Development of rabbit models of ventilator-associated bacterial pneumonia produced by carbapenem-resistant Pseudomonas aeruginosa.

Ventilator-associated bacterial pneumonia (VABP) is among the most intractable of carbapenem-resistant Gram-negative bacterial infections. New antimicrobial agents are critically needed for the treatment of VABP. However, current conventionally used animal model systems are inadequate to meet this challenge. We, therefore, developed rabbit models of VABP caused by carbapenem-resistant Pseudomonas aeruginosa. Persistently neutropenic New Zealand White rabbits were used throughout the study. The early-phase intubated model (0-24 h) received mechanical ventilation, while the late-phase intubated model (72-96 h) was ambulatory. The following outcome parameters were studied: survival, residual tissue bacterial burden (CFU/g), residual BAL bacterial burden (CFU/mL), lung weights, pulmonary lesion score, histology, O2 saturation, radiographic imaging, and histology. Each anesthetized rabbit received a predetermined endotracheal bacterial inoculum, and ventilators were set to FiO2 = 40% and PEEP = 8 mmHg. Within the first 12 h post-inoculation, mean bacterial burdens in lung tissue and BAL fluid, respectively, were established at approximately 107 CFU/g and 106 CFU/mL, persisted through 24 h in the early-phase model and increased in the late-phase model to approximately 108 CFU/g and 107 CFU/mL. Mean max SpO2 was ≥98 mmHg, and mean nadir SpO2 was ≥68 mmHg. Serial thoracic radiographs demonstrated progressive multilobar pneumonic infiltrates. Lung histology revealed progressive focal bronchopneumonia, coagulative necrosis, intra-alveolar hemorrhage, alveolar epithelial cell necrosis, and bacterial microcolonies. The new rabbit model of VABP produced by carbapenem-resistant Pseudomonas aeruginosa recapitulates the pathophysiological, microbiological, diagnostic imaging, and histological patterns of human disease by which to assess critically needed new antimicrobial agents against this lethal infection.

Osteoarticular Mycoses.

Osteoarticular mycoses are chronic debilitating infections that require extended courses of antifungal therapy and may warrant expert surgical intervention. As there has been no comprehensive review of these diseases, the International Consortium for Osteoarticular Mycoses prepared a definitive treatise for this important class of infections. Among the etiologies of osteoarticular mycoses are Candida spp., Aspergillus spp., Mucorales, dematiaceous fungi, non-Aspergillus hyaline molds, and endemic mycoses, including those caused by Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species. This review analyzes the history, epidemiology, pathogenesis, clinical manifestations, diagnostic approaches, inflammatory biomarkers, diagnostic imaging modalities, treatments, and outcomes of osteomyelitis and septic arthritis caused by these organisms. Candida osteomyelitis and Candida arthritis are associated with greater events of hematogenous dissemination than those of most other osteoarticular mycoses. Traumatic inoculation is more commonly associated with osteoarticular mycoses caused by Aspergillus and non-Aspergillus molds. Synovial fluid cultures are highly sensitive in the detection of Candida and Aspergillus arthritis. Relapsed infection, particularly in Candida arthritis, may develop in relation to an inadequate duration of therapy. Overall mortality reflects survival from disseminated infection and underlying host factors.

Synthesis of 1-(2-Hydroxyphenyl)- and (3,5-Dichloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives as Promising Scaffolds for the Development of Novel Antimicrobial and Anticancer Agents.

Increasing antimicrobial resistance among Gram-positive pathogens and pathogenic fungi remains one of the major public healthcare threats. Therefore, novel antimicrobial candidates and scaffolds are critically needed to overcome resistance in Gram-positive pathogens and drug-resistant fungal pathogens. In this study, we explored 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and its 3,5-dichloro-2-hydroxyphenyl analogue for their in vitro antimicrobial activity against multidrug-resistant pathogens. The compounds showed structure-dependent antimicrobial activity against Gram-positive pathogens (S. aureus, E. faecalis, C. difficile). Compounds 14 and 24b showed promising activity against vancomycin-intermediate S. aureus strains, and favorable cytotoxic profiles in HSAEC-1 cells, making them attractive scaffolds for further development. 5-Fluorobenzimidazole, having a 3,5-dichloro-2-hydroxyphenyl substituent, was found to be four-fold, and hydrazone, with a thien-2-yl fragment, was two-fold stronger than clindamycin against methicillin resistant S. aureus TCH 1516. Moreover, hydrazone, bearing a 5-nitrothien-2-yl moiety, showed promising activity against three tested multidrug-resistant C. auris isolates representing major genetic lineages (MIC 16 µg/mL) and azole-resistant A. fumigatus strains harboring TR34/L98H mutations in the CYP51A gene. The anticancer activity characterization demonstrated that the 5-fluorobenzimidazole derivative with a 3,5-dichloro-2-hydroxyphenyl substituent showed the highest anticancer activity in an A549 human pulmonary cancer cell culture model. Collectively these results demonstrate that 1-(2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives could be further explored for the development of novel candidates targeting Gram-positive pathogens and drug-resistant fungi.

Efficacy of Cefiderocol in Experimental Stenotrophomonas maltophilia Pneumonia in Persistently Neutropenic Rabbits.

Stenotrophomonas maltophilia is an important cause of pneumonia in immunocompromised patients. Cefiderocol is a parenteral siderophore cephalosporin with potent in vitro activity against S. maltophilia. We evaluated the efficacy of cefiderocol in a neutropenic rabbit model of S. maltophilia pneumonia in comparison to trimethoprim-sulfamethoxazole (TMP-SMX). The cefiderocol area under the plasma drug concentration-time curve extrapolated to 8 h (AUC0-8) was lower (423.0 ± 40.9 µg·h/mL versus 713.6 ± 40.1 µg·h/mL) and clearance higher (252.77 ± 38.9 mL/h/kg versus 142.6 ± 32.9 mL/h/kg) in infected versus noninfected rabbits. We studied a clinical bloodstream S. maltophilia isolate with an MIC of 0.03 µg/mL of cefiderocol. Time spent above the MIC of cefiderocol for the majority of S. maltophilia isolates in rabbits recapitulated the plasma concentration-time profile observed in adult humans at the licensed dose of 2 g given intravenously (i.v.). Experimental groups consisted of 120 mg/kg cefiderocol i.v. every 8 hours (q8h); TMP-SMX, 5 mg/kg i.v. Q12h, and untreated controls (UCs). Treatment was administered for 10 days. Survival in cefiderocol-treated rabbits (87%) was greater than that in TMP-SMX-treated (25%; P < 0.05) and UC (0%; P < 0.05) groups. There was no residual bacterial burden in lung tissue or bronchoalveolar lavage (BAL) fluid in the cefiderocol group. Residual bacterial burden was present in lung tissue and BAL fluid in the TMP-SMX group but was decreased in comparison to UCs (P < 0.001). Lung weights (markers of pulmonary injury) were decreased in cefiderocol-treated versus TMP-SMX (P < 0.001) and UC (P < 0.001) groups. Cefiderocol is highly active in treatment of experimental S. maltophilia pneumonia, laying the foundation for future clinical investigations against this lethal infection in immunocompromised patients.

Mechanistic Insights to Combating NDM- and CTX-M-Coproducing Klebsiella pneumoniae by Targeting Cell Wall Synthesis and Outer Membrane Integrity.

Metallo-ß-lactamase (MBL)-producing Gram-negative bacteria cause infections associated with high rates of morbidity and mortality. Currently, a leading regimen to treat infections caused by MBL-producing bacteria is aztreonam combined with ceftazidime-avibactam. The purpose of the present study was to evaluate and rationally optimize the combination of aztreonam and ceftazidime-avibactam with and without polymyxin B against a clinical Klebsiella pneumoniae isolate producing NDM-1 and CTX-M by use of the hollow fiber infection model (HFIM). A novel de-escalation approach to polymyxin B dosing was also explored, whereby a standard 0-h loading dose was followed by maintenance doses that were 50% of the typical clinical regimen. In the HFIM, the addition of polymyxin B to aztreonam plus ceftazidime-avibactam significantly improved bacterial killing, leading to eradication, including for the novel de-escalation dosing strategy. Serial samples from the growth control and monotherapies were explored in a Galleria mellonella virulence model to assess virulence changes. Weibull regression showed that low-level ceftazidime resistance and treatment with monotherapy resulted in increased G. mellonella mortality (P < 0.05). A neutropenic rabbit pneumonia model demonstrated that aztreonam plus ceftazidime-avibactam with or without polymyxin B resulted in similar bacterial killing, and these combination therapies were statistically significantly better than monotherapies (P < 0.05). However, only the polymyxin B-containing combination therapy produced a statistically significant decrease in lung weights (P < 0.05), indicating a decreased inflammatory process. Altogether, adding polymyxin B to the combination of aztreonam plus ceftazidime-avibactam for NDM- and CTX-M-producing K. pneumoniae improved bacterial killing effects, reduced lung inflammation, suppressed resistance amplification, and limited virulence changes.

Synthesis of Novel Aminothiazole Derivatives as Promising Antiviral, Antioxidant and Antibacterial Candidates.

It is well-known that thiazole derivatives are usually found in lead structures, which demonstrate a wide range of pharmacological effects. The aim of this research was to explore the antiviral, antioxidant, and antibacterial activities of novel, substituted thiazole compounds and to find potential agents that could have biological activities in one single biomolecule. A series of novel aminothiazoles were synthesized, and their biological activity was characterized. The obtained results were compared with those of the standard antiviral, antioxidant, antibacterial and anticancer agents. The compound bearing 4-cianophenyl substituent in the thiazole ring demonstrated the highest cytotoxic properties by decreasing the A549 viability to 87.2%. The compound bearing 4-trifluoromethylphenyl substituent in the thiazole ring showed significant antiviral activity against the PR8 influenza A strain, which was comparable to the oseltamivir and amantadine. Novel compounds with 4-chlorophenyl, 4-trifluoromethylphenyl, phenyl, 4-fluorophenyl, and 4-cianophenyl substituents in the thiazole ring demonstrated antioxidant activity by DPPH, reducing power, FRAP methods, and antibacterial activity against Escherichia coli and Bacillus subtilis bacteria. These data demonstrate that substituted aminothiazole derivatives are promising scaffolds for further optimization and development of new compounds with potential influenza A-targeted antiviral activity. Study results could demonstrate that structure optimization of novel aminothiazole compounds may be useful in the prevention of reactive oxygen species and developing new specifically targeted antioxidant and antibacterial agents.

Efficacy and Pharmacokinetics of Fosmanogepix (APX001) in the Treatment of Candida Endophthalmitis and Hematogenous Meningoencephalitis in Nonneutropenic Rabbits.

Candida endophthalmitis is a serious sight-threatening complication of candidemia that may occur before or during antifungal therapy. Hematogenous Candida meningoencephalitis (HCME) is also a serious manifestation of disseminated candidiasis in premature infants, immunosuppressed children, and immunocompromised adults. We evaluated the antifungal efficacy and pharmacokinetics of the prodrug fosmanogepix (APX001) in a rabbit model of endophthalmitis/HCME. Manogepix (APX001A), the active moiety of prodrug fosmanogepix, inhibits the fungal enzyme Gwt1 and is highly active in vitro and in vivo against Candida spp., Aspergillus spp., and other fungal pathogens. Plasma pharmacokinetics of manogepix after oral administration of fosmanogepix on day 6 at 25, 50, and 100 mg/kg resulted in maximum concentration of drug in plasma (Cmax) of 3.96 ± 0.41, 4.14 ± 1.1, and 11.5 ± 1.1 µg/ml, respectively, and area under the concentration-time curve from 0 to 12 h (AUC0-12) of 15.8 ± 3.1, 30.8 ± 5.0, 95.9 ± 14 µg·h/ml, respectively. Manogepix penetrated the aqueous humor, vitreous, and choroid with liquid-to-plasma ratios ranging from 0.19 to 0.52, 0.09 to 0.12, and 0.02 to 0.04, respectively. These concentrations correlated with a significant decrease in Candida albicans burden in vitreous (>101 to 103 log CFU/g) and choroid (>101 to 103 log CFU/g) (P ≤ 0.05 and P ≤ 0.001, respectively). The aqueous humor had no detectable C. albicans in treatment and control groups. The tissue/plasma concentration ratios of manogepix in meninges, cerebrum, cerebellum, and spinal cord were approximately 1:1, which correlated with a >102 to 104 decline of C. albicans in tissue versus control (P ≤ 0.05). Serum and cerebrospinal fluid (CSF) (1→3)-ß-d-glucan levels demonstrated significant declines in response to fosmanogepix treatment. These findings provide an experimental foundation for fosmanogepix in treatment of Candida endophthalmitis and HCME and derisk the clinical trials of candidemia and invasive candidiasis.

Pharmacodynamics of Posaconazole in Experimental Invasive Pulmonary Aspergillosis: Utility of Serum Galactomannan as a Dynamic Endpoint of Antifungal Efficacy.

Aspergillus galactomannan antigenemia is an accepted tool for the diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Little is known, however, about the utility of this biomarker to assess the efficacy of antifungal therapies. The pharmacokinetics (PK) and pharmacodynamics (PD) of posaconazole in treatment and prophylaxis were investigated in the persistently neutropenic rabbit model of Aspergillus fumigatus IPA at doses between 2 and 20 mg/kg per day. Sparse plasma sampling was used to obtain PK data at steady state, and the serum galactomannan index (GMI), as a dynamic endpoint of antifungal response, was obtained every other day, in addition to conventional outcome parameters including survival and fungal tissue burden. Nonparametric PK/PD model building was performed using the Pmetrics package in R. A one-compartment model with linear elimination best described the PK of posaconazole. The PD effect of posaconazole exposure in plasma on the GMI in serum was best described by dynamic Hill functions reflecting growth and killing of the fungus. Through calculations of the area under the concentration-time curve from 0 to 24 h (AUC0-24) at steady state, the exposure-response relationship between posaconazole and the GMI for treatment followed a sigmoidal function with an asymptote forming above an AUC0-24 of 30 mg · h/liter. All prophylactic doses were able to control the fungal burden. A nonparametric population PK/PD model adequately described the effect of posaconazole in prophylaxis and treatment of experimental IPA. An AUC0-24 greater than 30 mg · h/liter was associated with adequate resolution of the GMI, which well supports previously suggested exposure-response relationships in humans.

Pharmacokinetics, Tissue Distribution, and Efficacy of VIO-001 (Meropenem/Piperacillin/Tazobactam) for Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia in Immunocompetent Rabbits with Chronic Indwelling Vascular Catheters.

Methicillin-resistant Staphylococcus aureus (MRSA) infections of surgically implanted subcutaneous vascular catheters (SISVCs) cause serious morbidity in patients with chronic illnesses. Previous in vitro and murine models demonstrated the synergistic interaction of equimolar concentrations of meropenem/piperacillin/tazobactam (MPT) (VIO-001) against MRSA infection. We investigated the pharmacokinetics (PK) and efficacy of VIO-001 for the treatment of MRSA bacteremia in immunocompetent rabbits with SISVCs. In PK studies, we determined that optimal dosing to achieve a time above 4× MIC (T>4×MIC) of a duration of 3 to 3.30 h required a 1-h infusion with every-4-h (Q4h) dosing. Study groups in efficacy experiments consisted of MPT combinations of 100/150/100 mg/kg of body weight (MPT100), 200/300/200 mg/kg (MPT200), and 400/600/400 mg/kg (MPT400); vancomycin (VAN) at 15 mg/kg; and untreated controls (UC). The inoculum of MRSA isolate USA300-TCH1516 (1 × 103 organisms) was administered via the SISCV on day 1 and locked for 24 h. The 8-day therapy started at 24 h postinoculation. There was a significant reduction of MRSA in blood cultures from the SISVCs in all treatment groups, with full clearance on day 4, versus UCs (P < 0.05). Consistent with the clearance of SISVC-related infection, full eradication of MRSA was achieved in lungs, heart, liver, spleen, and kidneys at the end of the study versus UC (P < 0.01). These results strongly correlated with time-kill data, where MPT in the range of 4/6/4 µg/ml to 32/48/32 µg/ml demonstrated a significant 6-log decrease in the bacterial burden versus UC (P < 0.01). In summary, VIO-001 demonstrated a favorable PK/pharmacodynamic (PD) profile and activity against SISCV MRSA infection, bacteremia, and disseminated infection. This rabbit model provides a new system for understanding new antimicrobial agents against MRSA SISVC-related infection, and these data provide a basis for future clinical investigation.

Antifungal efficacy of isavuconazole and liposomal amphotericin B in a rabbit model of Exserohilum rostratum meningoencephalitis: A preclinical paradigm for management of CNS phaeohyphomycosis.

Treatment options for Exserohilum rostratum meningoencephalitis and other causes of phaeohyphomycosis of the central nervous system (CNS) are limited, while mortality and morbidity remain high. We therefore evaluated isavuconazole, a new antifungal triazole in comparison to liposomal amphotericin B (LAMB), in vitro and in the rabbit model of Exserohilum rostratum meningoencephalitis. We hypothesized that isavuconazole alone or in combination with LAMB or micafungin may be alternative options for treatment of CNS phaeohyphomycosis. We therefore investigated the in vitro antifungal activity of isavuconazole alone or in combination with amphotericin B deoxycholate (DAMB) or micafungin and efficacy of treatment with isavuconazole and LAMB in a rabbit model of experimental E. rostratum meningoencephalitis. Combination checkerboard plates were used to determine the minimum inhibitory concentrations, minimal lethal concentrations, fractional inhibitory concentration indices, and Bliss surface analysis of isavuconazole and amphotericin B deoxycholate (DAMB), either alone or in combination. As there were no in vitro synergistic or antagonistic interactions for either combination of antifungal agents against the E. rostratum isolates, in vivo studies were conducted with isavuconazole and LAMB as monotherapies. Rabbits were divided in following groups: treated with isavuconazole at 60 mg/kg/d (ISAV60), LAMB at 5.0 (LAMB5), 7.5 (LAMB7.5), and 10 mg/kg/d (LAMB10), and untreated controls (UC). In ISAV60-, LAMB5-, LAMB7.5-, and LAMB10-treated rabbits, significant reductions of fungal burden of E. rostratum in cerebral, cerebellar, and spinal cord tissues (P < 0.01) were demonstrated in comparison to those of UC. These antifungal effects correlated with significant reduction of CSF (1→3)-ß-D-glucan levels vs UC (P < 0.05). These data establish new translational insights into treatment of CNS phaeohyphomycosis.

Antifungal Activity of Gepinacin Scaffold Glycosylphosphatidylinositol Anchor Biosynthesis Inhibitors with Improved Metabolic Stability.

The glycosylphosphatidylinositol anchor biosynthesis inhibitor gepinacin demonstrates broad-spectrum antifungal activity and negligible mammalian toxicity in culture but is metabolically labile. The stability and bioactivity of 39 analogs were tested in vitro to identify LCUT-8, a stabilized lead with increased potency and promising single-dose pharmacokinetics. Unfortunately, no antifungal activity was seen at the maximum dosing achievable in a neutropenic rabbit model. Nevertheless, structure-activity relationships identified here suggest strategies to further improve compound potency, solubility, and stability.

Combination Therapy with Ibrexafungerp (Formerly SCY-078), a First-in-Class Triterpenoid Inhibitor of (1→3)-ß-d-Glucan Synthesis, and Isavuconazole for Treatment of Experimental Invasive Pulmonary Aspergillosis.

Ibrexafungerp (formerly SCY-078) is a semisynthetic triterpenoid and potent (1→3)-ß-d-glucan synthase inhibitor. We investigated the in vitro activity, pharmacokinetics, and in vivo efficacy of ibrexafungerp (SCY) alone and in combination with antimold triazole isavuconazole (ISA) against invasive pulmonary aspergillosis (IPA). The combination of ibrexafungerp and isavuconazole in in vitro studies resulted in additive and synergistic interactions against Aspergillus spp. Plasma concentration-time curves of ibrexafungerp were compatible with linear dose proportional profile. In vivo efficacy was studied in a well-established persistently neutropenic New Zealand White (NZW) rabbit model of experimental IPA. Treatment groups included untreated control (UC) rabbits and rabbits receiving ibrexafungerp at 2.5 (SCY2.5) and 7.5 (SCY7.5) mg/kg of body weight/day, isavuconazole at 40 (ISA40) mg/kg/day, or combinations of SCY2.5+ISA40 and SCY7.5+ISA40. The combination of SCY+ISA produced an in vitro synergistic interaction. There were significant in vivo reductions of residual fungal burden, lung weights, and pulmonary infarct scores in SCY2.5+ISA40, SCY7.5+ISA40, and ISA40 treatment groups versus those of the SCY2.5-treated, SCY7.5-treated, and UC (P < 0.01) groups. Rabbits treated with SCY2.5+ISA40 and SCY7.5+ISA40 had prolonged survival in comparison to that of the SCY2.5-, SCY7.5-, ISA40-treated, or UC (P < 0.05) groups. Serum galactomannan index (GMI) and (1→3)-ß-d-glucan levels significantly declined in animals treated with the combination of SCY7.5+ISA40 in comparison to those of animals treated with SCY7.5 or ISA40 (P < 0.05). Ibrexafungerp and isavuconazole combination demonstrated prolonged survival, decreased pulmonary injury, reduced residual fungal burden, and lower GMI and (1→3)-ß-d-glucan levels in comparison to those of single therapy for treatment of IPA. These findings provide an experimental foundation for clinical evaluation of the combination of ibrexafungerp and an antimold triazole for treatment of IPA.

Pharmacokinetics and Efficacy of Ceftazidime-Avibactam in the Treatment of Experimental Pneumonia Caused by Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae in Persistently Neutropenic Rabbits.

Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is an emerging global public health threat that causes life-threatening pneumonia and bacteremia. Ceftazidime-avibactam (CZA) represents a promising advance for the treatment of serious infections caused by KPC-Kp We investigated the pharmacokinetics and efficacy of ceftazidime-avibactam in the treatment of experimental KPC-Kp pneumonia in persistently neutropenic rabbits. For single-dose and multidose (administration every 8 h) pharmacokinetics, rabbits received ceftazidime-avibactam intravenous infusions at 60/15, 90/22.5, and 120/30 mg/kg of body weight. Ceftazidime mean area under the concentration-time curves (AUCs) ranged from 287 to 608 µg·h/ml for a single dose and from 300 to 781 µg·h/ml for multiple doses. Avibactam AUCs ranged from 21 to 48 µg·h/ml for a single dose and from 26 to 48 µg·h/ml for multiple doses. KPC-Kp pneumonia was established by direct endotracheal inoculation. Treatments consisted of ceftazidime-avibactam at 120/30 mg/kg every 6 h, a polymyxin B (PMB) loading dose of 2.5 mg/kg followed by 1.5 mg/kg every 12 h q12h, or no treatment (untreated controls [UC]). There were significant reductions in the residual bacterial burden, lung weights, and pulmonary hemorrhage scores in CZA- and PMB-treated rabbits for a 7-day or a 14-day (P ≤ 0.01) course in comparison with those in the UC. These results corresponded to significant decreases in the bacterial burden in bronchoalveolar lavage fluid after a 7-day or a 14-day treatment (P ≤ 0.01). The outcomes demonstrated an improved response at 14 days versus that at 7 days. There was significantly prolonged survival in rabbits treated with CZA for 14 days in comparison with that in the PMB-treated or UC rabbits (P ≤ 0.05). This study demonstrates that ceftazidime-avibactam displays linear dose-proportional exposures simulating those seen from human plasma pharmacokinetic profiles, is active for the treatment of experimental KPC-Kp pneumonia in persistently neutropenic rabbits, and provides an experimental foundation for the treatment of severely immunocompromised patients with this life-threatening infection.

Amphotericin B Penetrates into the Central Nervous System Through Focal Disruption of the Blood Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis.

Hematogenous Candida meningoencephalitis (HCME) is a life-threatening complication of neonates and immunocompromised children. Amphotericin B (AmB) shows poor permeability and low cerebrospinal fluid (CSF) concentrations, but is effective in treatment of HCME. In order to better understand the mechanism of CNS penetration of AmB, we hypothesized that AmB may achieve focally higher concentrations in infected CNS lesions. An in vitro BBB model was serially infected with C. albicans. Liposomal AmB (LAMB) or deoxycholate AmB (DAMB) at 5 µg/ml were then provided, vascular and CNS compartments were sampled 4h later. For in vivo correlation, rabbits with experimental HCME received a single dose of DAMB 1 mg/kg or LAMB 5 mg/kg, and were euthanized after 1, 3, 6 and 24h. Evans blue solution (2%) 2 ml/kg administered IV one hour prior to euthanasia stained infected regions of tissue but not histologically normal areas. AmB concentrations in stained and unstained tissue regions were measured using UPLC. For selected rabbits, MRI scans performed on days 1-7 postinoculation were acquired before and after IV bolus Gd-DTPA at 15min intervals through 2h post-injection. The greatest degree of penetration of DAMB and LAMB through the in vitro BBB occurred after 24h of exposure (P=0.0022). In vivo the concentrations of LAMB and DAMB in brain abscesses were 4.35±0.59 and 3.14±0.89-times higher vs. normal tissue (P≤0.019). MRI scans demonstrated that Gd-DTPA accumulated in infected areas with disrupted BBB. Localized BBB disruption in HCME allows high concentrations of AmB within infected tissues, despite the presence of low CSF concentrations.

Ceftolozane-Tazobactam in the Treatment of Experimental Pseudomonas aeruginosa Pneumonia in Persistently Neutropenic Rabbits: Impact on Strains with Genetically Defined Mechanisms of Resistance.

Ceftolozane-tazobactam (C/T) is a novel cephalosporin with in vitro activity against Pseudomonas aeruginosa that is resistant to extended-spectrum penicillins and antipseudomonal cephalosporins. In order to assess the antimicrobial effect of C/T in treatment of Pseudomonas pneumonia, we investigated the pharmacokinetics and efficacy of C/T in persistently neutropenic rabbits. Pseudomonas pneumonia was established by direct endotracheal inoculation. Treatment groups consisted of C/T, ceftazidime (CAZ), piperacillin-tazobactam (TZP), and untreated controls (UC). Rabbits received a dosage of C/T of 80 mg/kg every 4 h (q4h) intravenously (i.v.) (53 mg/kg ceftolozane/26 mg/kg tazobactam) to match the free drug time above the MIC as well as a comparable plasma area under the concentration-time curve (AUC) (humanized doses of ceftolozane-tazobactam of 3 g [2 g/1 g]) q8h, due to the more rapid elimination of ceftolozane in rabbits (0.75 h) than in humans (2.5 h). Four molecularly characterized clinical P. aeruginosa isolates from patients with pneumonia were studied, including one isolate from each classification group: pan-susceptible (PS), outer membrane porin D (OPRD) porin loss (OPRDPL), efflux pump expression (EPE), and AmpC hyperexpression (ACHE). Treatment was continued for 12 days. Treatment with ceftolozane-tazobactam resulted in a ≥105 reduction in residual pulmonary and bronchoalveolar lavage (BAL) fluid bacterial burdens caused by all 4 strains (P ≤ 0.01). This antibacterial activity coincided with reduction of lung weight (an organism-mediated pulmonary injury marker) (P < 0.05). CAZ was less active in ACHE-infected rabbits, and TZP had less activity against EPE, ACHE, and OPRDPL strains. Survival was prolonged in the C/T and CAZ treatment groups in comparison to the TZP and UC groups (P < 0.001). Ceftolozane-tazobactam is highly active in treatment of experimental P. aeruginosa pneumonia in persistently neutropenic rabbits, including infections caused by strains with the most common resistance mechanisms.

Molecular Characterization of Uropathogenic Escherichia coli Reveals Emergence of Drug Resistant O15, O22 and O25 Serogroups.

Background and Objectives: Uropathogenic Escherichia coli (UPEC) are common pathogens causing urinary tract infections (UTIs). We aimed to investigate the relationship among clinical manifestation, serogroups, phylogenetic groups, and antimicrobial resistance among UPEC. Materials and Methods: One-hundred Escherichia coli isolates recovered from urine and ureteral scrapings were used for the study. The prevalence of antimicrobial resistance was determined by using European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. E. coli serogroups associated with UTI, as well as phylogenetic diversity were analyzed using multiplex PCR reactions. Results: Eighty-seven strains (87%) were isolated from females, while 13 (13%) from males. A high frequency of resistance to cephalosporins (43%) and fluoroquinolones (31%) was observed. Among UTI-associated serogroups O15 (32.8%), O22 (23.4%), and O25 (15.6%) were dominant and demonstrated elevated resistance rates. The E. coli phylogenetic group B2 was most common. These observations extended to pregnant patients with asymptomatic bacteriuria. Conclusions: Due to high rates of resistance, strategies using empirical therapy of second-generation cephalosporins and fluoroquinolones should be reconsidered in this population.

Pharmacokinetics and Comprehensive Analysis of the Tissue Distribution of Eravacycline in Rabbits.

Eravacycline (7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline or TP-434) is a novel, fully synthetic broad-spectrum fluorocycline with potent activity against Gram-positive bacteria, anaerobes, and multidrug-resistant Enterobacteriaceae We characterized the plasma pharmacokinetics of eravacycline and conducted a comprehensive analysis of the eravacycline tissue distribution in rabbits after multiple-day dosing. For single-dose pharmacokinetic analysis, eravacycline was administered to New Zealand White (NZW) rabbits at 1, 2, 4, 8, and 10 mg/kg of body weight intravenously (i.v.) once a day (QD) (n = 20). For multidose pharmacokinetic analysis, eravacycline was administered at 0.5, 1, 2, and 4 mg/kg i.v. QD (n = 20) for 6 days. Eravacycline concentrations in plasma and tissues were analyzed by a liquid chromatography-tandem mass spectrometry assay. Mean areas under the concentration-time curves (AUCs) following a single eravacycline dose ranged from 5.39 µg · h/ml to 183.53 µg · h/ml. Within the multidose study, mean AUCs ranged from 2.53 µg · h/ml to 29.89 µg · h/ml. AUCs correlated linearly within the dosage range (r = 0.97; P = 0.0001). In the cardiopulmonary system, the concentrations were the highest in the lung, followed by the heart > pulmonary alveolar macrophages > bronchoalveolar lavage fluid; for the intra-abdominal system, the concentrations were the highest in bile, followed by the liver > gallbladder > spleen > pancreas; for the renal system, the concentrations were the highest in urine, followed by those in the renal cortex > renal medulla; for the musculoskeletal tissues, the concentrations were the highest in muscle psoas, followed by those in the bone marrow > adipose tissue; for the central nervous system, the concentrations were the highest in cerebrum, followed by those in the aqueous humor > cerebrospinal fluid > choroid > vitreous. The prostate and seminal vesicles demonstrated relatively high mean concentrations. The plasma pharmacokinetic profile of 0.5 to 4 mg/kg in NZW rabbits yields an exposure comparable to that in humans (1 or 2 mg/kg every 12 h) and demonstrates target tissue concentrations in most sites.

Combination Therapy with Isavuconazole and Micafungin for Treatment of Experimental Invasive Pulmonary Aspergillosis.

Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity and mortality in immunocompromised patients. We hypothesized that simultaneous inhibition of biosynthesis of ergosterol in the fungal cell membrane and (1→3)-ß-d-glucan in the cell wall, respectively, by the antifungal triazole isavuconazole (ISA) and the echinocandin micafungin (MFG) may result in improved outcomes in experimental IPA in persistently neutropenic rabbits. Treatments included ISA at 20 mg/kg of body weight/day (ISA20), 40 mg/kg/day (ISA40), and 60 mg/kg/day (ISA60); MFG at 2 mg/kg/day (MFG2); combinations of ISA20 and MFG2, ISA40 and MFG2, and ISA60 and MFG2; and no treatment (untreated controls [UC]). The galactomannan index (GMI) and (1→3)-ß-d-glucan levels in serum were measured. The residual fungal burden (number of CFU per gram) was significantly reduced in ISA20-, ISA40-, ISA60-, ISA20-MFG2-, ISA40-MFG2-, and ISA60-MFG2-treated rabbits compared with that in MFG2-treated or UC rabbits (P < 0.01). Measures of organism-mediated pulmonary injury, lung weights, and pulmonary infarct score were lower in ISA40-MFG2-treated rabbits than in rabbits treated with ISA40 or MFG2 alone (P < 0.01). Survival was prolonged in ISA40-MFG2-treated rabbits in comparison to those treated with ISA40 or MFG2 alone (P < 0.01). These outcome variables correlated directly with significant declines in GMI and serum (1→3)-ß-d-glucan levels during therapy. The GMI correlated with measures of organism-mediated pulmonary injury, lung weights (r = 0.764; P < 0.001), and pulmonary infarct score (r = 0.911; P < 0.001). In summary, rabbits receiving combination therapy with isavuconazole and micafungin demonstrated a significant dose-dependent reduction in the residual fungal burden, decreased pulmonary injury, prolonged survival, a lower GMI, and lower serum (1→3)-ß-d-glucan levels in comparison to rabbits receiving isavuconazole or micafungin as a single agent.

Successful treatment of Aspergillus ventriculitis through voriconazole adaptive pharmacotherapy, immunomodulation, and therapeutic monitoring of cerebrospinal fluid (1→3)-ß-D-glucan.

Aspergillus ventriculitis is an uncommon but often fatal form of invasive aspergillosis of the central nervous system (CNS). As little is known about the diagnosis, treatment, and outcome of this potentially lethal infection, we report the strategies used to successfully treat Aspergillus ventriculitis complicating a pineal and pituitary germinoma with emphasis on the critical role of adaptive pharmacotherapy of voriconazole and serial monitoring of (1→3)-ß-D-glucan in cerebrospinal fluid. We describe several rationally based therapeutic modalities, including adaptive pharmacotherapy, combination therapy, sargramostim-based immunomodulation, and biomarker-based therapeutic monitoring of the CNS compartment. Through these strategies, our patient remains in remission from both his germinoma and Aspergillus ventriculitis making him one of the few survivors of Aspergillus ventriculitis.

In vitro combination therapy with isavuconazole against Candida spp.

Combination therapy may be an alternative therapeutic approach for difficult-to-treat Candida infections with the aim of increasing efficacy of antifungal therapy. Whether isavuconazole, an extended-spectrum triazole, possesses synergistic activity in combination therapy with echinocandins or polyenes for the treatment of invasive candidiasis has not been studied. We used Bliss independence drug interaction analysis and time-kill assays to examine the in vitro interactions of isavuconazole with amphotericin B or micafungin, an echinocandin, against strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Candida krusei. The Bliss independence-based drug interactions modeling showed that the combination of isavuconazole and micafungin resulted in synergistic interactions against C. albicans, C. parapsilosis, and C. krusei. The degree of synergy ranged from 1.8% to 16.7% (mean %ΔΕ value) with the highest synergy occurring against C. albicans (⊙SYN% = 8.8%-110%). Time-kill assays showed that the isavuconazole-micafungin combination demonstrated concentration-depended synergy against C. albicans and C. parapsilosis. The combined interaction by Bliss analysis between isavuconazole and amphotericin B was indifferent for C. albicans, C. parapsilosis, and C. tropicalis while for C. glabrata was antagonistic (-2% to -6%) and C. krusei synergistic (3.4% to 7%). The combination of isavuconazole-amphotericin B by time-kill assay was antagonistic against C. krusei and C. glabrata. Collectively, our findings demonstrate that combinations of isavuconazole and micafungin are synergistic against Candida spp., while those of isavuconazole and amphotericin B are indifferent in vitro.