ethanol Precipitation Method, Followed by Additional Cleanup and Quantification Steps.

In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus strains using a chloroform extraction and ethanol precipitation method. This gDNA-isolation method is well-suited for downstream whole-genome sequencing applications when working with S. aureus strains that contain plasmids, as only a small amount of plasmid DNA is isolated along with the gDNA. Similar to other gDNA isolation methods for Gram-positive bacteria, the first step in the procedure is a mechanical lysis (e.g., using a bead beating grinder) or an enzymatic lysis step. In this protocol, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, gDNA can be purified using similar procedures as those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the aim is to use the gDNA for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.

Agar Plate-Based Method for the Selection of Antibiotic-Resistant Bacterial Strains.

Identifying the molecular mechanisms underlying antibiotic resistance is important, as it can reveal key information on the mode of action of a drug and provide insights for the development of novel or improved antimicrobials. Here, we describe an agar-based method for the selection of bacterial strains with increased antibiotic resistance, and how the increase in resistance can be confirmed by a spot-plating assay. As a specific example, we describe the selection of Staphylococcus aureus strains with increased resistance to oxacillin; however, the protocol can be easily adapted and used with other bacteria and antibiotics.

Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.

We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.

Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus.

Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in ß-lactam resistance, which will support efforts to identify new drug targets and reintroduce ß-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other ß-lactam resistant pathogens. Author summary: High-level resistance to penicillin-type (ß-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of ß-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Staphylococcus Great Britain and Ireland 2023 (StaphGBI 2023) Conference Report.

Since 1997, Staphylococcus Great Britain and Ireland (StaphGBI) conferences have brought together the Staphylococcus research community in the UK and Ireland. The 12th StaphGBI conference, hosted by University of Galway 22-23 June 2023, was co-chaired by Dr Merve S. Zeden and Professor James P. O'Gara, supported by a local organizing committee of Chloe Hobbs-Tobin, Dr Rakesh Roy, Órla Burke and Aaron Nolan. Anchored by keynote speaker Professor Vinai Thomas, all other StaphGBI 2023 oral and post presentations were delivered by early career researchers. The conference attracted approximately 100 delegates, including 72 MRes/PhD students and postdoctoral fellows, 22 principal investigators and 4 exhibitors. The mix of scientists, clinicians and early career researchers stimulated excellent discussions on key issues and challenges in the Staphylococcus field. Staphylococcus aureus interactions with the host immune system, antimicrobial resistance (AMR) and new therapeutic approaches using antimicrobial peptides or metabolites, chronic wound and device-associated infections, and improving our understanding of staphylococcal genomics were common themes at StaphGBI 2023.

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics.

Penicillin binding protein 2a (PBP2a)-dependent resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact ß-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to ß-lactam antibiotics. IMPORTANCEmecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to ß-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of ß-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome ß-lactam resistance.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.