Repurposed drugs block toxin-driven platelet clearance by the hepatic Ashwell-Morell receptor to clear Staphylococcus aureus bacteremia.

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a "toxin-platelet-AMR" regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

The Role of Antibiotic Stewardship in Promoting Appropriate Antibiotic Use.

Antibiotics are one of the most significant medical discoveries in human history. The widespread use of antibiotics has resulted in the emergence of antibiotic-resistant pathogens. This fact, coupled with the paucity of new antibiotic developments, has spurred efforts to combat antibiotic resistance. One of the most critical components of these efforts is antibiotic stewardship, a multidisciplinary endeavor, employing a collection of interventions in a variety of health care settings with the aim of promoting appropriate utilization of antibiotics. This article describes antibiotic stewardship programs and key practices used to minimize the development and spread of antibiotic-resistant pathogens including the optimization of antibiotic pharmacokinetics and pharmacodynamics, the application of rapid diagnostic tools, and the use of computerized provider order entry tools.

Rethinking Antimicrobial Prophylaxis in the Transplant Patient in the World of Emerging Resistant Organisms-Where Are We Today?

PURPOSE OF REVIEW: The use of prophylactic antibiotics during the neutropenic period in hematopoietic stem cell transplantation has been the standard of care at most institutions for the past 20 years. We sought to review the benefits and risks of this practice. RECENT FINDINGS: Emerging data has highlighted the potential costs of antibacterial prophylaxis, from selecting for antibiotic resistance to perturbing the microbiome and contributing to increase risk for Clostridium difficile and perhaps graft-versus-host-disease, conditions which may lead to poorer outcomes. Though in many studies prophylactic antibiotics improved morbidity and mortality outcomes, the potential harms including antibiotic resistance, Clostridium difficile infection, and alterations of the gut microbiome should be considered. Future studies aimed to better risk-stratify patients and limit the use of broad-spectrum antibiotics are warranted.

Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence.

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.

Marinopyrrole derivatives as potential antibiotic agents against methicillin-resistant Staphylococcus aureus (III).

The marine natural product, marinopyrrole A (1), was previously shown to have significant antibiotic activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Although compound (1) exhibits a significant reduction in MRSA activity in the presence of human serum, we have identified key modifications that partially restore activity. We previously reported our discovery of a chloro-derivative of marinopyrrole A (1a) featuring a 2-4 fold improved minimum inhibitory concentration (MIC) against MRSA, significantly less susceptibility to serum inhibition and rapid and concentration-dependent killing of MRSA. Here, we report a novel fluoro-derivative of marinopyrrole A (1e) showing an improved profile of potency, less susceptibility to serum inhibition, as well as rapid and concentration-dependent killing of MRSA.

Anthracimycin activity against contemporary methicillin-resistant Staphylococcus aureus.

Anthracimycin is a recently discovered novel marine-derived compound with activity against Bacillus anthracis. We tested anthracimycin against an expanded panel of Staphylococcus aureus strains in vitro and in vivo. All strains of S. aureus tested, including methicillin-susceptible, methicillin-resistant (MRSA) and vancomycin-resistant strains of S. aureus, were susceptible to anthracimycin at MIC values of ⩽0.25 mg l(-1). Although its postantibiotic effects were minimal, anthracimycin exhibited potent and rapid bactericidal activity, with a >4-log kill of USA300 MRSA within 3 h at five times its MIC. At concentrations significantly below the MIC, anthracimycin slowed MRSA growth and potentiated the bactericidal activity of the human cathelicidin, LL-37. The bactericidal activity of anthracimycin was somewhat mitigated in the presence of 20% human serum, and the compound was minimally toxic to human cells, with an IC50 (inhibitory concentration 50)=70 mg l(-1) against human carcinoma cells. At concentrations near the MIC, anthracimycin inhibited S. aureus nucleic acid synthesis as determined by optimized macromolecular synthesis methodology, with inhibition of DNA and RNA synthesis occurring in the absence of DNA intercalation. Anthracimycin at a single dose of 1 or 10 mg kg(-1) was able to protect mice from MRSA-induced mortality in a murine peritonitis model of infection. Anthracimycin provides an interesting new scaffold for future development of a novel MRSA antibiotic.

Medications at school: disposing of pharmaceutical waste.

BACKGROUND: This project quantified and categorized medications left unclaimed by students at the end of the school year. It determined the feasibility of a model medication disposal program and assessed school nurses' perceptions of environmentally responsible medication disposal. METHODS: At a large urban school district all unclaimed medications were collected at the end of a school year to determine the extent and nature of this problem. Nurses documented unclaimed medications and transported them to a central district location. An environmentally responsible medication disposal program, consisting of sealed containers bound for a local hospital's disposal system, was implemented. RESULTS: In a school district of approximately 133,000 students, there were 926 different medications abandoned at the end of a school year brought to a central disposal area. Nurses complied with the newly implemented protocol. Information collected from nurses indicates acceptance of the program. Disposal of unclaimed medications at a central location, use of secured containers, and transportation to a hospital for environmentally responsible disposal proved to be feasible and acceptable to the staff. CONCLUSIONS: Unclaimed medications at school each year pose a potentially huge environmental risk when disposed of improperly. It is feasible to implement an environmentally responsible medication disposal protocol at schools.

Activity of the thiopeptide antibiotic nosiheptide against contemporary strains of methicillin-resistant Staphylococcus aureus.

The rapid rise in antimicrobial resistance in bacteria has generated an increased demand for the development of novel therapies to treat contemporary infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). However, antimicrobial development has been largely abandoned by the pharmaceutical industry. We recently isolated the previously described thiopeptide antibiotic nosiheptide from a marine actinomycete strain and evaluated its activity against contemporary clinically relevant bacterial pathogens. Nosiheptide exhibited extremely potent activity against all contemporary MRSA strains tested including multiple drug-resistant clinical isolates, with MIC values 0.25 mg l(-1). Nosiheptide was also highly active against Enterococcus spp. and the contemporary hypervirulent BI/NAP1/027 strain of Clostridium difficile but was inactive against most Gram-negative strains tested. Time-kill analysis revealed nosiheptide to be rapidly bactericidal against MRSA in a concentration- and time-dependent manner, with a nearly 2-log kill noted at 6 h at 10 × MIC. Furthermore, nosiheptide was found to be non-cytotoxic against mammalian cells at >>100 × MIC, and its anti-MRSA activity was not inhibited by 20% human serum. Notably, nosiheptide exhibited a significantly prolonged post-antibiotic effect against both healthcare- and community-associated MRSA compared with vancomycin. Nosiheptide also demonstrated in vivo activity in a murine model of MRSA infection, and therefore represents a promising antibiotic for the treatment of serious infections caused by contemporary strains of MRSA.

Marinopyrrole derivatives as potential antibiotic agents against methicillin-resistant Staphylococcus aureus (I).

Infections caused by drug-resistant pathogens are on the rise. The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains exemplifies the urgent need for new antibiotics. The marine natural product, marinopyrrole A, was previously shown to have potent antibiotic activity against Gram-positive pathogens, including MRSA. However, its minimum inhibitory concentration (MIC) against MRSA was increased by >500 fold in the presence of 20% human serum, thus greatly limiting therapeutic potential. Here we report our discovery of a novel derivative of marinopyrrole A, designated 1a, featuring a 2-4 fold improved MIC against MRSA and significantly less susceptibility to serum inhibition. Importantly, compound 1a displayed rapid and concentration-dependent killing of MRSA. Compared to the natural product counterpart, compound 1a provides an important natural product based scaffold for further Structure Activity Relationship (SAR) and optimization.

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target.

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.

Bactericidal kinetics of marine-derived napyradiomycins against contemporary methicillin-resistant Staphylococcus aureus.

There is an urgent need for new antibiotics to treat hospital- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections. Previous work has indicated that both terrestrial and marine-derived members of the napyradiomycin class possess potential anti-staphylococcal activities. These compounds are unique meroterpenoids with unusual levels of halogenation. In this paper we report the evaluation of two previously described napyradiomycin derivatives, A80915A (1) and A80915B (2) produced by the marine-derived actinomycete, Streptomyces sp. strain CNQ-525, for their specific activities against contemporary and clinically relevant MRSA. Reported are studies of the in vitro kinetics of these chemical scaffolds in time-kill MRSA assays. Both napyradiomycin derivatives demonstrate potent and rapid bactericidal activity against contemporary MRSA strains. These data may help guide future development and design of analogs of the napyradiomycins that could potentially serve as useful anti-MRSA therapeutics.

Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry.

Microbial competition exists in the general environment, such as soil or aquatic habitats, upon or within unicellular or multicellular eukaryotic life forms. The molecular actions that govern microbial competition, leading to niche establishment and microbial monopolization, remain undetermined. The emerging technology of imaging mass spectrometry (IMS) enabled the observation that there is directionality in the metabolic output of the organism Bacillus subtilis when co-cultured with Staphylococcus aureus. The directionally released antibiotic alters S. aureus virulence factor production and colonization. Therefore, IMS provides insight into the largely hidden nature of competitive microbial encounters and niche establishment, and provides a paradigm for future antibiotic discovery.

Fijimycins A-C, three antibacterial etamycin-class depsipeptides from a marine-derived Streptomyces sp.

Three new depsipeptides, fijimycins A-C (1-3), together with the known etamycin A (4), were isolated and identified from the fermentation broth of strain CNS-575, a Streptomyces sp. cultured from a marine sediment sample collected off Nasese, Fiji. The planar structures of the new fijimycins were assigned by combined interpretation of NMR and MS/MS spectroscopic data. These assignments were complicated by the fact that 1-3 occurred as complex amide conformational mixtures. The absolute configurations of the component amino acids were established using the Marfey's method. Fijimycins A-C, and etamycin A, were shown to possess significant in vitro antibacterial activity against three methicillin-resistant Staphylococcus aureus (MRSA) strains with MIC(100) values between 4 and 16 µg mL(-1).

Pharmacological properties of the marine natural product marinopyrrole A against methicillin-resistant Staphylococcus aureus.

The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains in hospital and community settings presents a great challenge to public health and illustrates the urgency of discovering new antibiotics. Marinopyrrole A is a member of a structurally novel class of compounds identified from a species of marine-derived streptomycetes with evidence of antistaphylococcal activity. We show that marinopyrrole A has potent concentration-dependent bactericidal activity against clinically relevant hospital- and community-acquired MRSA strains, a prolonged postantibiotic effect superior to that of the current first-line agents vancomycin and linezolid, and a favorable resistance profile. Marinopyrrole A showed limited toxicity to mammalian cell lines (at >20× MIC). However, its antibiotic activity against MRSA was effectively neutralized by 20% human serum. A variety of marinopyrrole analogs were isolated from culture or synthetically produced to try to overcome the inhibitory effect of serum. While many of these compounds retained potent bactericidal effect against MRSA, their activities were also inhibited by serum. Marinopyrrole A has significant affinity for plastic and may therefore have potential as a potent anti-MRSA agent in cutaneous, intracatheter, or antibiotic-lock applications.

Total synthesis and biological evaluation of marinopyrrole A and analogues.

A five-step total synthesis of the antibiotic marinopyrrole A (1) is described. The developed synthetic technology enabled the synthesis of several marinopyrrole A analogues whose antibacterial properties against methicillin-resistant Staphylococcus aureus TCH1516 were evaluated.

Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps.

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis.

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC(50) to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

Activity of the streptogramin antibiotic etamycin against methicillin-resistant Staphylococcus aureus.

The alarming rise of hospital- and community-associated methicillin-resistant Staphylococcus aureus (HA- and CA-MRSA) infections has prompted a desperate search for novel antibiotics. We discovered the streptogramin etamycin produced by an actinomycete species isolated from the coast of Fiji, the first time this antibiotic has been identified from a marine microbe. Etamycin was extracted and purified from this strain (CNS-575) and identified as a three-rotamer species by 2D NMR spectroscopy. Etamycin demonstrated potent activity against HA- and CA-MRSA in microbroth dilution assays, with minimum inhibitory concentrations (MIC) as low as 1-2 mg l(-1) against HA- and CA-MRSA strains. Furthermore, etamycin was also active against other Gram-positive and several Gram-negative pathogens and was found to be non-cytotoxic at concentrations more than 20-fold above MIC. Etamycin displayed favorable time-kill kinetics compared with the first-line MRSA antibiotic, vancomycin, and also conferred significant protection from mortality in a murine model of systemic lethal MRSA infection. These data emphasize the utility of the marine environment as a relatively untapped source of antibiotics against major drug-resistant human pathogens. These studies will also guide future isolation and preclinical development of depsipeptide anti-MRSA compounds from marine-derived actinomycetes.

ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis.

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.