Loss of function of the gdpP protein leads to joint β-lactam/glycopeptide tolerance in Staphylococcus aureus

The genetic basis of tolerance to inhibitors of peptidoglycan biosynthesis in Staphylococcus aureus was investigated by generating tolerant mutants in vitro and characterizing them by comparative genome sequencing. Two independently selected tolerant mutants harbored nonsynonymous mutations in gdpP, a gene encoding a putative membrane-located signaling protein. Insertional inactivation of gdpP also conferred tolerance. Our findings further implicate altered signal transduction as a route to antibiotic tolerance in S. aureus.     

Exposure of Staphylococcus aureus to Subinhibitory Concentrations of β-Lactam Antibiotics Induces Heterogeneous Vancomycin-Intermediate Staphylococcus aureus

Glycopeptides are known to select for heterogeneous vancomycin-intermediate Staphylococcus aureus (h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 days in vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of ≥90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR, yvqF, SA1703, graRS, walKR, and rpoB) were PCR sequenced; no de novo mutations were observed upon beta-lactam exposure. To determine whether trfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, a trfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistance in vitro. This induction required an intact trfA locus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections.     

Influence of β-lactamase inhibitors on the activity of oxacillin against methicillin-resistant Staphylococcus aureus

We studied the in vitro susceptibility to oxacillin of 46 isolates of methicillin-resistant Staphylococcus aureus (MRSA) isolates with a minimum inhibitory concentration (MIC) >8 μg/ml of oxacillin, with and without adding clavulanic acid, sulbactam, or tazobactam in three different concentrations (2, 4, and 8 μg/ml). All 46 strains were found by the rapid chromogenic cephalosporin method to be β-lactamase producers. For those strains with low-level resistance (MIC of 16 or 32 μg/ml), the MICs of oxacillin decreased four- to 32-fold and two- to 32-fold after adding sulbactam and tazobactam, respectively. For those with high-level resistance (MIC of ⩾64 μg/ml), the MICs either did not change or decreased only two-fold after we added one of three β-lactamase inhibitors. The results suggest that β-lactamase production probably plays a role in resistance to oxacillin in those MRSA strains of low-level oxacillin resistance.     

Molecular epidemiology of β-lactamase production in penicillin-susceptible Staphylococcus aureus under high-susceptibility conditions

Little is known about the prevalence of β-lactamase production in penicillin-susceptible Staphylococcus aureus isolates under high-susceptibility conditions. We analyzed S. aureus isolates with penicillin G minimum inhibitory concentration (MIC) ≤ 0.12 μg/ml that were recovered from in-/outpatients (n = 108) between 2016 and 2017 in Japan. β-Lactamase production was detected by nitrocefin-based and Clinical and Laboratory Standards Institute penicillin zone edge testing and blaZ PCR. All isolates were categorized as having penicillin G MIC ≤0.03 μg/ml using an automated system; MICs determined based on the microdilution method were 0.016 μg/ml (2%), 0.03 μg/ml (44%), and 0.06 μg/ml (54%). Notably, no isolates harbored the blaZ gene. The results from the nitrocefin-based and zone edge tests were consistent with those obtained by PCR. S. aureus isolates with penicillin G MIC ≤0.03 μg/ml exhibited a low frequency of β-lactamase production. Thus, screening for β-lactamase production may be unnecessary for isolates showing such high susceptibility.     

The mecA Homolog mecC Confers Resistance against β-Lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background

In staphylococci, methicillin resistance is mediated by mecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded by mecC, which shares only 70% similarity to mecA. To prove that mecC is the genetic determinant that confers methicillin resistance in Staphylococcus aureus, a mecC knockout strain was generated. The S. aureus ΔmecC strain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 μg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistant S. aureus (MRSA) strain (8 and 16 μg/ml, respectively). Complementing the mutant in trans with wild-type mecC restored the resistance to oxacillin and cefoxitin. By expressing mecC and mecA in different S. aureus clonal lineages, we found that mecC mediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those with mecA. In addition, we showed that mecC expression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosome mec (SCCmec) type XI. In summary, we showed that mecC is inducible by oxacillin and mediates beta-lactam resistance in SCCmec type XI-carrying strains as well as in different S. aureus genetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinical mecC-harboring S. aureus isolates.     

Is Panton-Valentine leucocidin associated with the pathogenesis of Staphylococcus aureus bacteraemia in the UK?

BACKGROUND: The morbidity and mortality associated with Panton-Valentine leucocidin (PVL)-positive Staphylococcus aureus suggest that this toxin is a key marker of disease severity. Nevertheless, the importance of PVL in the pathogenesis of primary bacteraemia caused by S. aureus is uncertain. We have determined the prevalence of PVL-encoding genes among isolates of S. aureus from bacteraemic patients. METHODS: Consecutive bacteraemia isolates of S. aureus (n=244) from patients hospitalized in 25 centres in the UK and Ireland during 2005 were screened for PVL and mecA genes. PVL-positive isolates were characterized by toxin gene profiling, PFGE, spa-typing and MIC determinations for a range of antimicrobials. RESULTS: Four out of 244 isolates (1.6%) were PVL-positive and susceptible to oxacillin [methicillin-susceptible S. aureus (MSSA)]. Eighty-eight out of 244 (36%) were oxacillin-resistant (methicillin-resistant S. aureus), but none was PVL-positive. The four patients (two males: 30 and 33 years; two females: 62 and 80 years) had infection foci of: skin and soft tissue, unknown, indwelling line, and surgical site, and were located at one centre in Wales, one in England and two in Ireland. One of four PVL-positive isolates was resistant to penicillin and fusidic acid, the remainder were susceptible to all antibiotics tested. Genotypic analyses showed that the four isolates represented three distinct strains; the two isolates from Ireland were related. CONCLUSIONS: We found that 1.6% of S. aureus (all MSSA) from bacteraemic patients were PVL-positive. This low incidence suggests that PVL-positive S. aureus are of no particular significance as causative agents of S. aureus bacteraemia.     

Prevention and Treatment of Staphylococcus aureus Pneumonia with a β-Cyclodextrin Derivative

Staphylococcus aureus pneumonia is a common, potentially life-threatening infection caused by this human pathogen. The only therapies available to treat S. aureus pneumonia are antibiotics, a modality that is jeopardized by the organism''s remarkable ability to acquire antimicrobial resistance. S. aureus alpha-hemolysin is a pore-forming cytotoxin that is essential for the pathogenesis of pneumonia. Strains lacking this cytotoxin are avirulent in a murine model of pneumonia; likewise, vaccine-based strategies that antagonize the toxin afford protection against lethal disease. Disruption of the function of this toxin therefore provides a potent mechanism to prevent and/or treat S. aureus pneumonia. β-Cyclodextrin derivatives are small molecules with a sevenfold symmetry that mirrors the heptameric alpha-hemolysin. These compounds block the assembled alpha-hemolysin pore, compromising toxin function. We report that a modified β-cyclodextrin compound, IB201, prevents alpha-hemolysin-induced lysis of human alveolar epithelial cells. This protective effect does not result from the ability of the β-cyclodextrin to impair formation of the oligomeric alpha-hemolysin on the cell surface, supporting a role for this molecule in blockade of the lytic pore. An examination of IB201 in murine S. aureus pneumonia demonstrated that administration of this compound prevents and treats disease, protecting against mortality. Consistent with the vital importance of alpha-hemolysin in pneumonia caused by methicillin-sensitive and highly virulent methicillin-resistant S. aureus strains, IB201 protects against lethal challenge with both types of isolates. These observations, coupled with a favorable safety profile of β-cyclodextrin compounds, provide a novel strategy that may be developed to combat S. aureus pneumonia.Staphylococcus aureus is a gram-positive human pathogen that is able to cause a multitude of diseases ranging in severity (21). Of the many infections and toxinoses mediated by S. aureus, pneumonia is among the most prominent, accounting for an estimated 50,000 staphylococcal infections per year in the United States alone (18, 20). As one of the leading etiologic agents of ventilator-associated pneumonia, S. aureus has long plagued the intensive care environment (14, 19). Further, this pathogen is now increasingly recognized as an important cause of community-acquired pneumonia, displaying the capacity to infect a population of otherwise healthy adults and children (9, 10, 19). A number of these reported cases of community-acquired disease have occurred on the backdrop of an intercurrent respiratory viral infection, often caused by influenza (8, 9, 15). The fulminant nature of coinfection with influenza and S. aureus is evident in mortality rates that approach or exceed 50%, highlighting an apparent synergy of these pathogens in the lung environment. Both the reliance of an aging population on intensive care therapies and the current threat of epidemic or pandemic influenza underscore the large populations of diverse individuals that are at significant risk for the development of S. aureus pneumonia.Complicating the clinical management of staphylococcal pneumonia is the fact that over half of S. aureus isolates are currently classified as methicillin-resistant S. aureus (MRSA), harboring genes that render these isolates insensitive to a once potent class of antimicrobial agents (14). Recent clinical observations have documented that mortality from MRSA pneumonia can exceed 50%, defining the severity of disease caused by this organism (1). The estimated direct medical cost to treat a patient suffering with S. aureus pneumonia is in excess of $35,000, imparting a significant burden on the economy (30). The combined risks of a changing disease epidemiology and widespread drug resistance among S. aureus strains mandate the development of novel strategies to both prevent and treat disease. Recent investigations highlighting the pore-forming cytotoxin alpha-hemolysin (Hla) as essential for the pathogenesis of S. aureus pneumonia have provided opportunities to design and investigate new strategies to combat this disease (5, 6, 28).S. aureus strains lacking Hla display a profound defect in virulence in a murine model of pneumonia (4, 5). Consistent with this observation, antagonism of the toxin through a number of distinct immunization strategies has been demonstrated to provide protection against disease (6, 28). Hla displays a detrimental effect on the lung epithelium, resulting in cellular injury and death as well as the generation of proinflammatory mediators (23, 29). The toxin also has the capacity to target the pulmonary vascular endothelium, as treatment of isolated pulmonary arteries ex vivo results in increased vascular resistance and vascular leakage (31, 32). In addition to its role in the lung, this toxin plays a critical role in S. aureus pathogenesis in intraperitoneal, intramammary, and corneal models of infection (3, 7, 27). Hla is secreted by the majority of S. aureus strains as a water-soluble monomer (2, 26). This monomeric form binds to susceptible host cell membranes and through a well-detailed series of intra- and intermolecular interactions, subsequently assembles into a stable homoheptameric transmembrane pore with a 2-nm internal diameter (11, 17, 33).With the essential nature of alpha-toxin in mind, Karginov et al. used structure-inspired drug design to demonstrate that a hepta-6-substituted β-cyclodextrin derivative, termed IB201, is able to prevent alpha-toxin-mediated hemolysis of rabbit red blood cells (rRBCs), a cell type that is highly sensitive to the lytic action of the toxin (16). Previous investigations had demonstrated the utility of unsubstituted β-cyclodextrin as an adapter molecule, capable of lodging in the central pore of alpha-hemolysin and facilitating the use of the toxin as a biosensor (12, 13). An investigation of IB201 revealed that this molecule blocks ion conductance through the assembled hemolysin pore, consistent with the ability of the cyclodextrins to insert into the pore itself. The inhibitory effect of IB201 on ion conductance and red blood cell hemolysis are both observed in the low micromolar concentration range, demonstrating the potency of this molecule as an inhibitor of the S. aureus alpha-hemolysin. Interestingly, McCormick et al. have recently utilized methyl-β-cyclodextrin plus cholesterol to inhibit the activity of S. aureus alpha-hemolysin, revealing that this treatment affords protection against toxin-induced corneal erosions in a rabbit model of S. aureus keratitis (22). We report herein that the β-cyclodextrin derivative IB201 is able to prevent alpha-toxin-mediated alveolar epithelial cell lysis in vitro and is also able to prevent mortality associated with S. aureus pneumonia in a murine model of infection.     

Staphylococcus aureus Extracellular Vesicles Carry Biologically Active β-Lactamase

Gram-positive bacteria naturally produce extracellular vesicles. However, little is known regarding the functions of Gram-positive bacterial extracellular vesicles, especially in the bacterial community. Here, we investigated the role of Staphylococcus aureus extracellular vesicles in interbacterial communication to cope with antibiotic stress. We found that S. aureus liberated BlaZ, a β-lactamase protein, via extracellular vesicles. These extracellular vesicles enabled other ampicillin-susceptible Gram-negative and Gram-positive bacteria to survive in the presence of ampicillin. However, S. aureus extracellular vesicles did not mediate the survival of tetracycline-, chloramphenicol-, or kanamycin-susceptible bacteria. Moreover, S. aureus extracellular vesicles did not contain the blaZ gene. In addition, the heat-treated S. aureus extracellular vesicles did not mediate the survival of ampicillin-susceptible bacteria. The β-lactamase activities of S. aureus soluble and extracellular vesicle-associated BlaZ were similar, but only the extracellular vesicle-associated BlaZ was resistant to protease digestion, which suggests that the enzymatic activity of BlaZ in extracellular vesicles is largely protected by the vesicle structure. Our observations provide evidence of the important role of S. aureus extracellular vesicles in antibiotic resistance, which allows the polymicrobial community to continue to evolve and prosper against antibiotics.     

Treatment of severe infections caused by Staphylococcus aureus: a change in the vancomycin paradigm?

Staphylococcus aureus is a frequent agent of serious intensive care unit infections with high associated mortality rates. Resistance to methicillin is frequent, exceeding 50% in many countries, and for these strains, vancomycin is the agent of choice. Resistance to this drug is now frequent for Enterococcus, but low rates are reported for S. aureus. Nonetheless, treatment failures with vancomycin for infections caused by strains considered susceptible have been reported during recent years. This article aims to review the possible explanations for this phenomenon, which is challenging the old paradigm of S. aureus infections and vancomycin use.     

Insufficient β-lactam concentrations in the early phase of severe sepsis and septic shock

Introduction

Altered pharmacokinetics (PK) in critically ill patients can result in insufficient serum β-lactam concentrations when standard dosages are administered. Previous studies on β-lactam PK have generally excluded the most severely ill patients, or were conducted during the steady-state period of treatment. The aim of our study was to determine whether the first dose of piperacillin-tazobactam, ceftazidime, cefepime, and meropenem would result in adequate serum drug concentrations in patients with severe sepsis and septic shock.

Methods

Open, prospective, multicenter study in four Belgian intensive care units. All consecutive patients with a diagnosis of severe sepsis or septic shock, in whom treatment with the study drugs was indicated, were included. Serum concentrations of the antibiotics were determined by high-pressure liquid chromatography (HPLC) before and 1, 1.5, 4.5 and 6 or 8 hours after administration.

Results

80 patients were treated with piperacillin-tazobactam (n = 27), ceftazidime (n = 18), cefepime (n = 19) or meropenem (n = 16). Serum concentrations remained above 4 times the minimal inhibitory concentration (T > 4 × MIC), corresponding to the clinical breakpoint for Pseudomonas aeruginosa defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), for 57% of the dosage interval for meropenem (target MIC = 8 μg/mL), 45% for ceftazidime (MIC = 32 μg/mL), 34% for cefepime (MIC = 32 μg/mL), and 33% for piperacillin-tazobactam (MIC = 64 μg/mL). The number of patients who attained the target PK profile was 12/16 for meropenem (75%), 5/18 for ceftazidime (28%), 3/19 (16%) for cefepime, and 12/27 (44%) for piperacillin-tazobactam.

Conclusions

Serum concentrations of the antibiotic after the first dose were acceptable only for meropenem. Standard dosage regimens for piperacillin-tazobactam, ceftazidime and cefepime may, therefore, be insufficient to empirically cover less susceptible pathogens in the early phase of severe sepsis and septic shock.     

Staphylococcus aureus versus Staphylococcus epidermidis in periprosthetic joint infection—Outcome analysis of methicillin-resistant versus methicillin-susceptible strains

Periprosthetic joint infections (PJIs) are a major complication in total joint arthroplasty. Staphylococcus aureus and coagulase-negative staphylococci are known to cause the majority of all PJIs. This study aimed to analyze the eradication rates of S. aureus and S. epidermidis with methicillin susceptibility and methicillin resistance in a 2-stage therapy algorithm. Seventy-four patients with PJI caused by methicillin-resistant S. aureus (MRSA), methicillin-resistant coagulase-negative staphylococci (MRSE), methicillin-susceptible S. aureus (MSSA), and methicillin-susceptible coagulase-negative staphylococci (MSSE) were included, and the outcome was analyzed retrospectively. After a minimal follow-up of 2?years, n?=?56 patients (75.7%) were definitively free of infection. The analysis revealed significant differences between the groups, with eradication rates as follows: MSSA (92.6%), MSSE (95.2%), MRSA (80%), and MRSE (54.2%). MRSE showed a significantly lower rate of patients graded as “definitively free of infection” as compared to patients with infections caused by MSSA, MSSE, and MRSA.