Activity of Cefepime in Combination with the Novel ß-Lactamase Inhibitor Taniborbactam (VNRX-5133) against Extended-Spectrum-ß-Lactamase-Producing Isolates in In Vitro Checkerboard Assays.

Extended-spectrum-ß-lactamase (ESBL)-producing strains are increasing worldwide, limiting therapeutic options. Taniborbactam (VNRX-5133) is a newly developed ß-lactamase inhibitor with a wide spectrum of activity covering both serine and metallo enzymes. We therefore evaluated cefepime-taniborbactam activity against ESBL-producing isolates and determined the concentrations to be used in MIC determinations in the clinical laboratory. The in vitro activity of cefepime (0.06 to 256 mg liter-1) combined with taniborbactam (0.03 to 32 mg liter-1) against 129 clinically and molecularly well-documented ESBL-producing isolates (42 Escherichia coli, 39 Klebsiella pneumoniae, 28 Pseudomonas aeruginosa, 16 Enterobacter cloacae, 2 Citrobacter freundii, and 2 Enterobacter aerogenes) was tested with a broth microdilution checkerboard method based on the ISO standard. The MICs of cefepime alone and in combination, together with percentage resistance at different concentrations of taniborbactam, were calculated for each species and resistance mechanism. The median (range)/MIC90 of cefepime was 32 (0.125 to 256)/256 mg liter-1 for all Enterobacterales isolates (n = 101), with 72% being resistant, and 32 (8 to 256)/128 mg liter-1 for the 28 P. aeruginosa isolates, with 86% being resistant. The median (range)/90th percentile concentration of taniborbactam required to restore Enterobacterales susceptibility to cefepime (MIC ≤1 mg liter-1) was 0.06 (≤0.03 to 32)/4 mg liter-1 and P. aeruginosa susceptibility to increased exposure to cefepime (MIC ≤8 mg liter-1) 1 (≤0.032 to 32)/32 mg liter-1 At a fixed concentration of 4 mg liter-1 of taniborbactam, cefepime median (range)/MIC90 were reduced to 0.125 (0.06 to 4)/1 mg liter-1 for Enterobacterales with no resistant isolates found, and to 8 (2 to 64)/16 mg liter-1 for P. aeruginosa isolates, where 36% remained resistant. The combination cefepime-taniborbactam demonstrated a potent activity against ESBL isolates, restoring susceptibility of all Enterobacterales and two-thirds of P. aeruginosa isolates.

Amphotericin B and terbinafine but not the azoles prolong survival in Galleria mellonella larvae infected with Madurella mycetomatis.

Mycetoma is a tropical neglected disease characterized by large subcutaneous lesions in which the causative organisms reside in the form of grains. The most common causative agent is Madurella mycetomatis. Antifungal therapy often fails due to these grains, but to identify novel treatment options has been difficult since grains do not form in vitro. We recently used Galleria mellonella larvae to develop an in vivo grain model. In the current study, we set out to determine the therapeutic efficacy of commonly used antifungal agents in this larval model. Pharmacokinetics of ketoconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and terbinafine were determined in the hemolymph of G. mellonella larvae. Antifungal therapy was given either therapeutically or prophylactic on three consecutive days in therapeutically equivalent dosages. Survival was monitored for 10 days and colony-forming units (cfu) and melanization were determined on day 3. Measurable concentrations of antifungal agents were found in the hemolymph of the larvae. None of the azole antifungal agents prolonged survival when given therapeutically or prophylactically. Amphotericin B and terbinafine did prolong survival, even at concentrations below the minimal inhibitory concentration of M. mycetomatis. The cfu and melanization did not differ between any of the treated groups and phosphate-buffered saline (PBS) treated groups. Grains were still present in surviving larvae but appeared to be encapsulated. This study demonstrated for the first time a comparison between the efficacy of different antifungal agents toward grains of M. mycetomatis. It appeared that amphotericin B and terbinafine were able to prolong larval survival.

In vitro antifungal activity of isavuconazole against Madurella mycetomatis.

Currently, therapy of black-grain mycetoma caused by Madurella mycetomatis consists of extensive debridement of the infected tissue combined with prolonged antifungal therapy with ketoconazole or itraconazole. In the present study, the in vitro activity of the new triazole isavuconazole toward M. mycetomatis was evaluated. Isavuconazole appeared to have high activity against M. mycetomatis, with MICs ranging from ≤0.016 to 0.125 µg/ml. Due to its favorable pharmacokinetics, isavuconazole could be a promising antifungal agent in the treatment of mycetoma.

Faecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae among humans in Java, Indonesia, in 2001-2002.

OBJECTIVE: To characterise commensal Escherichia coli and other Enterobacteriaceae with reduced susceptibility to cefotaxime that were collected in a large survey carried out among 3995 patients and healthy persons in two urban regions on Java, Indonesia, in 2001-2002. METHODS: The putative extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae were analysed using double-disk synergy tests, isoelectric focusing, PCR assays, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). RESULTS: On the day of discharge after five or more days of hospitalisation, at least 95 of 999 (9.5%) patients carried ESBL-positive Enterobacteriaceae as dominant faecal flora. Six patients were simultaneously colonised with E. coli and Klebsiella pneumoniae isolates with ESBL activity. On admission, only 6 of 998 (0.6%) patients were colonised. Faecal carriage of ESBL-producing Enterobacteriaceae among healthy persons or persons visiting a public health centre was not detected. The 107 ESBL-positive strains included 68 E. coli, 35 K. pneumoniae, and four other Enterobacteriaceae. bla(CTX-M-15) was the most prevalent ESBL in both E. coli (47.1%) and K. pneumoniae (45.7%), but the E. coli O25b-ST131 clone was virtually absent. Other ESBL types found were: SHV-2, -2a, -5, -12, CTX-M-3, -9, -14, and TEM-19. PFGE revealed extensive genetic diversity among the isolates. CONCLUSIONS: In 2001-2002, faecal carriage of ESBL-producing Enterobacteriaceae as dominant flora in Indonesia was almost exclusively hospital-associated. The presence of various bla(ESBL) genes and the extensive genetic diversity among isolates argue against a single/dominant strain outbreak.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 ' ' )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Key process conditions for production of C(4) dicarboxylic acids in bioreactor batch cultures of an engineered Saccharomyces cerevisiae strain.

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter(-1) of malate at a yield of 0.42 mol (mol glucose)(-1) in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO(3), was required for efficient C(4) dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO(3) dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)(-1), whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO(2), calcium, and O(2)). Under optimized conditions, a malate yield of 0.48 +/- 0.01 mol (mol glucose)(-1) was obtained in bioreactors, a 19% increase over yields in shake flask experiments.

An alternative strategy for combination therapy: Interactions between polymyxin B and non-antibiotics.

Antimicrobial resistance is increasing and few new antibiotics are in the development pipeline. Alternative strategies to treat infectious diseases, such as combination therapy, are urgently needed. Polymyxin B is a neglected and disused antibiotic with moderate antibacterial activity. In this study, we aimed to find synergistic interactions between polymyxin B and a wide range of non-antibiotics (non-ABs) to improve its efficacy. Thirty non-AB compounds from various drug classes were screened for synergistic potential with sub-minimum inhibitory concentrations (MICs) of polymyxin B in an agar diffusion assay against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (3 isolates per species). Potential candidates were further studied in in vitro checkerboard assays, up to 5 isolates per species, using optical density to assess growth. Interactions were assessed with fractional inhibitory concentration index (FICi) analysis and surface response analysis with Loewe, Bliss and Highest Single Agent analysis using the Combenefit program. Twenty non-ABs enhanced polymyxin B activity in the agar diffusion test in one or more species. Of these, three showed a consistent synergistic effect (FICi ≤ 0.5) in the checkerboard assay for at least one species: citalopram, sertraline and spironolactone. Surface response analyses were largely in concordance, and further assessment showed only spironolactone was synergistic with polymyxin B at clinically relevant levels. The screening strategy used showed consistent synergism in vitro between polymyxin B and some non-ABs for A. baumannii, E. coli and K. pneumoniae. The synergistic interactions found merit further exploration as alternative strategies for difficult-to-treat infections.

Urinary antibacterial activity of fosfomycin and nitrofurantoin at registered dosages in healthy volunteers.

Given emerging uropathogen resistance to more recent antibiotics, old antibiotics used for uncomplicated urinary tract infection (UTI) warrant re-examination. In this study, the urinary antibacterial activities of fosfomycin and nitrofurantoin were investigated by determining the urinary inhibitory titre and urinary bactericidal titre against uropathogens in urine samples from female volunteers following administration of single-dose fosfomycin (3 g) or nitrofurantoin (50 mg q6h or 100 mg q8h). Urine samples were collected over 48 h (fosfomycin) or 6 or 8 h (nitrofurantoin), with drug levels quantified with every void. Fosfomycin concentrations ranged from <0.75 mg/L [lower limit of quantification (LLOQ)] to 5729.9 mg/L and nitrofurantoin concentrations ranged from <4 mg/L (LLOQ) to 176.3 mg/L (50 mg q6h) or 209.4 mg/L (100 mg q8h). There was discrepancy in the response to fosfomycin between Escherichia coli and Klebsiella pneumoniae, with fosfomycin displaying strong bactericidal activity for 48 h against E. coli but moderate bactericidal activity for 18 h against K. pneumoniae. This effect was not related to the strain's baseline minimum inhibitory concentration but rather to the presence of a resistant subpopulation. Maximum titres of nitrofurantoin were obtained during the first 2 h, but no antibacterial effect was found in most samples regardless of the dose. In the rare samples in which antibacterial activity was detectable, titres were comparable for both species tested. These findings confirm doubts regarding fosfomycin administration in UTIs caused by K. pneumoniae and reveal a discrepancy between nitrofurantoin's measurable ex vivo activity and its clinical effect over multiple dosing intervals.

A Madurella mycetomatis Grain Model in Galleria mellonella Larvae.

Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.

The in vitro antifungal activity of sudanese medicinal plants against Madurella mycetomatis, the eumycetoma major causative agent.

Eumycetoma is a debilitating chronic inflammatory fungal infection that exists worldwide but it is endemic in many tropical and subtropical regions. The major causative organism is the fungus Madurella mycetomatis. The current treatment of eumycetoma is suboptimal and characterized by low cure rate and high recurrence rates. Hence, an alternative therapy is needed to address this. Here we determined the antifungal activity of seven Sudanese medicinal plant species against Madurella mycetomatis. Of these, only three species; Boswellia papyrifera, Acacia nubica and Nigella sativa, showed some antifungal activity against M. mycetomatis and were further studied. Crude methanol, hexane and defatted methanol extracts of these species were tested for their antifungal activity. B. papyrifera had the highest antifungal activity (MIC50 of 1 ug/ml) and it was further fractionated. The crude methanol and the soluble ethyl acetate fractions of B. papyrifera showed some antifungal activity. The Gas-Liquid-Chromatography hybrid Mass-Spectrophotometer analysis of these two fractions showed the existence of beta-amyrin, beta-amyrone, beta-Sitosterol and stigmatriene. Stigmatriene had the best antifungal activity, compared to other three phytoconstituents, with an MIC-50 of 32 µg/ml. Although the antifungal activity of the identified phytoconstituents was only limited, the antifungal activity of the complete extracts is more promising, indicating synergism. Furthermore these plant extracts are also known to have anti-inflammatory activity and can stimulate wound-healing; characteristics which might also be of great value in the development of novel therapeutic drugs for this chronic inflammatory disease. Therefore further exploration of these plant species in the treatment of mycetoma is encouraging.

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization.

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.

Active matrix metalloprotease-9 is associated with the collagen capsule surrounding the Madurella mycetomatis grain in mycetoma.

Madurella mycetomatis is the main causative organism of eumycetoma, a persistent, progressive granulomatous infection. After subcutaneous inoculation M. mycetomatis organizes itself in grains inside a granuloma with excessive collagen accumulation surrounding it. This could be contributing to treatment failure towards currently used antifungal agents. Due to their pivotal role in tissue remodelling, matrix metalloproteinases-2 (MMP-2) and 9 (MMP-9) or tissue inhibitor of metalloproteinases (TIMP) might be involved in this process. Local MMP-2 and MMP-9 expression was assessed by immunohistochemistry while absolute serum levels of these enzymes were determined in mycetoma patients and healthy controls by performing ELISAs. The presence of active MMP was determined by gelatin zymography. We found that both MMP-2 and MMP-9 are expressed in the mycetoma lesion, but the absolute MMP-2, -9, and TIMP-1 serum levels did not significantly differ between patients and controls. However, active MMP-9 was found in sera of 36% of M. mycetomatis infected subjects, whereas this active form was absent in sera of controls (P<0.0001). MMP-2, MMP-9, and TIMP-1 polymorphisms in mycetoma patients and healthy controls were determined through PCR-RFLP or sequencing. A higher T allele frequency in TIMP-1 (+372) SNP was observed in male M. mycetomatis mycetoma patients compared to controls. The presence of active MMP-9 in mycetoma patients suggest that MMP-9 is activated or synthesized by inflammatory cells upon M. mycetomatis infection. Inhibiting MMP-9 activity with doxycycline could prevent collagen accumulation in mycetoma, which in its turn might make the fungus more accessible to antifungal agents.

Madurella mycetomatis is highly susceptible to ravuconazole.

The current treatment of eumycetoma utilizing ketoconazole is unsatisfactory because of high recurrence rates, which often leads to complications and unnecessary amputations, and its comparatively high cost in endemic areas. Hence, an effective and affordable drug is required to improve therapeutic outcome. E1224 is a potent orally available, broad-spectrum triazole currently being developed for the treatment of Chagas disease. E1224 is a prodrug that is rapidly converted to ravuconazole. Plasma levels of E1224 are low and transient, and its therapeutically active moiety, ravuconazole is therapeutically active. In the present study, the in vitro activity of ravuconazole against Madurella mycetomatis, the most common etiologic agent of eumycetoma, was evaluated and compared to that of ketoconazole and itraconazole. Ravuconazole showed excellent activity with MICs ranging between ≤ 0.002 and 0.031 µg/ml, which were significantly lower than the MICs reported for ketoconazole and itraconazole. On the basis of our findings, E1224 with its resultant active moiety, ravuconazole, could be an effective and affordable therapeutic option for the treatment of eumycetoma.