Vancomycin-arginine (STM-001) abrogates ESBL carrier and carbapenem-resistant Escherichia coli burden in a murine complicated urinary tract infection model.

OBJECTIVES: STM-001, a retargeted glycopeptide, is active against MDR E. coli expressing ESBLs including carbapenemases. Herein, we assessed its capability to combat E. coli complicated urinary tract infections (cUTI) in mice driven by clinically important serine (CTX-M-15) and metallo-ß-lactamases (NDM-1). METHODS: Plasma and urine pharmacokinetics following IV administration of STM-001 (1-50 mg/kg) were determined in mice via LC-MS/MS. The effects on bacterial burden (kidney, bladder and urine) were determined in a 7 day mouse cUTI model whereby STM-001 was administered q12h or q24h at 2-100 mg/kg/day from Day 4. Efficacy was assessed by the change in log10 cfu/g or log10 cfu/mL from vehicle-treated infected mice. RESULTS: MICs of STM-001 for CTX-M-15 and NDM-1 E. coli were 8 and 16 mg/L, respectively. Blood pharmacokinetic profile was linear and dose-dependent with low clearance of 9.49 ±â€Š0.31 mL/min/kg, V = 0.63 ±â€Š0.02 L/kg and t½â€Š= 1.16 ±â€Š0.03 h. High STM-001 concentrations were recovered in urine 0-8 h post-administration, reaching up to 120-fold above its MIC. In cUTI efficacy studies, STM-001 (1-50 mg/kg, q12h) reduced CTX-M-15 burden by log10 4.31 (kidney), 3.95 (bladder) and 4.82 (urine) compared with vehicle-treated animals (P < 0.0001). STM-001 also reduced NDM-1 burden by log10 3.89 (kidney), 3.76 (bladder) and 3.08 (urine) (P < 0.0001), with similar inhibitory effects following q24h dosing. CONCLUSIONS: STM-001 was highly effective in reducing E. coli burden in kidney, bladder and urine in mouse cUTI models. The observed efficacy with either dosing regimen indicates potential low humanized doses of 1-5 mg/kg. These data support further development of STM-001 as an innovative, carbapenem-sparing antibiotic to combat human cUTIs.

In Vivo Targeting of Escherichia coli with Vancomycin-Arginine.

The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to Gram-negative bacteria was investigated. Its MIC towards Escherichia coli, including ß-lactamase expressing Ambler classes A, B, and D, was 8 to 16 µg/ml. Addition of 8 times the MIC of V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (<2.32 × 10-10). In vivo, V-r markedly reduced E. coli burden by >7 log10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli, including resistant forms.

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Discovery of ANT3310, a Novel Broad-Spectrum Serine ß-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and Acinetobacter baumannii.

The diazabicyclooctanes (DBOs) are a class of serine ß-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of ß-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of ß-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

ANT2681: SAR Studies Leading to the Identification of a Metallo-ß-lactamase Inhibitor with Potential for Clinical Use in Combination with Meropenem for the Treatment of Infections Caused by NDM-Producing Enterobacteriaceae.

The clinical effectiveness of the important ß-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-ß-lactamase (MBL) enzymes. To address this resistance issue, multiple ß-lactam/ß-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.

Pharmacokinetic/pharmacodynamic considerations for new and current therapeutic drugs for uncomplicated gonorrhoea-challenges and opportunities.

BACKGROUND: Increasing multidrug resistance rates in Neisseria gonorrhoeae have raised concerns and an urgent call for new antibiotics for treatment of gonorrhoea. Several decades of subdued drug development in this field and the recent failures of two new antibiotics to show non-inferiority compared with the current first-line antibiotics ceftriaxone plus azithromycin highlight the need for improved preclinical tools to predict clinical outcome of new drugs in the development process. OBJECTIVES: To summarize current pharmacokinetic/pharmacodynamic (PK/PD) knowledge and dose-finding strategies for antibiotics against gonorrhoea. SOURCES: Literature review of published papers and discussions by global experts at a special workshop on this topic. CONTENT: We review current knowledge of gonococcal specific PK/PD principles and provide an update on new in vitro and in vivo models to correlate drug exposure with clinical outcome, and identify challenges and gaps in gonococcal therapeutic research. IMPLICATIONS: Identifying the ideal antimicrobial agent and dose for treating uncomplicated urogenital and pharyngeal gonococcal disease requires appropriate validated non-clinical PK/PD models. Recent advances in adapting in vitro and in vivo models for use in gonorrhoea are an important step for enabling the development of new drugs with reduced risk of failure in Phase 3 clinical development and diminish the risk of emergence of resistance.

Intermittent micafungin for prophylaxis in a rat model of chronic Candida albicans gut colonization.

BACKGROUND: During antifungal prophylaxis, micafungin is generally infused IV once daily over 1 h. In practice, less-frequent dosing could improve the quality of life in patients requiring long-term treatment or prophylaxis. The feasibility of this approach was assessed using humanized doses of daily or infrequent micafungin regimens. OBJECTIVES: To evaluate the effectiveness of intermittent high-dose micafungin, simulating human exposure, for prophylaxis of invasive candidiasis in a rat model of chronic Candida albicans gastrointestinal colonization and systemic dissemination. METHODS: Two weeks post-infection with an oral challenge of C. albicans, Sprague-Dawley rats were immunocompromised with a cytotoxic drug and a steroid. Rats received IV infusions of: daily vehicle control; daily subcutaneous micafungin (20 mg/kg SC); high-dose micafungin (20 mg/kg bolus SC + 80 mg/kg infusion/72 h, to simulate intermittent human dosing of 300 mg/72 h); or daily fluconazole by mouth (10 mg/kg PO). The effects of antifungal prophylaxis on faecal fungal burden and systemic C. albicans dissemination were evaluated. RESULTS: A rat model of chronic C. albicans gastrointestinal colonization and systemic dissemination was established, characterized by a sustained microbiological burden over 29 days and fungal recovery from normally sterile tissues. Using this model, intermittent high-dose micafungin (delivered via iPrecio pumps) to simulate humanized doses of 300 mg/72 h was significantly more effective than vehicle control, as effective as once-daily micafungin and similar to daily fluconazole at reducing faecal burden and preventing systemic dissemination. CONCLUSIONS: These data indicate that intermittent high-dose micafungin can be as effective as daily therapy, supporting clinical assessment in high-risk patients requiring long-term antifungal prophylaxis.

Pharmacokinetics-Pharmacodynamics of Enmetazobactam Combined with Cefepime in a Neutropenic Murine Thigh Infection Model.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum ß-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > CT ) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 ß-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 µg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log10 bioburden reduction of 8% and 44% fT > 2 µg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.

Infection Augments Expression of Mechanosensing Piezo1 Channels in Amyloid Plaque-Reactive Astrocytes.

A defining pathophysiological hallmark of Alzheimer's disease (AD) is the amyloid plaque; an extracellular deposit of aggregated fibrillar Aß1-42 peptides. Amyloid plaques are hard, brittle structures scattered throughout the hippocampus and cerebral cortex and are thought to cause hyperphosphorylation of tau, neurofibrillary tangles, and progressive neurodegeneration. Reactive astrocytes and microglia envelop the exterior of amyloid plaques and infiltrate their inner core. Glia are highly mechanosensitive cells and can almost certainly sense the mismatch between the normally soft mechanical environment of the brain and very stiff amyloid plaques via mechanosensing ion channels. Piezo1, a non-selective cation channel, can translate extracellular mechanical forces to intracellular molecular signaling cascades through a process known as mechanotransduction. Here, we utilized an aging transgenic rat model of AD (TgF344-AD) to study expression of mechanosensing Piezo1 ion channels in amyloid plaque-reactive astrocytes. We found that Piezo1 is upregulated with age in the hippocampus and cortex of 18-month old wild-type rats. However, more striking increases in Piezo1 were measured in the hippocampus of TgF344-AD rats compared to age-matched wild-type controls. Interestingly, repeated urinary tract infections with Escherichia coli bacteria, a common comorbidity in elderly people with dementia, caused further elevations in Piezo1 channel expression in the hippocampus and cortex of TgF344-AD rats. Taken together, we report that aging and peripheral infection augment amyloid plaque-induced upregulation of mechanoresponsive ion channels, such as Piezo1, in astrocytes. Further research is required to investigate the role of astrocytic Piezo1 in the Alzheimer's brain, whether modulating channel opening will protect or exacerbate the disease state, and most importantly, if Piezo1 could prove to be a novel drug target for age-related dementia.

Dihydroorotate dehydrogenase inhibitor olorofim exhibits promising activity against all clinically relevant species within Aspergillus section Terrei.

Objectives: In vitro and in vivo activity of the dihydroorotate dehydrogenase inhibitor olorofim (formerly F901318) (F2G Limited, UK) against clinically relevant species of the Aspergillus section Terrei was evaluated. Methods: A total of 92 clinical Aspergillus section Terrei isolates [42 Aspergillus terreus sensu stricto and 50 cryptic species: Aspergillus alabamensis (n = 8), Aspergillus citrinoterreus (n = 27), Aspergillus floccosus (n = 1), Aspergillus hortai (n = 13) and Aspergillus neoafricanus (n = 1)] were evaluated. MICs were determined using the CLSI M38-A2 method. MICs of olorofim were compared with those of posaconazole, voriconazole, itraconazole and amphotericin B. The in vivo efficacy of olorofim was determined in an immunosuppressed murine model of disseminated aspergillosis. Results: Olorofim was highly active against all tested Aspergillus section Terrei isolates, exhibiting an MIC range of 0.002-0.063 mg/L. Slightly higher MICs were observed for A. terreus cryptic species. Olorofim MICs were lower than those observed for the azoles. Selected strains with elevated MICs of azoles were highly susceptible to olorofim. Olorofim administered by oral and intravenous routes produced survival rates of 90%-100% in A. terreus-infected mice. Conclusions: Olorofim showed potent and consistent in vitro activity against all A. terreus strains tested, including those with elevated MICs of other antifungal substances. Overall, growth inhibition by olorofim was superior to that of azoles. In vivo data showed that olorofim was highly efficacious in prolonging survival of mice with disseminated aspergillosis due to A. terreus sensu stricto.

Discovery of a Novel Metallo-ß-Lactamase Inhibitor That Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacteriaceae.

Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are increasingly prevalent and have become a major worldwide threat to human health. Carbapenem resistance is driven primarily by the acquisition of ß-lactamase enzymes, which are able to degrade carbapenem antibiotics (hence termed carbapenemases) and result in high levels of resistance and treatment failure. Clinically relevant carbapenemases include both serine ß-lactamases (SBLs; e.g., KPC-2 and OXA-48) and metallo-ß-lactamases (MBLs), such as NDM-1. MBL-producing strains are endemic within the community in many Asian countries, have successfully spread worldwide, and account for many significant CRE outbreaks. Recently approved combinations of ß-lactam antibiotics with ß-lactamase inhibitors are active only against SBL-producing pathogens. Therefore, new drugs that specifically target MBLs and which restore carbapenem efficacy against MBL-producing CRE pathogens are urgently needed. Here we report the discovery of a novel MBL inhibitor, ANT431, that can potentiate the activity of meropenem (MEM) against a broad range of MBL-producing CRE and restore its efficacy against an Escherichia coli NDM-1-producing strain in a murine thigh infection model. This is a strong starting point for a chemistry lead optimization program that could deliver a first-in-class MBL inhibitor-carbapenem combination. This would complement the existing weaponry against CRE and address an important and growing unmet medical need.

Potentiation of Antibiotic Activity by a Novel Cationic Peptide: Potency and Spectrum of Activity of SPR741.

Novel approaches for the treatment of multidrug-resistant Gram-negative bacterial infections are urgently required. One approach is to potentiate the efficacy of existing antibiotics whose spectrum of activity is limited by the permeability barrier presented by the Gram-negative outer membrane. Cationic peptides derived from polymyxin B have been used to permeabilize the outer membrane, granting antibiotics that would otherwise be excluded access to their targets. We assessed the in vitro efficacies of combinations of SPR741 with conventional antibiotics against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii Of 35 antibiotics tested, the MICs of 8 of them were reduced 32- to 8,000-fold against E. coli and K. pneumoniae in the presence of SPR741. The eight antibiotics, azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, retapamulin, rifampin, and telithromycin, had diverse targets and mechanisms of action. Against A. baumannii, similar potentiation was achieved with clarithromycin, erythromycin, fusidic acid, retapamulin, and rifampin. Susceptibility testing of the most effective antibiotic-SPR741 combinations was extended to 25 additional multidrug-resistant or clinical isolates of E. coli and K. pneumoniae and 17 additional A. baumannii isolates in order to rank the potentiated antibiotics. SPR741 was also able to potentiate antibiotics that are substrates of the AcrAB-TolC efflux pump in E. coli, effectively circumventing the contribution of this pump to intrinsic antibiotic resistance. These studies support the further development of SPR741 in combination with conventional antibiotics for the treatment of Gram-negative bacterial infections.

A single dose of epidermicin NI01 is sufficient to eradicate MRSA from the nares of cotton rats.

Objectives: To investigate the efficacy of a potent novel antimicrobial protein of mass 6 kDa, epidermicin NI01, for eradicating the nasal burden of MRSA in a cotton rat ( Sigmodon hispidus ) model. Methods: MRSA strain ATCC 43300 was used to establish a robust colonization of cotton rat nares. This model was used to evaluate the efficacy of topical 0.04% and 0.2% epidermicin NI01, administered twice daily for 3 days consecutively, and topical 0.8% epidermicin NI01 administered once, for reducing nasal MRSA burden. Control groups remained untreated or were administered vehicle only (0.5% hydroxypropylmethylcellulose) or 2% mupirocin twice daily for 3 days. The experiment was terminated at day 5 and MRSA quantitative counts were determined. Tissues recovered from animals treated with 0.2% epidermicin twice daily for 3 days were examined for histological changes. Results: Mupirocin treatment resulted in a reduction in burden of log 10 (log R) of 2.59 cfu/nares compared with vehicle ( P < 0.0001). Epidermicin NI01 administered once at 0.8% showed excellent efficacy, resulting in a log R of 2.10 cfu/nares ( P = 0.0004), which was equivalent to mupirocin. Epidermicin NI01 administered at 0.2% or 0.04% twice daily for 3 days did not have a significant impact on the tissue burden recovered from the nares. Mild to marked histological abnormalities were noted, but these were determined to be reversible. Conclusion: A single dose of topical epidermicin NI01 was as effective as mupirocin administered twice daily for 3 days in eradication of MRSA from the nares of cotton rats. This justifies further development of epidermicin for this indication.

Disease Progression and Resolution in Rodent Models of Clostridium difficile Infection and Impact of Antitoxin Antibodies and Vancomycin.

Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.

Alternatives to antibiotics-a pipeline portfolio review.

Antibiotics have saved countless lives and enabled the development of modern medicine over the past 70 years. However, it is clear that the success of antibiotics might only have been temporary and we now expect a long-term and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. A broader approach to address bacterial infection is needed. In this Review, we discuss alternatives to antibiotics, which we defined as non-compound approaches (products other than classic antibacterial agents) that target bacteria or any approaches that target the host. The most advanced approaches are antibodies, probiotics, and vaccines in phase 2 and phase 3 trials. This first wave of alternatives to antibiotics will probably best serve as adjunctive or preventive therapies, which suggests that conventional antibiotics are still needed. Funding of more than £1·5 billion is needed over 10 years to test and develop these alternatives to antibiotics. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches in phase 2 trials, which would be a catalyst for active engagement and investment by the pharmaceutical and biotechnology industry. Only a sustained, concerted, and coordinated international effort will provide the solutions needed for the future.

SMT19969 for Clostridium difficile infection (CDI): in vivo efficacy compared with fidaxomicin and vancomycin in the hamster model of CDI.

OBJECTIVES: SMT19969 is a novel narrow-spectrum antimicrobial under development for the treatment of Clostridium difficile infection (CDI). The objectives were to assess the relative efficacies of SMT19969, vancomycin and fidaxomicin in the hamster model of CDI. METHODS: Hamsters were infected with either C. difficile BI1 (ribotype 027) or C. difficile 630 (ribotype 012) prior to treatment with vehicle, SMT19969, fidaxomicin or vancomycin for 5 days. Animals were further monitored through to day 28 and survival recorded. Plasma and gastrointestinal concentrations of SMT19969 following single and repeat administration in infected hamsters were determined. RESULTS: Following infection with C. difficile BI1, treatment with SMT19969, vancomycin and fidaxomicin resulted in 100% survival during the 5 day dosing period, with 90%-100% of animals receiving SMT19969 and fidaxomicin surviving during the post-dosing follow-up period. Whilst protective during treatment, onset of mortality was observed on day 11 in animals treated with vancomycin, with a 10% survival recorded by day 28. Similar results were observed for SMT19969 and vancomycin following infection with C. difficile 630, with day 28 survival rates of 80%-100% and 0%, respectively. Fidaxomicin protected animals infected with C. difficile 630 from mortality during dosing, although day 28 survival rates varied from 0% to 40% depending on dose. Plasma levels of SMT19969 were typically below the limit of quantification, but levels in the gastrointestinal tract remained far in excess of the MIC. CONCLUSIONS: These data show that SMT19969 is highly effective at treating both acute infection and preventing recurrent disease and support continued investigation of SMT19969 as a potential therapy for CDI.

Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibα.

OBJECTIVE: Bacterial infection contributes to diverse noninfectious diseases and worsens outcome after stroke. Streptococcus pneumoniae, the most common infection in patients at risk of stroke, is a major cause of prolonged hospitalization and death of stroke patients, but how infection impacts clinical outcome is not known. METHODS: We induced sustained pulmonary infection by a human S. pneumoniae isolate in naive and comorbid rodents to investigate the effect of infection on vascular and inflammatory responses prior to and after cerebral ischemia. RESULTS: S. pneumoniae infection triggered atherogenesis, led to systemic induction of interleukin (IL) 1, and profoundly exacerbated (50-90%) ischemic brain injury in rats and mice, a response that was more severe in combination with old age and atherosclerosis. Systemic blockade of IL-1 with IL-1 receptor antagonist (IL-1Ra) fully reversed infection-induced exacerbation of brain injury and functional impairment caused by cerebral ischemia. We show that infection-induced systemic inflammation mediates its effects via increasing platelet activation and microvascular coagulation in the brain after cerebral ischemia, as confirmed by reduced brain injury in response to blockade of platelet glycoprotein (GP) Ibα. IL-1 and platelet-mediated signals converge on microglia, as both IL-1Ra and GPIbα blockade reversed the production of IL-1α by microglia in response to cerebral ischemia in infected animals. INTERPRETATION: S. pneumoniae infection augments atherosclerosis and exacerbates ischemic brain injury via IL-1 and platelet-mediated systemic inflammation. These mechanisms may contribute to diverse cardio- and cerebrovascular pathologies in humans.

MRSA decolonization of cotton rat nares by a combination treatment comprising lysostaphin and the antimicrobial peptide ranalexin.

OBJECTIVES: To evaluate the in vivo effectiveness of a combination treatment containing ranalexin (a natural antimicrobial peptide) and lysostaphin (an antistaphylococcal endopeptidase) for reducing nasal burden of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The community-acquired MRSA strain S. aureus NRS384 (USA300-0114) was used in the present study because it is commonly isolated from human nares and it established consistent and reproducible colonization of cotton rat nares. This model was used to evaluate the efficacy of ranalexin/lysostaphin gels (0.1%-1% w/v; administered intranasally once or once per day for 3 consecutive days) for reducing nasal MRSA burden. Control animals were administered vehicle gel only (0.5% hydroxypropyl methylcellulose) or 2% mupirocin, which is used clinically for nasal decolonization of MRSA. Nasal MRSA burden was assessed at 192 h post-inoculation, which was at least 72 h after the final treatment had been administered. An additional study assessed the efficacy of 0.1% ranalexin/lysostaphin against a mupirocin-resistant MRSA strain (MUP20), which had been selected by serial passage of S. aureus NRS384 through subinhibitory concentrations of mupirocin. RESULTS: Gels containing 0.1% ranalexin/lysostaphin consistently reduced median nasal burden of MRSA to an extent similar to or greater than 2% mupirocin. Treatment with 0.1% ranalexin/lysostaphin was also effective against the MUP20 strain. There was evidence for only minimal irritancy in cotton rat nares administered three doses of 0.1% ranalexin/lysostaphin, suggesting that this agent is suitable for short-course therapy such as is employed currently for nasal decolonization with mupirocin. CONCLUSIONS: Ranalexin/lysostaphin could serve as an alternative to mupirocin for nasal decolonization of MRSA.

Pharmacokinetics and pharmacodynamics of fluconazole for cryptococcal meningoencephalitis: implications for antifungal therapy and in vitro susceptibility breakpoints.

Fluconazole is frequently the only antifungal agent that is available for induction therapy for cryptococcal meningitis. There is relatively little understanding of the pharmacokinetics and pharmacodynamics (PK-PD) of fluconazole in this setting. PK-PD relationships were estimated with 4 clinical isolates of Cryptococcus neoformans. MICs were determined using Clinical and Laboratory Standards Institute (CLSI) methodology. A nonimmunosuppressed murine model of cryptococcal meningitis was used. Mice received two different doses of fluconazole (125 mg/kg of body weight/day and 250 mg/kg of body weight/day) orally for 9 days; a control group of mice was not given fluconazole. Fluconazole concentrations in plasma and in the cerebrum were determined using high-performance liquid chromatography (HPLC). The cryptococcal density in the brain was estimated using quantitative cultures. A mathematical model was fitted to the PK-PD data. The experimental results were extrapolated to humans (bridging study). The PK were linear. A dose-dependent decline in fungal burden was observed, with near-maximal activity evident with dosages of 250 mg/kg/day. The MIC was important for understanding the exposure-response relationships. The mean AUC/MIC ratio associated with stasis was 389. The results of the bridging study suggested that only 66.7% of patients receiving 1,200 mg/kg would achieve or exceed an AUC/MIC ratio of 389. The potential breakpoints for fluconazole against Cryptococcus neoformans follow: susceptible, ≤ 2 mg/liter; resistant, >2 mg/liter. Fluconazole may be an inferior agent for induction therapy because many patients cannot achieve the pharmacodynamic target. Clinical breakpoints are likely to be significantly lower than epidemiological cutoff values. The MIC may guide the appropriate use of fluconazole. If fluconazole is the only option for induction therapy, then the highest possible dose should be used.

The Aspergillus fumigatus dihydroxyacid dehydratase Ilv3A/IlvC is required for full virulence.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.