Cell Wall-active Antibiotics Research Articles

Loratadine Combats Methicillin-Resistant Staphylococcus aureus by Modulating Virulence, Antibiotic Resistance, and Biofilm Genes.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved to become resistant to multiple classes of antibiotics. New antibiotics are costly to develop and deploy, and they have a limited effective lifespan. Antibiotic adjuvants are molecules that potentiate existing antibiotics through nontoxic mechanisms. We previously reported that loratadine, the active ingredient in Claritin, potentiates multiple cell-wall active antibiotics in vitro and disrupts biofilm formation through a hypothesized inhibition of the master regulatory kinase Stk1. Loratadine and oxacillin combined repressed the expression of key antibiotic resistance genes in the bla and mec operons. We hypothesized that additional genes involved in antibiotic resistance, biofilm formation, and other cellular pathways would be modulated when looking transcriptome-wide. To test this, we used RNA-seq to quantify transcript levels and found significant effects in gene expression, including genes controlling virulence, antibiotic resistance, metabolism, transcription (core RNA polymerase subunits and sigma factors), and translation (a plethora of genes encoding ribosomal proteins and elongation factor Tu). We further demonstrated the impacts of these transcriptional effects by investigating loratadine treatment on intracellular ATP levels, persister formation, and biofilm formation and morphology. Loratadine minimized biofilm formation in vitro and enhanced the survival of infected Caenorhabditis elegans. These pleiotropic effects and their demonstrated outcomes on MRSA virulence and survival phenotypes position loratadine as an attractive anti-infective against MRSA.

Evaluation of Cost-Conscious Course-Embedded Research Projects in a Freshman Biochemistry and Molecular Biology Course

Students who experience research as undergraduates tend to remain in science, technology, engineering, and mathematics fields. Since course-based undergraduate research experiences (CUREs) are often expensive to implement, we developed a relatively inexpensive first-semester research course for majors entitled “Freshman Research in Biochemistry,” in which approximately 100 students participated in an ongoing research project each year. During this course, students conducted laboratory work related to multiple research veins, and graduate teaching assistants (TAs) and the instructor ensured that sufficient data from the laboratory were made available to the students to analyze. During three unique course iterations, students worked on faculty research projects that included connecting contigs of the draft genome of a native Oklahoma bacterial Arhodomonas isolate capable of degrading petroleum hydrocarbons, cloning antibiotic resistance genes from the genus Elizabethkingia, and analyzing RNAseq data from Elizabethkingia anopheles challenged with cell wall-active antibiotics to determine gene function, gene differential expression, and relative gene genome location. During the course, students scored results, analyzed data, maintained laboratory notebooks, and presented posters in a symposium to faculty and graduate TAs. To determine positive impacts of the course on students, they completed the Laboratory Course Assessment Survey in two course sections. Survey data indicated/students agreed that the course included elements of Iteration, Collaboration, and Discovery and Relevance. Students also reported that they gained scientific skills but were ambivalent about whether instructor feedback was harsh. Examination of Student Survey of Instruction responses showed students thought positively about the course, but some considered it too difficult. Biochemistry and Molecular Biology and Biochemistry major cohort tracking revealed no effect of Freshman Research in Biochemistry on student persistence in the major or institution. Nonetheless, we consider the course a cost-conscious model for biochemistry and molecular biology departments for introducing research to first-year students.

Dissecting the roles of peptidoglycan synthetic and autolytic activities in the walled to L-form bacterial transition.

Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled "L-form" state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection. Recent work has revealed that inhibition of de novo PG precursor synthesis efficiently induces the L-form conversion in a wide range of bacteria, but the molecular mechanisms remain poorly understood. Growth of walled bacteria requires the orderly expansion of the PG layer, which involves the concerted action not just of synthases but also degradative enzymes called autolysins. Most rod-shaped bacteria have two complementary systems for PG insertion, the Rod and aPBP systems. Bacillus subtilis has two major autolysins, called LytE and CwlO, which are thought to have partially redundant functions. We have dissected the functions of autolysins, relative to the Rod and aPBP systems, during the switch to L-form state. Our results suggest that when de novo PG precursor synthesis is inhibited, residual PG synthesis occurs specifically via the aPBP pathway, and that this is required for continued autolytic activity by LytE/CwlO, resulting in cell bulging and efficient L-form emergence. The failure of L-form generation in cells lacking aPBPs was rescued by enhancing the Rod system and in this case, emergence specifically required LytE but was not associated with cell bulging. Our results suggest that two distinct pathways of L-form emergence exist depending on whether PG synthesis is being supported by the aPBP or RodA PG synthases. This work provides new insights into mechanisms of L-form generation, and specialisation in the roles of essential autolysins in relation to the recently recognised dual PG synthetic systems of bacteria.

Repurposing drugs with specific activity against L-form bacteria.

Cell wall deficient "L- form" bacteria are of growing medical interest as a possible source of recurrent or persistent infection, largely because of their complete resistance to cell wall active antibiotics such as β-lactams. Antibiotics that specifically kill L-forms would be of potential interest as therapeutics, but also as reagents with which to explore the role of L-forms in models of recurrent infection. To look for specific anti-L-form antibiotics, we screened a library of several hundred FDA-approved drugs and identified compounds highly selective for L-form killing. Among the compounds identified were representatives of two different classes of calcium channel blockers: dihydropyridines, e.g., manidipine; and diphenylmethylpiperazine, e.g., flunarizine. Mode of action studies suggested that both classes of compound work by decreasing membrane fluidity. This leads to a previously recognized phenotype of L-forms in which the cells can continue to enlarge but fail to divide. We identified a considerable degree of variation in the activity of different representatives of the two classes of compounds, suggesting that it may be possible to modify them for use as drugs for L-form-dependent infections.

Molecular Imaging of Isolated Escherichia coli DH5α Peptidoglycan Sacculi Identifies the Mechanism of Action of Cell Wall-Inhibiting Antibiotics.

Antibiotic resistance of pathogenic bacteria needs to be urgently addressed by the development of new antibacterial entities. Although the prokaryotic cell wall comprises a valuable target for this purpose, development of novel cell wall-active antibiotics is mostly missing today. This is mainly caused by hindrances in the assessment of isolated enzymes of the co-dependent murein synthesis machineries, e.g., the elongasome and divisome. We therefore present imaging methodologies to evaluate inhibitors of bacterial cell wall synthesis by high-resolution atomic force microscopy on isolated Escherichia coli murein sacculi. With the ability to elucidate the peptidoglycan ultrastructure of E. coli cells, unprecedented molecular insights into the mechanisms of antibiotics were established. The nanoscopic impairments introduced by ampicillin, amoxicillin, and fosfomycin were not only identified by AFM but readily correlated with their known mechanism of action. These valuable in vitro capabilities will facilitate the identification and evaluation of new antibiotic leads in the future.

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile.

Clostridioides difficile is a Gram-positive opportunistic pathogen responsible for 250,000 hospital-associated infections, 12,000 hospital-associated deaths, and $1 billion in medical costs in the United States each year. There has been recent interest in using a daptomycin analog, surotomycin, to treat C. difficile infections. Daptomycin interacts with phosphatidylglycerol and lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition, as it has no phosphatidylserine or phosphatidylethanolamine, and ~50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS), HexRK, that is required for C. difficile resistance to daptomycin. Using transcriptome sequencing (RNA-seq), we found that HexRK regulates expression of hexSDF, a three-gene operon of unknown function. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. Deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8- to 16-fold decrease in daptomycin MIC. The ΔhexSDF mutant is also 4-fold less resistant to bacitracin but no other cell wall-active antibiotics. Our data indicate that in the absence of HexSDF, the phospholipid membrane composition is altered. In wild-type (WT) C. difficile, the unique glycolipid HNHDRG makes up ~17% of the lipids in the membrane. However, in a ΔhexSDF mutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes to daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms. Here, we show that HexSDF and HexRK are required for synthesis of HNHDRG and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

Structural dynamics of the activation loop in the kinase domain of E.faecalis IreK.

IreK is a transmembrane Ser/Thr PASTA kinase found in Enterococcus faecalis, as well as most other Gram-positive bacteria, and plays a key role in resistance to cell wall-active antibiotics such as cephalosporins. IreK belongs to a family of bacterial transmembrane kinases that include multiple PASTA (Penicillin-and Ser/Thr-kinase Associated) domains in the extracellular segment and are ubiquitous in nearly all Gram-positive bacteria. PASTA kinases have conserved functions across Gram-positive bacteria involving monitoring and regulating processes associated with the cell wall. Enterococci are frequently found in the human gut and are a common cause of severe and difficult to treat hospital acquired infections, due to their intrinsic drug resistance. The underlying mechanisms of antibiotic resistance in enterococci are not well understood and are an excellent target for the creation of preventative treatments to eliminate the growth opportunity of resistant E. faecalis in the gut and transfer to other parts of the body. Based on the crystal structures of IreK kinase domain homologs, the activation loop structure is unknown and the phosphorylation sites within this loop are proposed to play a key regulatory role in IreK kinase activity. IreB has also been identified as one of the targets for phosphorylation by IreK. Here, continuous wave electron paramagnetic resonance (CW EPR) spectroscopy was applied to study the dynamics of the IreK activation loop, including the effects of phosphorylation and IreB binding on activation loop structure and dynamics. In addition, new functionally important non-phosphorylation sites in the activation loop that regulate IreK function in vivo were identified and characterized. This comprehensive investigation into the IreK activation loop structure and functional dynamics characterizes the role of this loop as a key regulatory domain in kinase function.

Bacillus subtilis forms twisted cells with cell wall integrity defects upon removal of the molecular chaperones DnaK and trigger factor.

The protein homeostasis network ensures a proper balance between synthesis, folding, and degradation of all cellular proteins. DnaK and trigger factor (TF) are ubiquitous bacterial molecular chaperones that assist in protein folding, as well as preventing protein misfolding and aggregation. In Escherichia coli, DnaK and TF possess partially overlapping functions. Their combined depletion results in proteostasis collapse and is synthetically lethal at temperatures above 30°C. To increase our understanding on how proteostasis is maintained in Gram-positive bacteria, we have investigated the physiological effects of deleting dnaK and tig (encoding for DnaK and TF) in Bacillus subtilis. We show that combined deletion of dnaK and tig in B. subtilis is non-lethal, but causes a severe pleiotropic phenotype, including an aberrant twisted and filamentous cell morphology, as well as decreased tolerance to heat and to cell wall active antibiotics and hydrolytic enzymes, indicative of defects in cell wall integrity. In addition, cells lacking DnaK and TF have a much smaller colony size due to defects in motility. Despite these physiological changes, we observed no major compromises in important cellular processes such as cell growth, FtsZ localization and division and only moderate defects in spore formation. Finally, through suppressor analyses, we found that the wild-type cell shape can be partially restored by mutations in genes involved in metabolism or in other diverse cellular processes.

Characterization of the L-form switch in the Gram-negative pathogen Streptobacillus moniliformis.

ABSTRACTAlmost all major classes of bacteria are surrounded by a peptidoglycan cell wall, which is a crucial target for antibiotics. It is now understood that many bacteria can tolerate loss of the cell wall provided that they are in an isotonic environment. Furthermore, in some cases the cells can continue to proliferate in a state known as the L-form. L-form proliferation occurs by an unusual blebbing or tubulation mechanism that is completely independent of the normally essential division machine or cell wall synthetic enzymes, and is resistant to cell wall-active antibiotics. However, the growth is limited by reactive oxygen species generated by the respiratory chain pathway. In this work, we examined the walled to L-form transition in a pathogenic Gram-negative bacterium, Streptobacillus moniliformis, which naturally lacks the respiratory chain pathway, under aerobic conditions. L-form-like cells often emerged spontaneously, but proliferation was not observed unless the cells were treated with cell wall-active antibiotics. Time-lapse imaging revealed that cell division of S. moniliformis L-forms involves unusual membrane dynamics with an apparent imbalance between outer membrane and cytoplasmic volume growth. The results suggest that outer membrane expansion may be an important general factor for L-form proliferation of diderm bacteria.

Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.

Understanding tolerance to cell wall-active antibiotics.

Antibiotic tolerance—the ability of bacteria to survive for an extended time in the presence of bactericidal antibiotics—is an understudied contributor to antibiotic treatment failure. Herein, I review the manifestations, mechanisms, and clinical relevance of tolerance to cell wall–active (CWA) antibiotics, one of the most important groups of antibiotics at the forefront of clinical use. I discuss definitions of tolerance and assays for tolerance detection, comprehensively discuss the mechanism of action of β‐lactams and other CWA antibiotics, and then provide an overview of how cells mitigate the potentially lethal effects of CWA antibiotic–induced cell damage to become tolerant. Lastly, I discuss evidence for a role of CWA antibiotic tolerance in clinical antibiotic treatment failure.

Bacterial Cell Wall Quality Control during Environmental Stress.

Single-celled organisms must adapt their physiology to persist and propagate across a wide range of environmental conditions. The growth and division of bacterial cells depend on continuous synthesis of an essential extracellular barrier: the peptidoglycan cell wall, a polysaccharide matrix that counteracts turgor pressure and confers cell shape. Unlike many other essential processes and structures within the bacterial cell, the peptidoglycan cell wall and its synthesis machinery reside at the cell surface and are thus uniquely vulnerable to the physicochemical environment and exogenous threats. In addition to the diversity of stressors endangering cell wall integrity, defects in peptidoglycan metabolism require rapid repair in order to prevent osmotic lysis, which can occur within minutes. Here, we review recent work that illuminates mechanisms that ensure robust peptidoglycan metabolism in response to persistent and acute environmental stress. Advances in our understanding of bacterial cell wall quality control promise to inform the development and use of antimicrobial agents that target the synthesis and remodeling of this essential macromolecule.IMPORTANCE Nearly all bacteria are encased in a peptidoglycan cell wall, an essential polysaccharide structure that protects the cell from osmotic rupture and reinforces cell shape. The integrity of this protective barrier must be maintained across the diversity of environmental conditions wherein bacteria replicate. However, at the cell surface, the cell wall and its synthesis machinery face unique challenges that threaten their integrity. Directly exposed to the extracellular environment, the peptidoglycan synthesis machinery encounters dynamic and extreme physicochemical conditions, which may impair enzymatic activity and critical protein-protein interactions. Biotic and abiotic stressors-including host defenses, cell wall active antibiotics, and predatory bacteria and phage-also jeopardize peptidoglycan integrity by introducing lesions, which must be rapidly repaired to prevent cell lysis. Here, we review recently discovered mechanisms that promote robust peptidoglycan synthesis during environmental and acute stress and highlight the opportunities and challenges for the development of cell wall active therapeutics.

Parenteral Fosfomycin for the Treatment of Multidrug Resistant Bacterial Infections: The Rise of the Epoxide.

Fosfomycin was initially discovered in 1969 but has recently gained renewed interest for the treatment of multidrug-resistant (MDR) bacterial infections, particularly in the United States. Its unique mechanism of action, bactericidal activity, broad spectrum of activity, and relatively safe and tolerable adverse effect profile make it a great addition to the dwindling antibiotic armamentarium. Fosfomycin contains a three-membered epoxide ring with a direct carbon to phosphorous bond that bypasses the intermediate oxygen bond commonly present in other organophosphorous compounds; this structure makes the agent unique from other antibiotics. Despite nearly 50 years of parenteral fosfomycin use in Europe, fosfomycin has retained stable activity against most pathogens. Furthermore, fosfomycin demonstrated in vitro synergy in combination with other cell wall-active antibiotics (e.g., β-lactams, daptomycin). These combinations may offer respite for severe infections due to MDR gram-positive and gram-negative bacteria. The intravenous (IV) formulation is currently under review in the United States, and apropos, this review collates more contemporary evidence (i.e., studies published between 2000 and early 2019) in anticipation of this development. The approval of IV fosfomycin provides another option for consideration in the management of MDR infections. Its unique structure will give rise to a promising epoxide epoch in the battle against MDR bacteria.

Influence of antibiotic treatment on the detection of S. aureus in whole blood following pathogen enrichment

BackgroundEarly pathogen detection and identification are crucial for an effective and targeted antibiotic therapy in patients suffering from blood stream infection. Molecular diagnostic methods can accelerate pathogen identification as compared to blood culture, but frequently suffer from the inhibition of polymerase chain reation (PCR) by sample matrix components, such as host DNA, anticoagulants, or plasma proteins. To overcome this limitation, molecular diagnostic methods commonly rely on pathogen enrichment by selective lysis of blood cells and pelleting of intact pathogens prior to analysis.ResultsHere, we investigated the impact of antibiotic treatment on the recovery of pathogen DNA using an established pathogen enrichment protocol. Based on the hypothesis that induction of bacterial cell wall disintegration following antibiotic administration leads to incomplete pelleting of pathogen DNA, S. aureus was grown in human whole blood with or without addition of cell wall active (vancomycin, piperacillin) or non cell wall active (ciprofloxacin, clindamycin) antibiotics at clinically relevant concentrations. Pathogen detection remained unaffected by non cell wall active antibiotics or even increased in the presence of cell wall active antibiotics, indicating improved accessibility of pathogen DNA. Likewise, mechanical lysis of S. aureus prior to pathogen enrichment resulted in increased recovery of pathogen DNA. Quantification of pathogen and human DNA after selective lysis of blood cells and pathogen enrichment confirmed partial depletion of human DNA, leading to a net enrichment of pathogen DNA over human DNA.ConclusionConcurrent antibiotic administration does not reduce the recovery of pathogen DNA during pathogen enrichment by selective lysis and centrifugation.Leads to a 10-fold human DNA depletion as compared to pathogen DNA. Moreover, we confirm that the recovery of pathogen DNA after pathogen enrichment is not negatively influenced by concurrent antibiotic administration.

A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

Regulation of virulence and antibiotic resistance in Gram-positive microbes in response to cell wall-active antibiotics.

Antibiotic stress can evoke considerable genotypic and phenotypic changes in Gram-positive bacteria. Here, we review recent studies describing altered virulence expression in response to cell wall-acting antibiotics and discuss mechanisms that coordinate regulation of the antibiotic response. Pleiotropic effects induced by antibiotic exposure include alterations to bacterial metabolism, cell wall structure and antibiotic resistance. In addition, subinhibitory concentrations of cell wall-active (CWA) antibiotics have increasingly been shown to induce the production of exotoxins and biofilm formation that may influence virulence. Remarkably, phenotypes associated with comparable antibiotic stresses can vary considerably, emphasizing the need to better understand the response to CWA antibiotics. Recent studies support both direct antibiotic recognition and recognition of antibiotic-induced stress to the bacterial cell wall. Specifically, bacterial two-component systems, penicillin-binding protein and serine/threonine kinase-associated kinases and conserved oxidative-stress sensors each contribute to modulating the antibiotic stress response. Bacterial sensory systems and global regulators coordinate signaling in response to CWA antibiotics. Regulation of the antibiotic response is complex and involves integration of signals from multiple response pathways. A better definition of the antibiotic stress response among Gram-positive pathogens may yield novel therapeutic targets to counter antibiotic resistance and virulence factor expression.

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Adjunctive gentamicin did not improve outcome of enterococcal bacteraemia in neutropenic patients: a propensity scored matched study

Background: Patients with haematologic malignancies receiving chemotherapy have a high risk of developing febrile neutropenia and bloodstream infections. The benefit of adjunctive gentamicin treatment for enterococcal bloodstream infections is debated. In this study, we compare the treatment outcome of a cell wall-active antibiotic with and without gentamicin for enterococcal bacteraemia in patients with neutropenia.Methods: The observational study was performed from 1999 through 2016. Patients with bacteraemia due to non-high level gentamicin-resistant enterococci were included. Analyses were performed in two data sets of episodes with enterococcal bacteraemia. One data set consisting of all included episodes (full cohort, n = 154) and one with propensity score-matched episodes (n = 96). The primary endpoint was death within 30 days, and the secondary outcomes were defervescence and persistence of enterococcal bloodstream infection after initiation of anti-enterococcal therapy.Results: Episodes with gentamicin treatment (n = 82, full cohort; n = 48, propensity score-matched cohort) were comparable with episodes without gentamicin treatment (n = 72, full cohort; n = 48, propensity score-matched cohort) with regard to patient- and disease-related characteristics. Enterococcus faecium (40.9%) was the most frequently isolated organism. In the propensity score-matched cohort, there was no difference in 30-days mortality (14.6% in episodes with gentamicin versus 16.7% in episodes without gentamicin, p = 1), median time to defervescence (1 versus 2 days, p = .37) or persistence of enterococcal bloodstream infection for ≥72 h (9.4% versus 7.5%, p = 1).Conclusions: In our study with neutropenic patients, treatment with a cell wall-active antibiotic without adjunctive gentamicin for episodes with enterococcal bloodstream infection was as effective as combination therapy with gentamicin.

The Role of Methionine Sulfoxide Reductases in Oxidative Stress Tolerance and Virulence of Staphylococcus aureus and Other Bacteria.

Methionine sulfoxide reductases (MSRA1 and MSRB) are proteins overproduced in Staphylococcus aureus during exposure with cell wall-active antibiotics. Later studies identified the presence of two additional MSRA proteins (MSRA2 and MSRA3) in S. aureus. These MSR proteins have been characterized in many other bacteria as well. This review provides the current knowledge about the conditions and regulatory network that mimic the expression of these MSR encoding genes and their role in defense from oxidative stress and virulence.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model

ObjectivesStreptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall–active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. MethodsWe investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. ResultsIn vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus–infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9–14.5) vs. 4 mm2 (0.8–10.5), subcutaneous: 10.3 mm2 (6.9–18.6) vs. 0.5 mm2 (0.1–6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. ConclusionsThe clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.