In Central African Republic, the conflict between armed groups is now compounded by a conflict between armed communities. The roadmap to end the crisis including elections late 2015 presents only a short-term answer and risks exacerbating existing tensions. The transitional authorities and their international partners must address crucial issues by implementing a comprehensive disarmament policy and reaffirming that Muslims belong within the nation.

Test-based accountability has not generated the significant gains in student achievement that proponents intended, Helen F. Ladd contends.

What should be included in a measure of school quality? If not by standardized test scores, how should we measure student achievement? Julian Vasquez Heilig and I each have some thoughts.

Federal law requires states to report student test scores in achievement levels, but leaders in the Golden State want to take a different approach.

The U.S. Education Department says the states need more evidence to use the popular admissions test to measure high school achievement.

More than four years after the passage of ESSA, English-language-learner education policies across the country remain "disjointed and inaccessible," a new report concludes.

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Objectives. The aim of this study was to assess the safety of early oral switch (EOS) prior to 14 days for low-risk Staphylococcus aureus bacteremia (LR-SAB), which is the primary treatment strategy employed at our institution. Usually recommended therapy is 14 days of intravenous (IV) antibiotics.

Methods. All patients with SAB at our hospital were identified between 1 January 2014 and 31 December 2018. Those meeting low-risk criteria (healthcare-associated, no evidence of deep infection or demonstrated involvement of prosthetic material, and no further positive blood cultures after 72-hours) were included in the study. The primary outcome was occurrence of a SAB-related complication within 90 days.

Results. There were 469 SAB episodes during the study period, 100 (21%) of whom met inclusion criteria. EOS was performed in 84 patients. In this group, line infection was the source in 79%, methicillin-susceptible S. aureus caused 95% of SABs and 74% of patients received IV flucloxacillin. The median duration of IV and oral antibiotics in the EOS group was 5 (IQR 4-6) and 10 days (IQR 9-14), respectively. Seventy-one percent of patients received flucloxacillin as their EOS agent. Overall, 86% of oral step-down therapy was with beta-lactams. One patient (1%) undergoing EOS had SAB relapse within 90 days. No deaths attributable to SAB occurred within 90 days.

Conclusions. In this low MRSA prevalence LR-SAB cohort, EOS was associated with a low incidence of SAB-related complications. This was achieved with oral beta-lactam therapy in most patients. Larger prospective studies are needed to confirm these findings.

Background: This study summarizes drug resistance analyses in 4 recent phase 2b trials of the respiratory syncytial virus (RSV) fusion inhibitor presatovir in naturally infected adults.

Methods: Adult hematopoietic cell transplant (HCT) recipients, lung transplant recipients, or hospitalized patients with naturally acquired, laboratory-confirmed RSV infection were enrolled in 4 randomized, double-blind, placebo-controlled studies with study-specific presatovir dosing. Full-length RSV F sequences amplified from nasal swabs obtained at baseline and postbaseline were analyzed by population sequencing. Substitutions at RSV fusion inhibitor resistance-associated positions are reported.

Results: Genotypic analyses were performed on 233 presatovir-treated and 149 placebo-treated subjects. RSV F variant V127A was present in 8 subjects at baseline. Population sequencing detected treatment-emergent substitutions in 10/89 (11.2%) HCT recipients with upper and 6/29 (20.7%) with lower respiratory tract infection, 1/35 (2.9%) lung transplant recipients, and 1/80 (1.3%) hospitalized patients treated with presatovir; placebo-treated subjects had no emergent resistance-associated substitutions. Subjects with substitutions at resistance-associated positions had smaller decreases in viral load during treatment relative to those without, but similar clinical outcomes.

Conclusions: Subject population type and dosing regimen may have influenced RSV resistance development during presatovir treatment. Subjects with vs without genotypic resistance development had decreased virologic responses but comparable clinical outcomes.

QPX7728 is a new ultra-broad-spectrum inhibitor of serine and metallo beta-lactamases from a class of cyclic boronates that gave rise to vaborbactam. The spectrum and mechanism of beta-lactamase inhibition by QPX7728 were assessed using purified enzymes from all molecular classes. QPX7728 inhibits class A ESBLs (IC₅₀ range 1-3 nM) and carbapenemases such as KPC (IC₅₀ 2.9±0.4 nM) as well as class C P99 (IC₅₀ of 22±8 nM) with a potency that is comparable or higher than recently FDA approved BLIs avibactam, relebactam and vaborbactam. Unlike those other BLIs, QPX7728 is also a potent inhibitor of class D carbapenemases such as OXA-48 from Enterobacteriaceae and OXA enzymes from A. baumannii (OXA-23/24/58, IC₅₀ range 1-2 nM) as well as MBLs such as NDM-1 (IC₅₀ 55±25 nM), VIM-1 (IC₅₀ 14±4 nM) and IMP-1 (IC₅₀ 610±70 nM). Inhibition of serine enzymes by QPX7728 is associated with progressive inactivation with a high efficiency k₂/K ranging from of 6.3 x 10⁴ (for P99) to 9.9 x 10⁵ M^-1 s^-1 (for OXA-23). This inhibition is reversible with variable stability of the QPX7728-beta-lactamase complexes with target residence time ranging from minutes to several hours: 5-20 minutes for OXA carbapenemases from A. baumanii, ~50 minutes for OXA-48 and 2-3 hours for KPC and CTX-M-15. QPX7728 inhibited all tested serine enzymes at 1:1 molar ratio. Metallo-beta-lactamases NDM, VIM, and IMP were inhibited by a competitive mechanism with fast-on-fast-off kinetics, with K_is of 7.5±2.1 nM, 32±14 nM and 240±30 nM for VIM-1, NDM-1 and IMP-1, respectively. QPX7728 ultra-broad-spectrum of BLI inhibition combined with its high potency enables combinations with multiple different beta-lactam antibiotics.

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC50 concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, reversing acquired resistance to the antimalarial compound blasticidin S that is transported through PSAC. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

Continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRNI-A/FGKGS/T pfdhfr/pfdhps haplotypes including the pfdhps A581G and A613S/T mutations was high at delivery among post-SP treatment isolates (18.2%) compared to those of first-antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP); 6.1%; p = 0.03). Regarding the pfk13 marker gene, two non-synonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast-Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance on additional mutations associated with increased SP resistance as well as emergence of resistance against artemesinin derivatives.

Lipid II is an essential precursor of the bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target lipid II with lanthionine-stabilized lipid II-binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct C-terminal lipid II binding motif of one peptide of the two-component haloduracin (i.e. HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against E. faecium than nisin (1-22), which has only one lipid II binding site, and (ii) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different lipid II binding motifs to treat specific antibiotic-resistant pathogens.

The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct acting-antivirals (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with an EC₅₀ of 9±0.3 μM and a maximum HCV RNA level reduction of approximatively 1 Log₁₀. MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonists SR2640 increased HCV-SGR RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

Since 2012, single low dose of primaquine (SLDPQ, 0.25mg/kg) has been recommended with artemisinin-based combination therapies, as first-line treatment of acute uncomplicated Plasmodium falciparum malaria, to interrupt its transmission, especially in low transmission settings of multidrug, including artemisinin, resistance. Policy makers in Cambodia have been reluctant to implement this recommendation due to primaquine safety concerns and lack of data on its efficacy.

In this randomized controlled trial, 109 Cambodians with acute uncomplicated P. falciparum malaria received dihydroartemisinin-piperaquine (DP) alone or combined with SLDPQ on the first treatment day. Transmission-blocking efficacy of SLDPQ was evaluated on Days 0, 1, 2, 3, 7, 14, 21, 28 and recrudescence by reverse transcriptase polymerase chain reaction (RT-PCR) (gametocyte prevalence) and membrane-feeding assays with Anopheles minimus mosquitoes (gametocyte infectivity). Without the influence of recrudescent infections, DP+SLDPQ reduced gametocyte carriage 3 fold compared to DP. Of 48 patients tested on Day 0, only three patients were infectious to mosquitoes (~6%). Post-treatment, three patients were infectious: on D14 (3.5%, 1/29), and on the first and seventh day of recrudescence (8.3%, 1/12 for each); this overall low infectivity precluded our ability to assess its transmission blocking efficacy.

Our study confirms effective gametocyte clearance of SLDPQ when combined with DP in multidrug resistant P. falciparum and the negative impact of recrudescent infections due to poor DP efficacy. Artesunate-mefloquine (ASMQ) has replaced DP and ASMQ-SLDPQ has been deployed to treat all P. falciparum symptomatic patients to further support the elimination of multidrug resistant P. falciparum in Cambodia.

Objectives: Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Yet, data of which combinations are most effective is lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa.

Methods: We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, meropenem, minocycline, rifampicin, temocillin, thiamphenicol or trimethoprim by automated time-lapse microscopy using predefined cut-off values indicating inhibition of growth (≤10⁶ CFU/mL) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments.

Results: All strains were intermediate or resistant to polymyxin B, anti-pseudomonal β-lactams, ciprofloxacin and amikacin. Genes encoding β-lactamases (e.g., bla_PAO and bla_OXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol and trimethoprim. Time kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem.

Conclusion: Positive interactions were frequently found with the tested combinations, also against strains that harboured several resistance mechanisms to the single drugs and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

β-lactam resistance in Staphylococcus aureus limits treatment options. Stp1 and Stk1, a serine-threonine phosphatase and kinase respectively, mediate serine-threonine kinase (STK) signaling. Loss of function point mutations in stp1 were detected among laboratory passaged, β-lactam resistant S. aureus strains lacking mecA and blaZ, the major determinants of β-lactam resistance in the bacteria. Loss of Stp1 function facilitates β-lactam resistance of the bacteria.

Carbapenems are currently the preferred agents for the treatment of serious Acinetobacter infections. However, whether cefepime/cefpirome can be used to treat Acinetobacter bloodstream infection (BSI) if it is active against the causative pathogens is not clear. This study aimed to compare the efficacy of cefepime/cefpirome and carbapenem monotherapy in patients with Acinetobacter BSI. The population included 360 patients with monomicrobial Acinetobacter BSI receiving appropriate antimicrobial therapy admitted to four medical centres in Taiwan in 2012–2017. The predictors of 30-day mortality were determined by Cox regression analysis. The overall 30-day mortality rate in the appropriate antibiotic treatment group was 25.0% (90/360 patients), respectively. The crude 30-day mortality rates for cefepime/cefpirome and carbapenem therapy were 11.5% (7/61 patients) and 26.3% (21/80 patients), respectively. The patients receiving cefepime/cefpirome/carbapenem therapy were infected by Acinetobacter nosocomialis (51.8%), A. baumannii (18.4%) and A. pittii (12.1%). After adjusting for age, Sequential Organ Failure Assessment (SOFA) score, invasive procedures, and underlying diseases, cefepime/cefpirome therapy was not independently associated with a higher or lower 30-day mortality compared to the carbapenem therapy. SOFA score (hazard ratio [HR], 1.324; 95% confidence interval [CI], 1.137–1.543; P < 0.001) and neutropenia (HR, 7.060; 95% CI, 1.607–31.019; P = 0.010) were independent risk factors for 30-day mortality of patients receiving cefepime/cefpirome or carbapenem monotherapy. The incidence density of 30-day mortality for cefepime/cefpirome versus carbapenem therapy was 0.40% versus 1.04%. The therapeutic response of cefepime/cefpirome therapy was comparable to that of carbapenems among patients with Acinetobacter BSI receiving appropriate antimicrobial therapy.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo β-lactamase (MBL) families. The recent introduction of SBL carbapenemase-inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n=234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ~30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modelling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor, capable of operating in a functional space not presently filled by any clinically approved compound.

Ibrexafungerp (formerly SCY-078) is a semisynthetic triterpenoid and potent (1->3)-β-D-glucan synthase inhibitor. We investigated the in vitro activity, pharmacokinetics, and in vivo efficacy of ibrexafungerp (SCY) alone and in combination with anti-mould triazole isavuconazole (ISA) against invasive pulmonary aspergillosis (IPA). The combination of ibrexafungerp and isavuconazole in in vitro studies resulted in an 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 NZW rabbit model of experimental IPA. Treatment groups included untreated rabbits (UC) and rabbits receiving ibrexafungerp at 2.5(SCY2.5) and 7.5(SCY7.5) mg/kg/day, isavuconazole at 40(ISA40) mg/kg/day, or combinations of SCY2.5+ISA40 and SCY7.5+ISA40. The combination of SCY+ISA produced in vitro synergistic interaction. There was significant in vivo reduction of residual fungal burden, lung weights, and pulmonary infarct scores in SCY2.5+ISA40, SCY7.5+ISA40, and ISA40-treatment groups vs that of SCY2.5-treated, SCY7.5-treated and UC (p<0.01). Rabbits treated with SCY2.5+ISA40 and SCY7.5+ISA40 had prolonged survival in comparison to that of SCY2.5-, SCY7.5-, ISA40-treated or UC (p<0.05). Serum GMI and (1->3)-β-D-glucan levels significantly declined in animals treated with the combination of SCY7.5+ISA40 in comparison to those treated with SCY7.5 or ISA40 (p<0.05). Ibrexafungerp and isavuconazole combination demonstrated prolonged survival, decreased pulmonary injury, reduced residual fungal burden, lower GMI and (1->3)-β-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 anti-mould triazole for treatment of IPA.

Ceftobiprole medocaril is an advanced-generation cephalosporin prodrug that has qualified infectious disease product status granted by the US-FDA and is currently being evaluated in phase 3 clinical trials in patients with acute bacterial skin and skin structure infections (ABSSSIs) and in patients with Staphylococcus aureus bacteremia. In this study, the activity of ceftobiprole and comparators was evaluated against more than 7,300 clinical isolates collected in the United States from 2016 through 2018 from patients with skin and skin structure infections. The major species/pathogen groups were S. aureus (53%), Enterobacterales (23%), Pseudomonas aeruginosa (7%), β-hemolytic streptococci (6%), Enterococcus spp. (4%), and coagulase-negative staphylococci (2%). Ceftobiprole was highly active against S. aureus (MIC_50/90, 0.5/1 mg/L; 99.7% susceptible by EUCAST criteria; 42% methicillin-resistant S. aureus [lsqb]MRSA[rsqb]). Ceftobiprole also exhibited potent activity against other Gram-positive cocci. The overall susceptibility of Enterobacterales to ceftobiprole was 84.8% (>99.0% susceptible for isolate subsets that exhibited a non-extended-spectrum β-lactamase [lsqb]ESBL[rsqb]-phenotype). A total of 74.4% of P. aeruginosa, 100% of β-hemolytic streptococci and coagulase-negative staphylococci, and 99.6% of Enterococcus faecalis isolates were inhibited by ceftobiprole at ≤4 mg/L. As expected, ceftobiprole was largely inactive against Enterobacterales that contained ESBL genes and Enterococcus faecium. Overall, ceftobiprole was highly active against most clinical isolates from the major Gram-positive and Gram-negative skin and skin structure pathogen groups collected at U.S. medical centers participating in the SENTRY Antimicrobial Surveillance Program during 2016–2018. The broad-spectrum activity of ceftobiprole, including potent activity against MRSA, supports its further evaluation for the potential ABSSSI indication.

Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans. At high concentrations, rhSAA1 disrupts C. albicans membrane integrity and induces rapid fungal cell death. In the current study, we find that rhSAA1 promotes cell aggregation and targets the C. albicans cell wall adhesin Als3. Inactivation of ALS3 in C. albicans leads to a striking decrease in cell aggregation and cell death upon rhSAA1 treatment, suggesting that Als3 plays a critical role in SAA1 sensing. We further demonstrate that deletion of the transcriptional regulators controlling the expression of ALS3, such as AHR1, BCR1, and EFG1 in C. albicans results in similar effects to that of the als3/als3 mutant upon rhSAA1 treatment. Global gene expression profiling indicates that rhSAA1 has a discernible impact on the expression of cell wall- and metabolism-related genes, suggesting that rhSAA1 treatment could lead to a nutrient starvation effect on C. albicans cells.

Background: Intensive care unit (ICU) patients may experience ceftriaxone underexposure but clinical outcomes data are lacking. The objective of this study was to determine the impact of ceftriaxone dosing on clinical outcomes amongst ICU patients without central nervous system (CNS) infection.

Methods: A retrospective study of ICU patients receiving intravenous, empiric ceftriaxone for non-CNS infections was conducted. Patients ≥18 years of age who received ≤2 grams of ceftriaxone daily for ≥72 hours were included and categorized as receiving ceftriaxone 1 gram or 2 grams daily. The primary, composite outcome was treatment failure: inpatient mortality and/or antibiotic escalation due to clinical worsening. Propensity score matching was performed based on the probability of receiving ceftriaxone 2 grams daily. Multivariable logistic regression determined the association between ceftriaxone dose and treatment failure in a propensity-matched cohort.

Results: A total of 212 patients were included in the propensity-matched cohort. The most common diagnoses (83.0%) were pneumonia and urinary tract infection. Treatment failure occurred in 17.0% and 5.7% of patients receiving 1 gram and 2 grams daily, respectively (p=0.0156). Overall inpatient mortality was 8.5%. Ceftriaxone 2 gram dosing was associated with a reduced likelihood of treatment failure (adjusted odds ratio=0.190; 95% confidence interval: 0.059 – 0.607). Other independent predictors of treatment failure included sequential organ failure assessment score (aOR 1.440, 95% CI 1.254 – 1.653) and creatinine clearance at 72 hours from ceftriaxone initiation (aOR 0.980, 95% CI (0.971 – 0.999).

Conclusions: Ceftriaxone 2 grams daily when used as appropriate antimicrobial coverage may be appropriate for ICU patients with lower mortality risk.

Multi-drug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely-used antibiotic class, the β-lactams, including the most recent-generation carbapenems. Interspecies spread renders these enzymes a serious clinical threat and there are no clinically-available inhibitors. We present crystal structures of IMP-13, a structurally-uncharacterized MBL from Gram-negative Pseudomonas aerugionasa found in clinical outbreaks globally, and characterize the binding using solution NMR-spectroscopy and molecular-dynamics simulations. Crystal structures of apo IMP-13 and bound to four clinically-relevant carbapenem antibiotics (doripenem, ertapenem, imipenem and meropenem) are presented. Active site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

Mucormycosis is a life-threatening infection with high mortality that occurs predominantly in immunocompromised patients. Manogepix (MGX) is a novel antifungal that targets Gwt1, an early step in the conserved glycosylphosphotidyl inositol (GPI) post-translational modification pathway of surface proteins in eukaryotic cells. Inhibition of inositol acylation by MGX results in pleiotropic effects including inhibition of maturation of GPI-anchored proteins necessary for growth and virulence. MGX has been previously shown to have in vitro activity against some strains of Mucorales. Here we assessed the in vivo activity of the prodrug fosmanogepix, currently in clinical development for the treatment of invasive fungal infections, against two Rhizopus arrhizus strains with high (4.0 μg/ml) and low (0.25 μg/ml) minimum effective concentration (MEC) values. In both invasive pulmonary infection models, treatment of mice with 78 mg/kg or 104 mg/kg fosmanogepix, along with 1-aminobenzotriazole to enhance the serum half-live of MGX in mice, significantly increased median survival time and prolonged overall survival by day 21 post infection when compared to placebo. In addition, administration of fosmanogepix resulted in a 1-2 log reduction in both lung and kidney fungal burden. For the 104 mg/kg fosmanogepix dose, tissue clearance and survival were comparable to clinically relevant doses of isavuconazole (ISA), which is FDA approved for the treatment of mucormycosis. These results support continued development of fosmanogepix as a first in class treatment for invasive mucormycosis.

Telacebec (Q203) is a new anti-tubercular drug with extremely potent activity against Mycobacterium ulcerans. Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture-negative in 2 weeks. Combining Q203 with rifampin resulted in relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF+clarithromycin, the current standard of care, for 4 weeks.

The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for ≥ 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2-10 mg/kg were culture-negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2 and 10 mg/kg were culture-negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics sub-study revealed that Q203 doses of 2-10 mg/kg in mice produce plasma concentrations similar to those produced by 100-300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations.

These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for cure to ≤ 1 week (or 5 doses of 2-10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lies behind the ongoing outbreak of coronavirus disease 2019 (COVID-19). There is a growing understanding of SARS-CoV-2 in the virology, epidemiology and clinical management strategies. However, no anti-SARS-CoV-2 drug or vaccine has been officially approved due to the absence of adequate evidence. Scientists are racing towards the development of treatment for COVID-19. Recent studies have revealed many attractive threptic options, even if some of them remain to be further confirmed in rigorous preclinical models and clinical trials. In this minireview, we aim to summarize the updated potential approaches against SARS-CoV-2. We emphasize that further efforts are warranted to develop the safest and most effective approach.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity 23% (78/339)) but improved when genetic features associated with penicillinase hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. We investigated two potential causes; the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Objectives: Carbapenemase-producing Enterobacterales and specifically KPC-producing Klebsiella pneumoniae (KPC-Kp) are rapidly spreading worldwide. The prognosis of ventilator-associated pneumonia (VAP) caused by KPC-producing Klebsiella pneumoniae (KPC-Kp) is not well known. Our study tries to assess whether ventilator-associated pneumonia caused by a KPC-Kp strain is associated with higher all-cause mortality than if caused by carbapenem-susceptible isolates.

Study design and methods: This is a retrospective cohort study of patients with VAP due to K. pneumoniae from a 35-bed polyvalent Intensive Care Unit in a university hospital (> 40,000 annual admissions) between January 2012 and December 2016. Adjusted multivariate analysis was used to study the association of KPC-Kp with 30-day all-cause mortality (Cox regression).

Results. We analyze 69 cases of K. pneumoniae VAP of which 39 were produced by a KPC-Kp strain with high-level resistance to meropenem (MIC > 16 mg/mL). All-cause mortality at 30 days was 41% in the KPC-Kp group (16/39) and 33.3% in the carbapenem-susceptible cases (10/30). KPC-Kp etiology was not associated with higher mortality when controlled for confounders (adjusted hazard ratio [lsqb]HR[rsqb] 1.25; 95% CI: 0.46–3.41). Adequate targeted therapy (HR 0.03; 95% CI: <0.01–0.23) was associated with all-cause mortality.

Conclussion. Assuming the limitations due to the available sample size, the prognosis of VAP caused by KPC-Kp is similar to VAPs caused by carbapenem-susceptible K. pneumoniae when appropriate treatment is used.

Highly conserved PenI-type class A β-lactamase in pathogenic members of Burkholderia can evolve to extended-spectrum β-lactamase (ESBL), which exhibits hydrolytic activity towards third-generation cephalosporins, while losing its activity towards the original penicillin substrates. We describe three single-amino-acid-substitution mutations in the ArgS arginine-tRNA synthetase that confer extra antibiotic tolerance protection to ESBL-producing Burkholderia thailandensis. This pathway can be exploited to evade antibiotic tolerance induction in developing therapeutic measures against Burkholderia species, targeting their essential aminoacyl-tRNA synthetases.

Periodontitis as a biofilm-associated inflammatory disease is highly prevalent worldwide. It severely affects oral health and yet closely links to systemic diseases like diabetes and cardiovascular disease. Porphyromonas gingivalis as a ‘keystone' periodontopathogen drives the shift of microbe-host symbiosis to dysbiosis, and critically contributes to the pathogenesis of periodontitis. Persisters are a tiny subset of biofilm-associated microbes highly tolerant to lethal treatment of antimicrobials, and notably metronidazole-tolerant P. gingivalis persisters have recently been identified by our group. This study further explored the interactive profiles of metronidazole-treated P. gingivalis persisters (M-PgPs) with human gingival epithelial cells (HGECs). P. gingivalis cells (ATCC 33277) at stationary phase were treated with lethal dosage of metronidazole (100 μg/ml, 6 hours) for generating M-PgPs. The interaction of M-PgPs with HGECs was assessed by microscopy, flow cytometry, cytokine profiling and qPCR. We demonstrated that the overall morphology and ultra-cellular structure of M-PgPs remained unchanged. Importantly, M-PgPs maintained the capabilities to adhere to and invade into HGECs. Moreover, M-PgPs significantly suppressed pro-inflammatory cytokine expression in HGECs at a comparable level with the untreated P. gingivalis cells, through the thermo-sensitive components. The present study reveals that P. gingivalis persisters induced by lethal treatment of antibiotics could maintain their capabilities to adhere to and invade into human gingival epithelial cells, and perturb the innate host responses. Novel strategies and approaches need to be developed for tackling P. gingivalis and favourably modulating the dysregulated immuno-inflammatory responses for oral/periodontal health and general wellbeing.

Background. CLSI and EUCAST susceptibility breakpoints for voriconazole and C. albicans differ by one dilution (≤0.125 and ≤0.06 mg/l, respectively) whereas the epidemiological cutoff values (ECOFF/ECV) with both methodologies are the same (0.03 mg/L). We therefore determined the pharmacokinetic-pharmacodynamic (PK/PD) breakpoints of voriconazole against C. albicans for both methodologies with an in vitro PK/PD model, which was validated using existing animal PK/PD data.

Methods. Four clinical wild-type and non-wild-type C. albicans isolates (voriconazole MICs 0.008-0.125 mg/l) were tested in an in vitro PK/PD model. For validation purposes, mouse PK were simulated and in vitro PD were compared with in vivo outcome. Human PK were simulated and the exposure-effect relationship fAUC_0-24/MIC was described for EUCAST and CLSI24/48h methods. PK/PD breakpoints were determined using the fAUC_0-24/MIC associated with half-maximal activity (EI₅₀) and Monte Carlo simulation analysis.

Results. The in vitro 24h-PD EI₅₀ of voriconazole against C. albicans were 2.5-5 (1.5-17) fAUC/MIC. However, the 72h-PD were higher, 133 (51-347) fAUC/MIC for EUCAST and 94 (35-252) fAUC/MIC for CLSI. The mean (95% confidence interval) probability of target attainment (PTA) was 100(95-100)%, 97(72-100)%, 83(35-99)%, and 49(8-91)% and 100(97-100)%, 99(85-100)%, 91(52-100)% and 68(17-96)% for EUCAST and CLSI MICs 0.03, 0.06, 0.125, and 0.25 mg/L, respectively. Significantly, >95% PTAs were found for EUCAST/CLSI MICs ≤0.03 mg/ll. For MICs 0.06-0.125 mg/l trough levels 1-4 mg/ll would be required.

Conclusion. A PK/PD breakpoint of C. albicans voriconazole at the ECOFF/ECV of 0.03 mg/L was determined for both EUCAST/CLSI methods, indicating the need for breakpoint harmonization for the reference methodologies.

QPX7728 is an ultra-broad-spectrum boronic acid beta-lactamase inhibitor with potent inhibition of key serine and metallo beta-lactamases observed in biochemical assays. Microbiological studies using characterized strains were used to provide a comprehensive characterization of the spectrum of beta-lactamase inhibition by QPX7728. The MIC of multiple IV only (ceftazidime, piperacillin, cefepime, ceftolozane and meropenem) and orally bioavailable (ceftibuten, cefpodoxime, tebipenem) antibiotics alone and in combination with QPX7728 (4 μg/ml), as well as comparator agents, were determined against the panels of laboratory strains of P. aeruginosa and K. pneumoniae expressing over 55 diverse serine and metallo beta-lactamases. QPX7728 significantly enhanced the potency of antibiotics against the strains expressing Class A extended spectrum beta-lactamases (CTX-M, SHV, TEM, VEB, PER) and carbapenemases (KPC, SME, NMC-A, BKC-1), consistent with beta-lactamase inhibition demonstrated in biochemical assays. It also inhibits both plasmidic (CMY, FOX, MIR, DHA) and chromosomally encoded (P99, PDC, ADC) Class C beta-lactamases and Class D enzymes including carbapenemases such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/72/58). QPX7728 is also a potent inhibitor of many class B metallo beta-lactamases (NDM, VIM, CcrA1, IMP, GIM but not SPM or L1). Addition of QPX7728 (4 μg/ml) reduced the MICs in a majority of strains to the level observed for the vector alone control, indicative of complete beta-lactamase inhibition. The ultra-broad-spectrum beta-lactamase inhibition profile makes QPX7728 a viable candidate for further development.

The Cpx stress response is widespread among Enterobacteriaceae. We have previously reported a mutation in cpxA in a multidrug resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields to a single amino acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxA^Y144N is an activated allele that confers resistance to β-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the intimate interconnection between Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC β-lactamase by using imipenem as a cell wall active antibiotic or inactivation of penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increase phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to causes abnormal accumulation of muropeptides (disaccharide-pentapeptide, N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycan caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular the interaction between CpxA and CpxP, as a promising therapeutic target.

QPX7728 is an ultra-broad-spectrum boronic acid beta-lactamase inhibitor that demonstrates inhibition of key serine and metallo beta-lactamases at a nano molar range in biochemical assays with purified enzymes. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments translates into enhancement of the potency of many beta-lactams against strains of target pathogens producing beta-lactamases. The impact of bacterial efflux and permeability on inhibitory potency were determined using isogenic panels of KPC-3 producing isogenic strains of K. pneumoniae and P. aeruginosa and OXA-23-producing strains of A. baumannii with various combinations of efflux and porin mutations. QPX7728 was minimally affected by multi-drug resistance efflux pumps in either Enterobacteriaceae, or in non-fermenters such as P. aeruginosa or A. baumannii. In P. aeruginosa, the potency of QPX7728 was further enhanced when the outer membrane is permeabilized. The potency of QPX7728 in P. aeruginosa is not affected by inactivation of the carbapenem porin OprD. While changes in OmpK36 (but not OmpK35) reduced the potency of QPX7728 (8-16-fold), QPX7728 (4 μg/ml) nevertheless completely reversed KPC-mediated meropenem resistance in strains with porin mutations, consistent with a lesser effect of these mutations on the potency of QPX7728 compared to other agents. The ultra-broad-spectrum beta-lactamase inhibition profile combined with enhancement of the activity of multiple beta-lactam antibiotics with varying sensitivity to the intrinsic resistance mechanisms of efflux and permeability indicate QPX7728 is a useful inhibitor for use with multiple beta-lactam antibiotics.

There is an urgent need for new, potent anti-tuberculosis (TB) drugs with novel mechanisms of action that can be included in new regimens to shorten the treatment period for TB. After screening a library of carbostyrils, we optimized 3, 4-dihydrocarbostyril derivatives and identified OPC-167832 as having potent anti-tuberculosis activity. The minimum inhibitory concentrations of the compound for Mycobacterium tuberculosis ranged from 0.00024 to 0.002 μg/mL. It had bactericidal activity against both growing and intracellular bacilli, and the frequency of spontaneous resistance for Mycobacterium tuberculosis H37Rv was less than 1.91 x 10^-7. It did not show antagonistic effects with other anti-TB agents in an in vitro checkerboard assay. Whole genome and targeted sequencing of resistant isolates to OPC-167832 identified the decaprenylphosphoryl-β-D-ribose 2'-oxidase (DprE1), an essential enzyme for cell wall biosynthesis, as the target of this compound, and further studies demonstrated inhibition of the DprE1 enzymatic activity by OPC-167832. In a mouse model of chronic TB, OPC-167832 showed potent bactericidal activities starting at a dose of 0.625 mg/kg. Further, it exhibited significant combination effects in 2-drug combinations with delamanid, bedaquiline, or levofloxacin. Finally, 3-4 drug regimens comprised of delamanid and OPC-167832 as the core along with bedaquiline, moxifloxacin, or linezolid showed superior efficacy in reducing bacterial burden and preventing relapse compared to the standard treatment regimen. In summary, these results suggest that OPC-167832 is a novel and potent anti-TB agent and regimens containing OPC-167832 and new or repurposed anti-TB drugs may have the potential to shorten the duration of treatment for TB.

The biologic Griffithsin (GRFT) has recently emerged as a candidate to safely prevent sexually transmitted infections (STIs) including human immunodeficiency virus (HIV-1) and herpes simplex virus 2 (HSV-2). However, to date, there are few delivery platforms that are available to effectively deliver biologics to the female reproductive tract (FRT). The goal of this work was to evaluate rapid-release polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) fibers, that incorporate GRFT, in in vitro (HIV-1 and HSV-2) and in vivo (HSV-2) infection models. GRFT loading was determined via ELISA, and the bioactivity of GRFT fibers was assessed using in vitro HIV-1 pseudovirus and HSV-2 plaque assays. Afterwards, the efficacy of GRFT fibers was assessed in a murine model of lethal HSV-2 infection. Finally, murine reproductive tracts and vaginal lavages were evaluated for histology and cytokine expression, 24 and 72 hr after fiber administration, to determine safety. All rapid-release formulations achieved high levels of GRFT incorporation and were completely efficacious against in vitro HIV-1 and HSV-2 infections. Importantly, all rapid-release GRFT fibers provided potent protection in a murine model of HSV-2 infection. Moreover, histology and cytokine levels, evaluated from collected murine reproductive tissues and vaginal lavages treated with blank fibers, showed no increased cytokine production or histological aberrations, demonstrating the preliminary safety of rapid-release GRFT fibers in vaginal tissue.

Contezolid (MRX-I), a new oxazolidinone, is an antibiotic in development for treating complicated skin and soft tissue infections (cSSTI) caused by resistant Gram-positive bacteria. This was a thorough QT study conducted in 52 healthy subjects who were administered oral contezolid at a therapeutic (800 mg) dose, a supratherapeutic (1600 mg) dose, placebo, and oral moxifloxacin 400 mg in 4 separate treatment periods. The pharmacokinetic profile of contezolid was also evaluated. Time-point analysis indicated that the upper bounds of the two-sided 90% confidence interval (CI) for placebo-corrected change-from-baseline QTc (QTc) were <10 ms for the contezolid therapeutic dose at each time point. The upper bound of the 90% CI for QTc were slightly more than 10 ms with the contezolid supratherapeutic dose at 3 and 4 hours postdose, and the prolongation effect on the QT/QTc interval was less than that of the positive control, moxifloxacin 400 mg. At 3 and 4 h after the moxifloxacin dose, the moxifloxacin group met the assay sensitivity criteria outlined in ICH Guidance E14 with having a lower confidence bound ≥5 ms. The results of a linear exposure-response model which were similar to that of a time point analysis demonstrated a slightly positive relationship between contezolid plasma levels and QTcF interval with a slope of 0.227 ms per mg/L (90% CI: 0.188 to 0.266). In summary, contezolid did not prolong the QT interval at a therapeutic dose and may have a slight effect on QT interval prolongation at a supratherapeutic dose.

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually for the last few years. Although studies on mechanisms of polymyxin are expanding, system-wide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapt to colistin (polymyxin E) pressure, we carried out proteomic analysis of Klebsiella pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in Klebsiella pneumoniae involving several pathways, including (i) gluconeogenesis and TCA cycle; (ii) arginine biosynthesis; (iii) porphyrin and chlorophyll metabolism; and (iv) enterobactin biosynthesis. Interestingly, decreased abundance of class A β-lactamases including TEM, SHV-11, SHV-4 were observed in cells treated with colistin. Moreover, we also present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant Klebsiella pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of Klebsiella pneumoniae ATCC BAA 2146, showed missense mutation in crrB. The crrB mutant Ci, which displayed lipid A modification with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and palmitoylation, showed striking increases of CrrAB, PmrAB, PhoPQ, ArnBCADT and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediate colistin resistance, which may further offer valuable information to manage polymyxin resistance.

Antibiotic resistance is a global concern; however, data on antibiotic-resistant Ureaplasma spp. and Mycoplasma hominis are limited in comparison to similar data on other microbes. A total of 492 Ureaplasma spp. and 13 M. hominis strains obtained in Hangzhou, China, in 2018, were subjected to antimicrobial susceptibility testing for levofloxacin, moxifloxacin, erythromycin, clindamycin, and doxycycline using the broth microdilution method. The mechanisms underlying quinolone and macrolide resistance were determined. Meanwhile, a model of the topoisomerase IV complex bound to levofloxacin in wild-type Ureaplasma spp. was built to study the quinolone resistance mutations. For Ureaplasma spp., the levofloxacin, moxifloxacin and erythromycin resistance rates were 84.69%, 51.44% and 3.59% in U. parvum and 82.43%, 62.16% and 5.40% in U. urealyticum, respectively. Of the 13 M. hominis strains, 11 were resistant to both levofloxacin and moxifloxacin, and five strains showed clindamycin resistance. ParC S83L was the most prevalent mutation in levofloxacin-resistant Ureaplasma strains, followed by ParE R448K. The two mutations GyrA S153L and ParC S91I were commonly identified in quinolone-resistant M. hominis. A molecular dynamics-refined structure revealed that quinolone resistance-associated mutations inhibited the interaction and reduced affinity with gyrase or topoisomerase IV and quinolones. The novel mutations S21A in the L4 protein and G2654T and T2245C in 23S rRNA and ermB gene were identified in erythromycin-resistant Ureaplasma spp. Fluoroquinolone resistance in Ureaplasma spp. and Mycoplasma hominis remains high in China, the rational use of antibiotics needs to be further enhanced.

Multidrug resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV and C-VIII, have increasingly emerged as a leading cause of healthcare-associated infections, with colistin used as one of the last line of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hormaechei subsp. steigerwaltii. An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. Wild-type mgrB gene from Eh22, as well as that of a clinical strain of Klebsiella pneumoniae used as controls, were cloned and the corresponding recombinant plasmids were used for complementation assays. Results showed a fully restored susceptibility to colistin, and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains.

OptrA is an ATP-binding cassette (ABC)-F protein that confers resistance to oxazolidinones and phenicols, and can be either plasmid or chromosomally encoded. We isolated 13 Enterococcus faecalis strains possessing linezolid MIC ≥ 4 mg/L from nursery pigs in swine herds located across Brazil. Genome sequence comparison showed that these strains possess optrA in different genetic contexts occurring in 5 different E. faecalis sequence type backgrounds. The optrA gene invariably occurred in association with an araC regulator and a gene encoding a hypothetical protein. In some contexts, this genetic island was able to excise and form a covalently closed circle within the cell which appeared to occur in high abundance, and to be transmissible by co-resident plasmids.

There are limited treatment options for enterococcal urinary tract infections, especially vancomycin-resistant Enterococcus (VRE). Oral fosfomycin is a potential option, although limited data are available guiding dosing and susceptibility. We undertook pharmacodynamic profiling of fosfomycin against E. faecalis and E. faecium isolates using a dynamic in vitro bladder infection model. Eighty-four isolates underwent fosfomycin agar dilution susceptibility testing (E. faecalis MIC_50/90 32/64 μg/mL; E. faecium MIC_50/90 64/128 μg/mL). Sixteen isolates (including E. faecalis ATCC 29212 and E. faecium ATCC 35667) were chosen to reflect the MIC range and tested in the bladder infection model with synthetic human urine (SHU). Under drug-free conditions, E. faecium demonstrated greater growth restriction in SHU compared to E. faecalis (E. faecium maximal growth 5.8 ± 0.6 log₁₀ CFU/mL; E. faecalis 8.0 ± 1.0 log₁₀ CFU/mL). Isolates were exposed to high and low fosfomycin urinary concentrations after a single dose, and two-doses given daily with low urinary exposure. Simulated concentrations closely matched the target (bias 2.3%). E. faecalis isolates required greater fosfomycin exposure for 3 log₁₀ kill from the starting inoculum compared with E. faecium. The fAUC_0-72/MIC and f%T > MIC_0-72 for E. faecalis was 672 and 70%, compared to 216 and 51% for E. faecium, respectively. There was no rise in fosfomycin MIC post-exposure. Two doses of fosfomycin with low urinary concentrations resulted in equivalent growth inhibition to a single dose with high urinary concentrations. With this urinary exposure, fosfomycin was effective in promoting suppression of regrowth (>3 log₁₀ kill) in the majority of isolates.

Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is the modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT (9) family or by the dual-specificity ANT(3") (9) family that also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3") (9) AadA from Salmonella enterica. ANT (9) from Enterococcus faecalis adenylates the 9-hydroxyl of spectinomycin. We here present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT (9) in apo form, in complex with ATP, spectinomycin and magnesium or in complex with only spectinomycin. ANT (9) shows similar overall structure as AadA with an N-terminal nucleotidyltransferase domain and a C-terminal α-helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates by 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight α₅ helix and a shorter α_5-α₆ loop that would clash with the larger streptomycin substrate. In the active site, we observe two magnesium ions, one of them in a previously un-observed position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that also spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, will be subjected to modification by ANT (9) and ANT(3") (9) enzymes.

Carbapenem pharmacokinetic profiles are significantly changed in critically ill patients because of the drastic variability of the patients' physiological parameters. Published population PK studies have mainly focused on specific diseases and the majority of these studies had small sample sizes. The aim of this study was to develop a population PK model of imipenem in critically ill patients that estimated the influence of various clinical and biological covariates and the use of Extracorporeal Membrane Oxygenation (ECMO) and Continuous Renal Replacement Therapy (CRRT). A two-compartment population PK model with Creatinine clearance (CrCL), body weight (WT), and ECMO as fixed effects was developed using the non-linear mixed effect model (NONMEM). A Monte Carlo simulation was performed to evaluate various dosing schemes and different levels of covariates based on the pharmacokinetic/pharmacodynamic index (f%T>MIC) for the range of clinically relevant minimum inhibitory concentrations(MICs). The results showed that there may be insufficient drug use in the clinical routine drug dose regimen, and 750mg Q6h could achieve a higher treatment success rate. The blood concentrations of imipenem in ECMO patients were lower than that of non-ECMO patients, therefore dosage may need to be increased. The dosage may need adjustment for patients with CrCL ≤ 70ml/min, but dose should be lowered carefully to avoid the insufficient drug exposure. Dose adjustment is not necessary for patients within the WT ranging from 50-80 kg. Due to the large variation in PK profile of imipenem in critically ill patients, TDM should be carried out to optimize drug regimens.

Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at the concentrations of 0.5 μg/mL and 2 μg/mL, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3-log₁₀ within six hours. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficile in vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to β-lactams in Enterobacteriaceae and Pseudomonas aeruginosa. The new β-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn³⁴⁶ of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the N³⁴⁶Y amino acid substitution in Citrobacter freundii AmpC was previously shown to confer resistance to the ceftazidime-avibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the N³⁴⁶Y substitution in various AmpC backgrounds. Introduction of N³⁴⁶Y into Enterobacter cloacae AmpC (AmpC_cloacae), plasmid-mediated DHA-1, and P. aeruginosa PDC-5, led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k₂/K_i) of avibactam, respectively. The kinetic parameters of AmpC_cloacaeand DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpC_cloacaeand DHA-1 harboring N³⁴⁶Y conferred CAZ-AVI resistance (MIC of ceftazidime of 16 µg/ml in the presence of 4 µg/ml of avibactam). In contrast, production of PDC-5 N³⁴⁶Y was associated with a lower MIC (4 µg/ml) since this β-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpC_cloacaeN³⁴⁶Y. For FOX-3, the I³⁴⁶Y substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for β-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpC_cloacaeand DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn³⁴⁶ and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and there is a necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeting local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food-additive, with a probiotic and investigated its antifungal activity against Candida albicans. Lactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1%). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic/gellan formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacteria-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic/gellan formulations provided a release of L. paracasei cells over 24 hours that was sufficient to inhibit the growth of C. albicans with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic/gellan formulations also had inhibitory activity against Candida spp. biofilms by reducing the number of Candida spp. cells (p < 0.0001), decreasing the total biomass (p = 0.0003), and impairing hyphae formation (p = 0.0002), compared to the control group which received no treatment. Interestingly, probiotic formulation of 1% w/v gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log of CFU/mL after 10 days. This formulation inhibited the C. albicans growth (p < 0.0001), prevented the development of candidiasis lesions (p = 0.0013), and suppressed inflammation (p = 0.0006) when compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Brain infections with Cryptococcus neoformans are associated with significant morbidity and mortality. Cryptococcosis typically presents as meningoencephalitis or fungal mass lesions called cryptococcomas. Despite frequent in vitro discoveries of promising novel antifungals, the clinical need for drugs that can more efficiently treat these brain infections remains. A crucial step in drug development is the evaluation of in vivo drug efficacy in animal models. This mainly relies on survival studies or post-mortem analyses in large groups of animals, but these techniques only provide information on specific organs of interest at predefined time points. In this proof-of-concept study, we validated the use of non-invasive preclinical imaging to obtain longitudinal information on the therapeutic efficacy of amphotericin B or fluconazole monotherapy in meningoencephalitis and cryptococcoma mouse models. Bioluminescence imaging (BLI) enabled the rapid in vitro and in vivo evaluation of drug efficacy while complementary high-resolution anatomical information obtained by magnetic resonance imaging (MRI) of the brain allowed a precise assessment of the extent of infection and lesion growth rates. We demonstrated a good correlation between both imaging readouts and the fungal burden in various organs. Moreover, we identified potential pitfalls associated with the interpretation of therapeutic efficacy based solely on post-mortem studies, demonstrating the added value of this non-invasive dual imaging approach compared to standard mortality curves or fungal load endpoints. This novel preclinical imaging platform provides insights in the dynamic aspects of the therapeutic response and facilitates a more efficient and accurate translation of promising antifungal compounds from bench to bedside.

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (Coxsackievirus, rhinovirus), flavirivuses (Zika), and coronaviruses (HCoV-229E and MERS-CoV). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.

Importance No antivirals are approved for the treatment of bunyavirus infection. The ability to rapidly screen compounds and identify novel antivirals is one means to accelerate drug discovery for viruses with no approved treatments. We used this approach to screen hundreds of compounds against La Crosse virus, an emerging bunyavirus that causes significant disease, including encephalitis. We identified several known and previously unidentified antivirals. We focused on a potassium ionophore, valinomycin, due to its promising in vitro antiviral activity. We demonstrate that valinomycin, as well as a selection of other ionophores, exhibits activity against La Crosse virus as well as several other distantly related bunyaviruses. We finally observe that valinomycin has activity against a wide array of human viral pathogens, suggesting that disrupting potassium ion homeostasis with valinomycin may be a potent host pathway to target to quell virus infection.