Leonurine alleviates vancomycin nephrotoxicity via activating PPARγ and inhibiting the TLR4/NF-κB/TNF-α pathway.

Vancomycin (VCM) is the first-line antibiotic for severe infections, but nephrotoxicity limits its use. Leonurine (Leo) has shown protective effects against kidney damage. However, the effect and mechanism of Leo on VCM nephrotoxicity remain unclear. In this study, mice and HK-2 cells exposed to VCM were treated with Leo. Biochemical and pathological analysis and fluorescence probe methods were performed to examine the role of Leo in VCM nephrotoxicity. Immunohistochemistry, q-PCR, western blot, FACS, and Autodock software were used to verify the mechanism. The present results indicate that Leo significantly alleviates VCM-induced renal injury, morphological damage, and oxidative stress. Increased intracellular and mitochondrial ROS in HK-2 cells and decreased mitochondrial numbers in mouse renal tubular epithelial cells were reversed in Leo-administrated groups. In addition, molecular docking analysis using Autodock software revealed that Leo binds to the PPARγ protein with high affinity. Mechanistic exploration indicated that Leo inhibited VCM nephrotoxicity via activating PPARγ and inhibiting the TLR4/NF-κB/TNF-α inflammation pathway. Taken together, our results indicate that the PPARγ inhibition and inflammation reactions were implicated in the VCM nephrotoxicity and provide a promising therapeutic strategy for renal injury.

Time-Dependent Differences in Vancomycin Sensitivity of Macrophages Underlie Vancomycin-Induced Acute Kidney Injury.

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1ß expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1ß and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

In Vitro Evaluation of Vancomycin-Induced Toxicity in Human Primary Knee Chondrocytes.

Septic arthritis as a complication of orthopaedic joint surgery can have catastrophic outcomes for patients. To minimise infection risk associated with elective orthopaedics, topical vancomycin during surgery has become increasingly common. Evidence suggests that high concentrations of vancomycin, following direct application of the drug to the joint, are toxic towards various local cell types in the joint, including chondrocytes. However, the mechanism of this vancomycin tissue toxicity is yet to be determined. The aim of this study was to evaluate the toxicity of vancomycin on chondrocytes and the mechanisms of cell death involved. Human primary knee chondrocytes were exposed to vancomycin (1.25-10 mg/mL) for 24 h and their viability assessed using the resazurin reduction assay in vitro. Specific cell death mechanisms and their contributors, including reactive oxygen species (ROS) production and apoptosis, were measured. This study showed that high concentrations of vancomycin (5 and 10 mg/mL) were toxic towards human primary knee chondrocyte cells, while lower concentrations (1.25 and 2.5 mg/mL) were not. Cell death studies found that this occurred through an apoptotic pathway. This study provides additional support that vancomycin in high doses is toxic towards chondrocytes and preliminary evidence that this toxicity occurs via apoptotic cell death mechanisms.

Vancomycin Pretreatment on MPTP-Induced Parkinson's Disease Mice Exerts Neuroprotection by Suppressing Inflammation Both in Brain and Gut.

A growing body of evidence implies that gut microbiota was involved in pathogenesis of Parkinson's disease (PD), but the mechanism is still unclear. The aim of this study is to investigate the effects of antibiotics pretreatment on the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD mice. In this study, vancomycin pretreatment was given by gavage once daily with either vancomycin or distilled water for 14 days to mice, then mice were administered with MPTP (20 mg/kg, i.p) for four times in one day to establish an acute PD model. Results show that vancomycin pretreatment significantly improved motor dysfunction of mice in pole and traction tests. Although vancomycin pretreatment had no effect on dopamine (DA) or the process of DA synthesis, it inhibited the metabolism of DA by suppressing the expression of striatal monoamine oxidase B (MAO-B). Furthermore, vancomycin pretreatment reduced the number of astrocytes and microglial cells in the substantia nigra pars compacta (SNpc) to alleviate neuroinflammation, decreased the expression of TLR4/MyD88/NF-κB/TNF-α signaling pathway in both brain and gut. Meanwhile, vancomycin pretreatment changed gut microbiome composition and the levels of fecal short chain fatty acids (SCFAs). The abundance of Akkermansia and Blautia increased significantly after vancomycin pretreatment, which might be related to inflammation and inhibition of TLR4 signaling pathway. In summary, these results demonstrate that the variation of gut microbiota and its metabolites induced by vancomycin pretreatment might decrease dopamine metabolic rate and relieve inflammation in both gut and brain via the microbiota-gut-brain axis in MPTP-induced PD mice. The neuroprotection of vancomycin pretreatment on MPTP-induced Parkinson's disease mice The alterations of gut microbiota and SCFAs induced by vancomycin pretreatment might not only improve motor dysfunction, but also decrease dopamine metabolism and relieve inflammation in both brain and gut via TLR4/MyD88/NF-κB/TNF-α pathway in MPTP-induced PD mice.

RoboCap: Robotic mucus-clearing capsule for enhanced drug delivery in the gastrointestinal tract.

Oral drug delivery of proteins is limited by the degradative environment of the gastrointestinal tract and poor absorption, requiring parenteral administration of these drugs. Luminal mucus represents the initial steric and dynamic barrier to absorption. To overcome this barrier, we report the development of the RoboCap, an orally ingestible, robotic drug delivery capsule that locally clears the mucus layer, enhances luminal mixing, and topically deposits the drug payload in the small intestine to enhance drug absorption. RoboCap's mucus-clearing and churning movements are facilitated by an internal motor and by surface features that interact with small intestinal plicae circulares, villi, and mucus. Vancomycin (1.4 kilodaltons of glycopeptide) and insulin (5.8 kilodaltons of peptide) delivery mediated by RoboCap resulted in enhanced bioavailability 20- to 40-fold greater in ex vivo and in vivo swine models when compared with standard oral delivery (P < 0.05). Further, insulin delivery via the RoboCap resulted in therapeutic hypoglycemia, supporting its potential to facilitate oral delivery of drugs that are normally precluded by absorption limitations.

N-acetylcysteine Ameliorates Vancomycin-induced Nephrotoxicity by Inhibiting Oxidative Stress and Apoptosis in the in vivo and in vitro Models.

Background: Oxidative stress-related apoptosis is considered as the key mechanism implicated in the pathophysiology of nephrotoxicity with vancomycin (VCM) therapy. We evaluated the possible effects of N-acetylcysteine (NAC) on VCM-induced nephrotoxicity and the underlying mechanism. Methods: VCM-induced nephrotoxicity was established using HK-2 cells and SD rats and observed by measuring cell survival, kidney histological changes, renal function and kidney injury related markers (KIM-1 and NGAL). Oxidative stress, renal cell apoptosis and the involved signaling pathways were also evaluated. Results: In model rats, NAC could protect against VCM-induced acute kidney injury with histological damage, renal dysfunction, and increased Cre and BUN levels. In HK-2 cells, VCM-induced decreased cell viability was restored by NAC. In addition, increased expression of caspase-3, KIM-1 and NGAL suffering from VCM was also reversed by NAC in vivo and in vitro. NAC inhibited ROS production, decreased cell apoptosis by decreasing the Bax/Bcl-2 ratio and caspase-3 expression in HK-2 cells and regulated oxidative stress indicators in the kidney by decreasing GSH, SOD and CAT activity and increasing MDA levels. Furthermore, NAC could effectively reverse VCM-associated increased P38 MAPK/JNK phosphorylation. Conclusions: The results demonstrated that NAC had a protective effect against nephrotoxicity from VCM by inhibiting oxidative stress and apoptosis via P38 MAPK/JNK.

A Meta-Analysis on the Performance of Cystatin C- versus Creatinine-based eGFR Equations in Predicting Vancomycin Clearance.

BACKGROUND: The objective of this study was to compare the performance of cystatin C- and creatinine-based estimated glomerular filtration rate (eGFR) equations in predicting the clearance of vancomycin. METHODS: MEDLINE and Embase databases were searched from inception up to September 2019 to identify all studies that compared the predictive performance of cystatin C- and/or creatinine-based eGFR in predicting the clearance of vancomycin. The prediction errors (PEs) (the value of eGFR equations minus vancomycin clearance) were quantified for each equation and were pooled using a random-effects model. The root mean squared errors were also quantified to provide a metric for imprecision. RESULTS: This meta-analysis included evaluations of seven different cystatin C- and creatinine-based eGFR equations in total from 26 studies and 1,234 patients. The mean PE (MPE) for cystatin C-based eGFR was 4.378 mL min-1 (95% confidence interval [CI], -29.425, 38.181), while the creatinine-based eGFR provided an MPE of 27.617 mL min-1 (95% CI, 8.675, 46.560) in predicting clearance of vancomycin. This indicates the presence of unbiased results in vancomycin clearance prediction by the cystatin C-based eGFR equations. Meanwhile, creatinine-based eGFR equations demonstrated a statistically significant positive bias in vancomycin clearance prediction. CONCLUSION: Cystatin C-based eGFR equations are better than creatinine-based eGFR equations in predicting the clearance of vancomycin. This suggests that utilising cystatin C-based eGFR equations could result in better accuracy and precision to predict vancomycin pharmacokinetic parameters.

Pillar[5]arene-based, dual pH and enzyme responsive supramolecular vesicles for targeted antibiotic delivery against intracellular MRSA.

A rationally designed mannosylated amphiphilic pillar[5]arene (Man@AP5) self-assembles into supramolecular vesicles with encapsulated vancomycin (Man@AP5-Van), which target macrophages, respond to both acid and cathepsin B, and release vancomycin (Van) rapidly inside macrophages. Man@AP5-Van significantly increases the intracellular concentration of Van, enhancing its antibacterial efficacy against intracellular MRSA.

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria.

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, ß-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.

Do Prophylactic Antibiotics Reach the Operative Site Adequately?: A Quantitative Analysis of Serum and Wound Concentrations of Systemic and Local Prophylactic Antibiotics in Spine Surgery.

STUDY DESIGN: Prospective cohort study. OBJECTIVE: To analyze the serum and drain concentrations of antibiotics administered by two different routes and compare the results. SUMMARY OF BACKGROUND DATA: Systemic antibiotics are expected to reach the surgical site and maintain adequate concentrations of the drug to prevent infection. However, it is unknown whether systemically administered antibiotics reach and maintain such adequate concentrations at the surgical wound or not. METHODS: Forty patients undergoing elective spine surgery received intra-wound Vancomycin (1 GM) before the wound closure and single dose of intravenous Gentamycin (80MG) immediately after surgery. Blood and drain samples were collected postoperatively to estimate serum and drain concentrations of Gentamycin and Vancomycin. Drug Estimation Protocol: Drug concentrations were estimated by ADVIA Centaur CP immunoassay (direct chemiluminescence). Gentamycin and vancomycin in the test samples competes with their respective acridinium ester-labeled gentamicin and vancomycin derivatives for monoclonal mouse anti-gentamycin and anti-vancomycin antibodies which are covalently coupled to paramagnetic particles in the solid phase. RESULTS: Gentamycin attained peak serum levels at 6 hours following administration with an average value of 9.90 ±â€Š3.1 µg/mL which was decreased to 6.76 ±â€Š2.6 µg/mL at 12 hours and steadily declining thereafter. Even though, the drug concentrations in the drain collection from the wound also attained peak levels at 6 hours, the drug concentrations were lower (3.75 ±â€Š1.4 µg/mL) than that of serum concentrations and inadequately attained the recommended target peak of Gentamycin (4-12 µg/mL).Wound levels of local vancomycin were significantly higher at 6 hours (413.4 ±â€Š217.3 µg/mL) and well maintained even at 72 hours. Serum vancomycin levels were observed to be highest at 6 hours in negligible concentrations of 6.06 ±â€Š2.2 µg/mL. CONCLUSION: After prophylactic systemic administration of the antibiotics, the antibiotic drug concentrations in the wound are much lower than the serum concentrations at any given time. After local intra-wound application of antibiotics, the drug concentrations in the wound are well maintained even after 72 hours. LEVEL OF EVIDENCE: 3.

Vancomycin and its crystalline degradation products released from bone grafts and different types of bone cement.

Vancomycin is often used in orthopedic surgery as a local prophylaxis of bacterial infection. The aim of this work was to compare the release of vancomycin and its biologically inactive crystalline degradation products (CDP-1) during in vitro experiments from different types of local antibiotic delivery systems (bone grafts and bone cements). The concentrations of vancomycin and its crystalline degradation products were determined by high-performance liquid chromatography. Each experiment was performed in a phosphate buffer solution over 21 days. Morselized bone grafts, synthetic bone cements Palacos and Copal, and synthetic bone grafts were tested as local carriers of vancomycin. The highest concentration approximately 670 mg/L of vancomycin was released from synthetic bone grafts Actifuse. Even after 21 days, the concentration of vancomycin was still above the minimum inhibitory concentration (MIC). The maximum concentration of vancomycin released in two experiments with human bone grafts exceeded 600 mg/L during the first day and was still above MIC level 21 days later when the experiment was concluded. By comparing the synthetic bone cements Palacos and Copal, Copal had the average maximum concentration of only 32.4 mg/L and Palacos 35.7 mg/L. The concentration of vancomycin fell below the MIC for vancomycin-resistant Staphylococcus aureus (VRSA) on the seventh day with Palacos and the ninth day with Copal. This study showed the insufficient concentration of released vancomycin from synthetic bone cements at the end of the experiment. For improvement of local prophylaxis, it would be beneficial to increase the amount of vancomycin in bone cements.

Improved Intestinal Mucus Permeation of Vancomycin via Incorporation Into Nanocarrier Containing Papain-Palmitate.

The aim of this study was to improve intestinal mucus permeation of a peptide antibiotic via incorporation into papain-palmitate-modified self-emulsifying drug delivery systems (SEDDS) as nanocarrier. Vancomycin as a peptide antibiotic was lipidized by hydrophobic ion pair formation using sodium bis-2-ethylhexyl-sulphosuccinate before incorporation in SEDDS comprising Capmul MCM, propylenglycol, and Kolliphor EL (2:1:2). As mucolytic agent, 0.5% papain-palmitate was introduced in SEDDS formulation containing the vancomycin-sodium bis-2-ethylhexyl-sulphosuccinate ion pair. The formulation was evaluated regarding droplet size, zeta potential, and cytotoxicity using Caco-2 cells previous to intestinal mucus permeation studies using Transwell diffusion and rotating tube method. The hydrophobic ion pair product yielded from surfactant to drug ratio of 3:1 provided a 25-fold increase in lipophilicity, drug payload in SEDDS of 5%, and log DSEDDS/release medium of 2.2. The formulation exhibited a droplet size and zeta potential of 221.5 ± 14.8 nm and -4.2 ± 0.8 mV, respectively. Cytotoxicity study showed that SEDDS formulations were not toxic. Introducing 0.5% papain-palmitate increased the mucus permeability of SEDDS 2.8-fold and 3.3-fold in Transwell diffusion and rotating tube studies, respectively. According to these results, papain decorated SEDDS might be a potential strategy to improve the mucus permeating properties of peptide antibiotics.

Proteomic analysis of small intestinal epithelial cells in antibiotic-treated mice: Changes in drug transporters and metabolizing enzymes.

Antibiotics act on bacterial flora originally present in the intestine, and changes in the intestinal flora have various effects on the host. This study investigated changes in the protein levels of drug transporters and metabolizing enzymes in the small intestines of antibiotic-treated mice by proteomic analysis. After the oral administration of non-absorbable antibiotics (vancomycin and polymyxin B) for 5 days, 15 drug transporter or metabolizing enzyme proteins had significantly changed levels among 1780 proteins identified in small intestinal epithelial cells. Of these, the levels of peptide transporter 1 (Pept1), multidrug resistance protein 1a (Mdr1a), and multidrug resistance-associated protein 2 (Mrp2) were increased approximately 2-fold. In addition, the levels of two Cyp4f proteins were decreased and those of Cyp4b1, Ces1d, and three glutathione S-transferase (Gst) proteins were increased. Our results indicate that the oral administration of antibiotics changes the levels of proteins related to the absorption and metabolism of drugs in the small intestine, and suggest that substrate drugs of these proteins have a risk for indirect drug interactions with antibacterial drugs via the intestinal flora.

A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins.

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.

Tunable sequential drug delivery system based on chitosan/hyaluronic acid hydrogels and PLGA microspheres for management of non-healing infected wounds.

Treatment of non-healing infected wounds is an arduous task in clinical practice. Early antibacterial strategy and subsequent promotion of granulation tissue growth facilitate to cure the wounds. For this purpose, we fabricated a sequential drug delivery system by incorporation of an injectable hydrogel with porous PLGA microspheres. Vancomycin was linked to the injectable hydrogel via the reversible Schiff's base reaction, and VEGF were encapsulated into PLGA microspheres. After adding vancomycin, the strength and elasticity of the hydrogel were improved, and the gelation time was shortened. The results also demonstrated that the releasing profile of vancomycin was pH-dependent and the VEGF's profile was adjustable by changing the pore sizes of PLGA microspheres. The duration of VEGF release was longer than vancomycin. This hybrid system was valid to inhibit bacteria growth and accelerate vein endothelial cell proliferation in vitro. In rat models, it was effective to manage non-healing infected wounds by reducing inflammation and promoting angiogenesis. In conclusion, this sequential delivery system is promoting to manage non-healing infected wounds, and also provides a new thought to realize the staged drug release.

The levels of vancomycin in the blood and the wound after the local treatment of bone and soft-tissue infection with antibiotic-loaded calcium sulphate as carrier material.

AIMS: Calcium sulphate (CaSO4) is a resorbable material that can be used simultaneously as filler of a dead space and as a carrier for the local application of antibiotics. Our aim was to describe the systemic exposure and the wound fluid concentrations of vancomycin in patients treated with vancomycin-loaded CaSO4 as an adjunct to the routine therapy of bone and joint infections. PATIENTS AND METHODS: A total of 680 post-operative blood and 233 wound fluid samples were available for analysis from 94 implantations performed in 87 patients for various infective indications. Up to 6 g of vancomycin were used. Non-compartmental pharmacokinetic analysis was performed on the data from 37 patients treated for an infection of the hip. RESULTS: The overall systemic exposure remained within a safe range, even in patients with post-operative renal failure, none requiring removal of the pellets. Local concentrations were approximately ten times higher than with polymethylmethacrylate (PMMA) as a carrier, but remained below reported cell toxicity thresholds. Decreasing concentrations in wound fluid were observed over several weeks, but remained above the common minimum inhibitory concentrations for Staphylococcus up to three months post-operatively. CONCLUSION: This study provides the first pharmacokinetic description of the local application of vancomycin with CaSO4 as a carrier, documenting slow release, systemic safety and a release profile far more interesting than from PMMA. In particular, considering in vitro data, concentrations of vancomycin active against staphylococcal biofilm were seen for several weeks. Cite this article: Bone Joint J 2017;99-B:1537-44.

Genetic alterations responsible for reduced susceptibility to vancomycin in community-associated MRSA strains of ST72.

Objectives: We previously reported the first case of vancomycin treatment failure due to development of vancomycin-intermediate resistance in a patient with an MRSA of ST72, a community genotype in Korea. We investigated two isogenic MRSA strains from this patient, who experienced treatment failure with vancomycin and rifampicin. Methods: We tracked the genetic alterations that confer reduced susceptibility to vancomycin on those two isogenic MRSA strains by WGS. Results: Five non-synonymous mutations were identified, including rpoB (H481Y), dprA (G196C), femA (F92C), vraR (E127K) and agrC (E391stop). We further studied the role of a mutation of vraR in reduced susceptibility to vancomycin. Introduction of the mutated vraR (E127K) into a vancomycin-susceptible Staphylococcus aureus strain resulted in an increase in vraSR mRNA expression and vancomycin MIC and development of the hetero-VISA phenotype, which was confirmed by the population analysis profile (PAP)/AUC. Electron microscopy showed increased cell wall thickness in the strains with mutated vraR. Conclusions: Based on the genomic data, molecular experiments and PAP and cell wall analyses, we propose that a single mutation of vraR is associated with the reduced susceptibility to vancomycin in MRSA and further treatment failure.

Vancomycin-Associated Cast Nephropathy.

Vancomycin is a widely prescribed antibiotic, but the exact nature of vancomycin-associated nephrotoxicity is unclear, in particular when considering the frequent coadministration of aminoglycosides. We describe here the initial case of a 56-year-old woman with normal renal function developing unexplained ARF without hypovolemia after administration of vancomycin without coadministration of aminoglycosides. Studying the patient's renal biopsy specimen, we ascertained that obstructive tubular casts composed of noncrystal nanospheric vancomycin aggregates entangled with uromodulin explained the vancomycin-associated ARF. We developed in parallel a new immunohistologic staining technique to detect vancomycin in renal tissue and confirmed retrospectively that deleterious vancomycin-associated casts existed in eight additional patients with acute tubular necrosis in the absence of hypovolemia. Concomitant high vancomycin trough plasma levels had been observed in each patient. We also reproduced experimentally the toxic and obstructive nature of vancomycin-associated cast nephropathy in mice, which we detected using different in vivo imaging techniques. In conclusion, the interaction of uromodulin with nanospheric vancomycin aggregates represents a new mode of tubular cast formation, revealing the hitherto unsuspected mechanism of vancomycin-associated renal injury.

Two small (p)ppGpp synthases in Staphylococcus aureus mediate tolerance against cell envelope stress conditions.

The stringent response is a conserved global regulatory mechanism that is related to the synthesis of (p)ppGpp nucleotides. Gram-positive bacteria, such as Staphylococcus aureus, possess three (p)ppGpp synthases: the bifunctional RSH (RelA/SpoT homolog) protein, which consists of a (p)ppGpp synthase and a (p)ppGpp hydrolase domain, and two truncated (p)ppGpp synthases, designated RelP and RelQ. Here, we characterized these two small (p)ppGpp synthases. Biochemical analyses of purified proteins and in vivo studies revealed a stronger synthetic activity for RelP than for RelQ. However, both enzymes prefer GDP over GTP as the pyrophosphate recipient to synthesize ppGpp. Each of the enzymes was shown to be responsible for the essentiality of the (p)ppGpp hydrolase domain of the RSH protein. The staphylococcal RSH-hydrolase is an efficient enzyme that prevents the toxic accumulation of (p)ppGpp. Expression of (p)ppGpp synthases in a hydrolase-negative background leads not only to growth arrest but also to cell death. Transcriptional analyses showed that relP and relQ are strongly induced upon vancomycin and ampicillin treatments. Accordingly, mutants lacking relP and relQ showed a significantly reduced survival rate upon treatments with cell wall-active antibiotics. Thus, RelP and RelQ are active (p)ppGpp synthases in S. aureus that are induced under cell envelope stress to mediate tolerance against these conditions.

Sensitization of Staphylococcus aureus to methicillin and other antibiotics in vitro and in vivo in the presence of HAMLET.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex from human milk with both tumoricidal and bactericidal activities. HAMLET exerts a rather specific bactericidal activity against some respiratory pathogens, with highest activity against Streptococcus pneumoniae, but lacks activity against most other bacterial pathogens, including Staphylococci. Still, ion transport associated with death in S. pneumoniae is also detected to a lower degree in insensitive organisms. In this study we demonstrate that HAMLET acts as an antimicrobial adjuvant that can increase the activity of a broad spectrum of antibiotics (methicillin, vancomycin, gentamicin and erythromycin) against multi-drug resistant Staphylococcus aureus, to a degree where they become sensitive to those same antibiotics, both in antimicrobial assays against planktonic and biofilm bacteria and in an in vivo model of nasopharyngeal colonization. We show that HAMLET exerts these effects specifically by dissipating the proton gradient and inducing a sodium-dependent calcium influx that partially depolarizes the plasma membrane, the same mechanism induced during pneumococcal death. These effects results in an increased cell associated binding and/or uptake of penicillin, gentamicin and vancomycin, especially in resistant stains. Finally, HAMLET inhibits the increased resistance of methicillin seen under antibiotic pressure and the bacteria do not become resistant to the adjuvant, which is a major advantageous feature of the molecule. These results highlight HAMLET as a novel antimicrobial adjuvant with the potential to increase the clinical usefulness of antibiotics against drug resistant strains of S. aureus.