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.

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.

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.

Linezolid is the first synthetic oxazolidone agent to treat infections caused by Gram-positive pathogens. Infected patients with liver dysfunction (LD) are more likely to suffer from adverse reactions such as thrombocytopenia when standard-dose linezolid is used than patients with LD who didn't use linezolid. Currently, pharmacokinetics data of linezolid in patients with LD are limited. The study aimed to characterize pharmacokinetics parameters of linezolid in patients with LD, identify the factors influencing the pharmacokinetics, and propose an optimal dosage regimen. We conducted a prospective study and established population pharmacokinetics model with the Phoenix NLME. The final model was evaluated by goodness-of-fit plots, bootstrap analysis, and prediction corrected-visual predictive check. A total of 163 concentration samples from 45 patients with LD were adequately described by a one-compartment model with first-order elimination along with prothrombin activity (PTA) and creatinine clearance as significant covariates. Linezolid clearance (CL) was 2.68 L/h (95% confidence interval [CI]: 2.34-3.03 L/h); the volume of distribution (Vd) was 58.34 L (95% CI: 48.00-68.68 L). Model-based simulation indicated that the conventional dose was at risk for overexposure in patients with LD or severe renal dysfunction; reduced dosage (300 mg/12 h) would be appropriate to achieve safe (C_min, ss at 2-8 ug/mL) and effective targets (the ratio of AUC_0-24 at steady state to MIC, 80-100). In addition, for patients with severe LD (PTA <= 20%), the dosage (400 mg/24 h) was sufficient at an MIC <= 2 ug/mL. This study recommended therapeutic drug monitoring for patients with LD.

Recent studies highlight the abundance of commensal coagulase-negative staphylococci (CoNS) on healthy skin. Evidence suggests that CoNS actively shape the skin immunological and microbial milieu to resist colonization or infection by opportunistic pathogens, including methicillin resistant Staphylococcus aureus (MRSA), in a variety of mechanisms collectively termed colonization resistance. One potential colonization resistance mechanism is the application of quorum sensing, also called the Accessory Gene Regulator (agr) system, which is ubiquitous among staphylococci. Common and rare CoNS make autoinducing peptides (AIPs) that function as MRSA agr inhibitors, protecting the host from invasive infection. In a screen of CoNS spent media we found that Staphylococcus simulans, a rare human skin colonizer and frequent livestock colonizer, released potent inhibitors of all classes of MRSA agr signaling. We identified three S. simulans agr classes, and have shown intraspecies cross-talk between non-cognate S. simulans agr types for the first time. The S. simulans AIP-I structure was confirmed, and the novel AIP-II and AIP-III structures were solved via mass spectrometry. Synthetic S. simulans AIPs inhibited MRSA agr signaling with nanomolar potency. S. simulans in competition with MRSA reduced dermonecrotic and epicutaneous skin injury in murine models. Addition of synthetic AIP-I also effectively reduced MRSA dermonecrosis and epicutaneous skin injury in murine models. These results demonstrate potent anti-MRSA quorum sensing inhibition by a rare human skin commensal, and suggest that cross-talk between CoNS and MRSA may be important in maintaining healthy skin homeostasis and preventing MRSA skin damage during colonization or acute infection.

Klebsiella pneumoniae that produce extended spectrum beta lactamases (ESBLs) are a persistent public health threat. There are relatively few therapeutic options and there is undue reliance on carbapenems. Alternative therapeutic options are urgently required. A combination of cefepime and the novel beta lactamase inhibitor enmetazobactam is being developed for treatment of serious infections caused by ESBL-producing organisms. The pharmacokinetics-pharmacodynamics (PK-PD) of cefepime-enmetazobactam against ESBL-producing K. pneumoniae was studied in a neutropenic murine pneumonia model. Dose ranging studies were performed. Dose fractionation studies were performed to define the relevant PD index for the inhibitor. The partitioning of cefepime and enmetazobactam into the lung was determined by comparing area under the concentration time curve (AUC) in plasma and epithelial lining fluid. The magnitude of drug exposure for cefepime-enmetazobactam required for logarithmic killing in the lung was defined using 3 ESBL-producing strains. Cefepime 100 mg/kg q8h i.v. had minimal antimicrobial effect. When this background regimen of cefepime was combined with enmetazobactam half-maximal effect was induced with enmetazobactam 4.71 mg/kg q8h i.v. The dose fractionation study suggest both fT>threshold and fAUC:MIC are potentially relevant PD indices. The AUC_ELF:AUC_plasma for cefepime and enmetazobactam was 73.4% and 61.5%, respectively. A ≥2-log kill in the lung was achieved with a plasma and ELF cefepime fT>MIC of ≥20% and enmetazobactam fT>2 mg/L of ≥20% of the dosing interval. These data and analyses provide the underpinning evidence for the combined use of cefepime and enmetazobactam for nosocomial pneumonia.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and C. gattii are associated with high indices of mortality, and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anti-cryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anti-cryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anti-cryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anti-cryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mice model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

In vitro and in vivo interactions of minocycline and azoles including itraconazole, voriconazole, and posaconazole against filamentous pathogenic fungi were investigated. A total of 56 clinical isolates were studied in vitro via broth microdilution checkerboard technique, including 20 strains of Aspergillus fumigatus, 7 strains of A. flavus, 16 strains of Exophiala dermatitidis, 10 strains of Fusarium solani and 3 strain s of F. oxysporum. The results revealed that minocycline individually did not exhibit any significant antifungal activity against all tested strains. However, favorable synergy of minocycline with itraconazole, voriconazole, or posaconazole were observed against 34 (61%), 28 (50%), and 38 (69%) isolates, respectively, including azole resistant A. fumigatus and Fusarium spp. with inherently high MICs of azoles. Synergistic combinations resulted in 4 fold to 16-fold reduction of effective MICs of minocycline and azoles. No antagonism was observed. In vivo effect of minocycline-azole combinations were evaluated by survival assay in Galleria mellonella model infected with E. dermatitidis strain BMU00034, F. solani strain FS9, A. fumigatus strain AF293, AFR1 and AFR2 . Minocycline acted synergistically with azoles and significantly increased larvae survival in all isolates (P<0.001), including azole resistant A. fumigatus and azole-inactive Fusarium spp.. In conclusion, the results suggested that minocycline combined with azoles may help to enhance the antifungal susceptibilities of azoles against pathogenic fungi and had the potential to overcome azole resistance issues.

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.

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.

Durlobactam (DUR, also known as ETX2514) is a novel β-lactamase inhibitor with broad activity against Ambler class A, C, and D β-lactamases. Addition of DUR to sulbactam (SUL) in vitro restores SUL activity against clinical isolates of Acinetobacter baumannii. The safety and pharmacokinetics (PK) of DUR alone and with SUL and/or imipenem/cilastatin (IMI/CIL) were evaluated in healthy subjects. This was a randomized, placebo-controlled study. In Part A, subjects including an elderly cohort (DUR 1 g) received single ascending doses of DUR 0.25-8 g. In Part B, multiple ascending dose of DUR 0.25-2 g were administered every 6 hours (q6h) for 29 doses. In Parts C and D, the drug-drug interaction (DDI) potential, including safety, of DUR (1 g) with SUL (1 g) and/or IMI/CIL (0.5/0.5 g) was investigated after single and multiple doses. Plasma and urine concentrations of DUR, SUL, and IMI/CIL were determined. Among 124 subjects, DUR was generally safe and well tolerated either alone or in combination with SUL and/or IMI/CIL. After single and multiple doses, DUR demonstrated linear dose proportional exposure across the studied dose ranges. Renal excretion was a predominant clearance mechanism. No drug:drug interaction potential was identified between DUR and SUL and/or IMI/CIL. SUL-DUR, 1 g (of each component) administered q6h with a 3 hour IV infusion, is under development for the treatment of serious infections due to A. baumannii.

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.

Ceftazidime–avibactam and cefiderocol are two of the latest generation β-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. Here we show that structural changes in AmpC β-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime–avibactam and cefiderocol without prior exposure to either agent. The AmpC β-lactamase of Ent385 (AmpC^Ent385) contained an alanine–proline deletion at positions 294–295 (A294_P295del) in the R2 loop. AmpC^Ent385 conferred reduced susceptibility to ceftazidime–avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpC^Ent385 showed increased hydrolysis of ceftazidime and cefiderocol compared with AmpC^Ent385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpC^Ent385 and AmpC^P99, the canonical AmpC of E. cloacae, revealed that the two-residue deletion in AmpC^Ent385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime–avibactam and cefiderocol requires close monitoring.

Importance Ceftazidime–avibactam and cefiderocol are newly approved β-lactam agents that possess broad spectrum activity against multidrug-resistant (MDR) Gram-negative bacteria. We show here that a two amino-acid deletion in the chromosomal AmpC β-lactamase, identified in a clinical strain of Enterobacter cloacae, confers reduced susceptibility to both agents. By crystallographic studies of free and drug-bound forms of enzyme, we demonstrate that this deletion in AmpC induces slanting of the H-9 helix that is directly connected with the R2 loop, and disappearance of the H-10 helix, is directly responsible for increased hydrolysis of ceftazidime and cefiderocol. These findings provide novel insights into how MDR Gram-negative bacteria may evolve their β-lactamases to survive selective pressure from these newly developed β-lactam agents.

Gepotidacin, a triazaacenaphthylene bacterial type II topoisomerase inhibitor, is in development for treatment of uncomplicated urinary tract infection (uUTI). This Phase 2a study in female participants with uUTI evaluated the pharmacokinetics (primary objective), safety, and exploratory efficacy of gepotidacin. Eligible participants (N = 22) were confined to the clinic at baseline, received oral gepotidacin 1,500 mg twice daily for 5 days (on-therapy; Days 1 to 5), and returned to the clinic for test-of-cure (Days 10 to 13) and follow-up (Day 28±3). Pharmacokinetic, safety, clinical, and microbiological assessments were performed. Maximum plasma concentrations were observed approximately 1.5 to 2 hours postdose. Steady state was attained by Day 3. Urinary exposure over the dosing interval increased from 3,742 μg.h/ml (Day 1) to 5,973 μg.h/ml (Day 4), with trough concentrations of 322 to 352 μg/ml from Day 3 onward. Gepotidacin had an acceptable safety-risk profile with no treatment-limiting adverse events and no clinically relevant safety trends. Clinical success was achieved in 19 (86%) and 18 (82%) of 22 participants at test-of-cure and follow-up, respectively. Eight participants had a qualifying baseline uropathogen (growth; ≥10⁵ CFU/ml). A therapeutic (combined clinical and microbiological [no growth; <10³ CFU/ml]) successful response was achieved in 6 (75%) and 5 (63%) of 8 participants at test-of-cure and follow-up, respectively. Plasma area under the free-drug concentration-time curve over 24 hours at steady state divided by the MIC (fAUC_0-24/MIC) and urine AUC_0-24/MIC ranged from 6.99 to 90.5 and 1,292 to 121,698, respectively. Further evaluation of gepotidacin in uUTI is warranted. (NCT03568942)

We report the first clinical Escherichia. coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, 6-11(RPISLR), in pmrB contributing to colistin resistance was verified using recombinant DNA techniques. Although decreased fitness compared to the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

Treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP+Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) at 4-day intervals, plus allopurinol at 30 mg/kg/12 h p.o. during 130 days; LC+Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose), plus allopurinol during 130 days; Allop, treated with allopurinol only; non-treated control. Parasite loads were evaluated by quantitative PCR in liver, spleen and bone marrow and by immunohistochemistry in the ear skin, before, just after treatment and 4 months later. LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen and bone marrow 4 months after treatment, compared to the pre-treatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin, in comparison to the control group. On the basis of clinical staging and parasitological evaluations, LCP formulation exhibited a more favorable therapeutic profile, when compared to LC one, being therefore promising for treatment of canine visceral leishmaniasis.

The rapid evolution of resistance in the malaria parasite to every single drug developed against it calls for the urgent identification of new molecular targets. Using a stain specific for the detection of intracellular amyloid deposits in live cells we have detected the presence of abundant protein aggregates in Plasmodium falciparum blood stages and female gametes cultured in vitro, in the blood stages of mice infected by Plasmodium yoelii, and in the mosquito stages of the murine malaria species Plasmodium berghei. Aggregated proteins could not be detected in early rings, the parasite form that starts the intraerythrocytic cycle. A proteomics approach was followed to pinpoint actual aggregating polypeptides in functional P. falciparum blood stages, which resulted in the identification of 369 proteins, with roles particularly enriched in nuclear import-related processes. Five aggregation-prone short peptides selected from this protein pool exhibited different aggregation propensity according to Thioflavin-T fluorescence measurements, and were observed to form amorphous aggregates and amyloid fibrils in transmission electron microscope images. The results presented suggest that generalized protein aggregation might have a functional role in malaria parasites. Future antimalarial strategies based on the upsetting of the pathogen's proteostasis and therefore affecting multiple gene products could represent the entry to new therapeutic approaches.

Omadacycline is a novel aminomethylcycline with activity against Gram-positive and -negative organisms, including Haemophilus influenzae, which is one of the leading causes of community-acquired bacterial pneumonia (CABP). The evaluation of antimicrobial agents against H. influenzae using standard murine infection models is challenging due to the low pathogenicity of this species in mice. Therefore, 24-hour dose-ranging studies using a one-compartment in vitro infection model were undertaken with the goal of characterizing the magnitude of the ratio of the area under the concentration-time curve (AUC) to the MIC (AUC/MIC ratio) associated with efficacy for a panel of five clinical H. influenzae isolates. These five isolates, which had MIC values of 1 or 2 mg/L, were exposed to omadacycline total-drug epithelial lining fluid (ELF) concentration-time profiles based on those observed in healthy volunteers following intravenous omadacycline administration. Relationships between change in log₁₀ colony forming units (CFU) from baseline at 24 hours and total-drug ELF AUC/MIC ratio for each isolate and the isolates pooled together were evaluated using Hill-type models and non-linear least squares regression. As evidenced by the high coefficient of determination (r²) of 0.88 to 0.98, total-drug ELF AUC/MIC ratio described the data well for each isolate and the isolates pooled together. The median total-drug ELF AUC/MIC ratio associated with net bacterial stasis and 1- and 2-log₁₀ CFU/mL reductions from baseline at 24 hours was 6.91, 8.91, and 11.1, respectively. These data were useful to support the omadacycline dosing regimens selected for the treatment of patients with CABP, as well as susceptibility breakpoints for H. influenzae.

The SHV β-lactamases (BLs) have undergone strong allele diversification that changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, in silico mathematical models predicted five positions as relevant for the β-lactamase inhibitor (BLI) resistant (2br) phenotype, 12 as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and two positions were related to solely the narrow spectrum (2b) phenotype. These positions and additional 6 positions described in other studies (including one promoter mutation), were systematically substituted and investigated for their substrate specificities in a BL-free E. coli background, representing, to our knowledge, the most comprehensive substrate and substitution analysis for SHV alleles to date. An in vitro analysis confirmed the essentiality of the positions 238 and 179 for the 2be phenotype and 69 for the 2br phenotype. The substitutions E240K and E240R, which do not occur alone in known 2br SHV variants, led to a 2br phenotype, indicating a latent BLI-resistance potential of these substitutions. The substitutions M129V, A234G, S271I and R292Q conferred latent resistance to cefotaxime. In addition, 7 positions that were found to be not always associated with the ESBL phenotype resulted in increased resistance to ceftaroline. We also observed that coupling of a strong promoter (IS26) to a A146V mutant with the 2b phenotype resulted in a highly increased resistance to BLIs, cefepime and ceftaroline but not to 3^rd generation cephalosporins, indicating that SHV enzymes represent an underestimated risk for empirical therapies that use piperacillin/tazobactam or cefepime to treat different infectious diseases caused by gram-negatives.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used non-human primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in IFN-α showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and severity of symptoms in the early stage. This study also showed the pathologically severe lung injury states in the cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and the need for further studies on the virus pathogenicity and new antiviral therapies.

A total of 2,283 Salmonella spp. isolates were recovered from 18,334 samples including patients with diarrhea, food of animal origin and pets across 5 provinces of China. The highest prevalence of Salmonella spp. was detected in chicken meats (39.3%, 486/1,237). Fifteen serogroups and 66 serovars were identified, with Typhimurium and Enteritidis being the most dominant. Most (85.5%, 1,952/2,283) isolates exhibited resistant to ≥ 1 antimicrobial and 56.4% were multi-drug resistant (MDR). A total of 222 isolates harbored extended-spectrum β-lactamases (ESBLs), 200 of which were CTX-M-type that were mostly detected from chicken meat and turtle fecal. Overall, eight bla_CTX-M genes were identified, with bla_CTX-M-65, bla_CTX-M-123, bla_CTX-M-14, bla_CTX-M-79, and bla_CTX-M-130 being the most prevalent. Totally, 166 of the 222 ESBL-producing isolates had amino acid substitutions in GyrA (S83Y, S83F, D87G, D87N, and D87Y) and ParC (and S80I), whilst the PMQR-encoding genes oqxA/B, qepA, and qnrB/S were detected in almost all isolates. Of the fifteen sequence types (STs) identified in the 222 ESBLs, ST17, ST11, ST34, and ST26 ranked among the top 5 in the number of isolates. Our study revealed considerable serovars diversity, high prevalence of co-occurrence of MDR determinants, including CTX-M-type ESBLs, QRDRs mutations and PMQR genes. This is the first report of CTX-M-130 Salmonella spp. from patients with diarrhea and QRDRs mutations from turtle fecal samples. Our study emphasizes the importance of actions, both in the health care settings and in the veterinary medicine sector, to control the dissemination of MDR, especially the CTX-M Salmonella spp. isolates.

Baloxavir marboxil, a prodrug of cap-dependent endonuclease inhibitor, baloxavir acid, reduces the time to improvement of influenza symptoms in patients infected with type A or B influenza virus. To characterize its pharmacokinetics, a population pharmacokinetic model for baloxavir acid was developed using 11846 plasma concentration data items from 1827 subjects including 2341 plasma concentration data items from 664 patients at high risk of influenza complications. A three-compartment model with first-order elimination and first-order absorption with lag time well described the plasma concentration data. Body weight and race were found to be the most important factors influencing clearance and volume of distribution. The exposures in high-risk patients were similar to those in otherwise healthy patients, and no pharmacokinetic difference was identified regarding any risk factors for influenza complications.

Exposure-response analyses were performed regarding the time to improvement of symptoms and the reduction in the influenza virus titer in high-risk patients. The analyses suggested that body weight-based dosage, 40 mg for patients weighing < 80 kg and 80 mg for patients weighing ≥ 80 kg, can shorten the time to improvement of influenza symptoms and reduce virus titer for both type A and B influenza virus regardless of the exposure levels of the high-risk patients as well as for the otherwise healthy influenza patients.

The results of our population pharmacokinetic and exposure-response analyses in patients with risk factors of influenza complications should provide useful information on the pharmacokinetic and pharmacodynamic characteristics of baloxavir marboxil and also for the optimization of dose regimens.

Treatment of Mycobacterium avium complex pulmonary disease (MAC-PD) is challenging partly due to high efflux pump expression. Thioridazine might block these efflux pumps. We explore thioridazine's efficacy against M. avium using minimum inhibitory concentrations (MICs), time-kill combination assays, ex vivo macrophage infection assays and efflux assays. Thioridazine is bactericidal against M. avium, inhibits intracellular growth at 2x MIC and blocks ethidium bromide efflux. However, its toxicity and low plasma concentrations, make it unlikely to add efficacy to MAC-PD therapy.

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug-resistance to specific anti-influenza drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug nitazoxanide with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza potentials with 10-fold improvement, compared with nitazoxanide, and were effective against a variety of influenza subtypes including oseltamivir-resistant strains. Notably, the combination using of compounds 4a/4d and oseltamivir carboxylate or zanamivir displayed synergistic antiviral effect against oseltamivir-resistant strain. Mode of action analysis demonstrated that compounds 4a/4d acted at the late phase of viral infection cycle through inhibiting viral RNA transcription and replication. Further experiments showed that treatment with compounds 4a/4d significantly inhibited influenza virus infection in human lung organoids, suggesting the druggability of the novel thiazolides. In-depth transcriptome analysis revealed a series of up-regulated cellular genes that may contribute to the antiviral activities of 4a/4d. Together, our study pointed the optimization direction of nitazoxanide as anti-influenza drug, and discovered two novel-structured candidates 4a/4d with relatively broad-spectrum antiviral potential.

Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) are associated with substantial morbidity and mortality. Monotherapy with first-line antimicrobials such as vancomycin (VAN; glycopeptide) and daptomycin (DAP; lipopeptide) are inadequate in some cases due to reduced antibiotic susceptibilities or therapeutic failure. In recent years, β-lactam antibiotics have emerged as a potential option for combination therapy with VAN/DAP that may meet an unmet therapeutic need for MRSA BSI. Ceftaroline (CPT), the only commercially available β-lactam in the United States with intrinsic in vitro activity against MRSA, has been increasingly studied in the setting of VAN and DAP failures. Novel combinations of first-line agents (VAN and DAP) with β-lactams have been the subject of many recent investigations due to in vitro findings such as the "see-saw effect", where β-lactam susceptibility may be improved in the presence of decreased glycopeptide and lipopeptide susceptibility. The combination of CPT and DAP, in particular, has become the focus of many scientific evaluations, due to intrinsic anti-MRSA activities and potent in vitro synergistic activity against various MRSA strains. This article reviews the available literature describing these innovative therapeutic approaches for MRSA BSI, focusing on preclinical and clinical studies, and evaluates the potential benefits and limitations of each strategy.

Globally, mutations in the katG gene account for the majority of isoniazid-resistant strains of Mycobacterium tuberculosis. Buyankhishig et al analyzed a limited number of Mycobacterium tuberculosis strains in Mongolia and found that isoniazid resistance was mainly attributable to inhA mutations. The GenoType® MTBDRplus assay was performed for isolates collected in the First National Tuberculosis Prevalence Survey and the Third Anti-Tuberculosis Drug Resistance Survey to investigate genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis in Mongolia. Of the 409 isoniazid-resistant isolates detected by the GenoType® MTBDRplus assay, 127 (31.1%) were resistant to rifampicin, 294 (71.9%) had inhA mutations without katG mutations, 113 (27.6%) had katG mutations without inhA mutations, and two (0.5%) strains had mutations in both the inhA and katG genes. Of the 115 strains with any katG mutation, 114 (99.1%) had mutations in codon 315 (S315T). Of the 296 trains with any inhA mutation, 290 (98.0%) had a C–15T mutation. The proportion of isoniazid-resistant strains with katG mutations was 25.3% among new cases and 36.2% among retreatment cases (p=0.03), as well as 17.0% among rifampicin-susceptible strains and 52.8% among rifampicin-resistant strains (p<0.01). Rifampicin resistance was significantly associated with the katG mutation (adjusted odds ratio 5.36, 95% CI 3.3–8.67, p<0.001). Mutations in inhA predominated in isoniazid-resistant tuberculosis in Mongolia. However, the proportion of katG mutations in isolates from previously treated cases was higher than that among new cases, and that in cases with rifampicin resistance was higher than that in cases without rifampicin resistance.

The currently unfolding coronavirus pandemic threatens health systems and economies worldwide....

Dihydroartemisinin-piperaquine has shown excellent efficacy and tolerability in malaria treatment. However, concerns have been raised of potentially harmful cardiotoxic effects associated with piperaquine. The population pharmacokinetics and cardiac effects of piperaquine were evaluated in 1,000 patients, mostly children enrolled in a multicentre trial from 10 sites in Africa. A linear relationship described the QTc-prolonging effect of piperaquine, estimating a 5.90ms mean QTc-prolongation per 100ng/mL increase in piperaquine concentration. The effect of piperaquine on absolute QTc-interval estimated a mean maximum QTc-interval of 456ms (EC₅₀=209ng/mL). Simulations from the pharmacokinetic-pharmacodynamic models predicted 1.98-2.46% risk of having QTc-prolongation > 60ms in all treatment settings. Although piperaquine administration resulted in QTc-prolongation, no cardiovascular adverse events were found in these patients. Thus, the use of dihydroartemisinin-piperaquine should not be limited by this concern.

Meropenem/vaborbactam resistance in Klebsiella pneumoniae is associated with loss of function mutations in the OmpK35 and OmpK36 porins. Here we identify two previously unknown loss of function mutations that confer cefuroxime resistance in K. pneumoniae. The proteins lost were NlpD and KvrA; the latter is a transcriptional repressor controlling capsule production. We demonstrate that KvrA loss reduces OmpK35 and OmpK36 porin production, which confers reduced susceptibility to meropenem/vaborbactam in a KPC-3 producing K. pneumoniae isolate.

Objectives: Empiric antibiotic prescribing can be supported by guidelines and/or local antibiograms, but these have limitations. We sought to use data from a comprehensive electronic health record to use statistical learning to develop predictive models for individual antibiotics that incorporate patient-, and hospital-specific factors. This paper reports on the development and validation of these models on a large retrospective cohort.

Methods: This is a retrospective cohort study including hospitalized patients with positive urine cultures in the first 48 hours of hospitalization at a 1500 bed, tertiary care hospital over a 4.5 year period. All first urine cultures with susceptibilities were included. Statistical learning techniques, including penalized logistic regression, were used to create predictive models for cefazolin, ceftriaxone, ciprofloxacin, cefepime, and piperacillin-tazobactam. These were validated on a held-out cohort.

Results: The final dataset used for analysis included 6,366 patients. Final model covariates included demographics, comorbidity score, recent antibiotic use, recent antimicrobial resistance, and antibiotic allergies. Models had acceptable to good discrimination in the training dataset and acceptable performance in the validation dataset, with a point estimate for area under the receiver operating characteristic curve (AUC) that ranged from 0.65 for ceftriaxone to 0.69 for cefazolin. All models had excellent calibration.

Conclusion: In this study we used electronic health record data to create predictive models to estimate antibiotic susceptibilities for UTIs in hospitalized patients. Our models had acceptable performance in a held-out validation cohort.

Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathologic bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal's "quorum-quenching" activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard of care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis, and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo. Similarly, locally-delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of sub-therapeutic vancomycin. However, we also found that the resorbable polyurethane foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard of care antibiotic therapy for S. aureus osteomyelitis, but also highlight potential pitfalls encountered with local drug delivery.

One of the reasons for the lengthy tuberculosis (TB) treatment is the difficult to treat non-multiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our pre-clinical drug development pipeline. In most available dormancy models it takes a long time to create a dormant state and it is difficult to identify and quantify this non-multiplying condition.

The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes non-multiplying after 10 days of streptomycin starvation, but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetic model to assess the difference in the activity of isoniazid, rifampicin, moxifloxacin and bedaquiline against log-phase growth compared to the non-multiplying M. tuberculosis subpopulation by CFU counting including a novel AUC-based approach as well as time-to-positivity (TTP) measurements.

We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampicin and high dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value as it identifies a specific mycobacterial subpopulation that is non-culturable on solid media.

In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline.

A case of M. leprae rifampicin resistance after irregular anti-leprosy treatments since 1971 is reported. Whole-genome sequencing from four longitudinal samples indicated relapse due to acquired rifampicin resistance and not to reinfection with another strain. A putative compensatory mutation in rpoC was also detected. Clinical improvement was achieved using an alternative therapy.

Background: MRSA pose significant therapeutic challenges, related to their: frequency in clinical infections; innate virulence properties; and propensity for multi-antibiotic resistance. MRSA are among the most common causes of endovascular infections, including infective endocarditis (IE).

Objective: To employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE.

Study Design: TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (vs daptomycin alone). Primary intravegetation outcomes were: maximum size; weights at sacrifice; and MRSA counts at infection baseline vs after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment Day 1).

Results: A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase + daptomycin vs the daptomycin-alone therapy groups (p = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower vs daptomycin-alone (p < 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (p = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower vs untreated controls (p<0.0001) and the daptomycin-alone group (p<0.0001).

Conclusion: This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size and weight. Moreover, TTE appears to be an efficient non-invasive tool to assess therapeutic efficacies in experimental MRSA IE.

Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. We here aim to identify the target of a novel class of CHIKV inhibitors i.e. CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low micromolar range. A CHVB-resistant variant (CHVB^res) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2 and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve ~18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVB^res was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.

Introduction: This study was performed to evaluate the impacts of vanA-positivity of Enterococcus faecium (EFM) exhibiting diverse susceptibility phenotypes to glycopeptides on clinical outcomes in patients with a bloodstream infection (BSI) through a prospective, multicenter, observational study.

Methods: A total of 509 patients with an EFM BSI from eight sentinel hospitals in South Korea during a two-year period were enrolled in this study. Risk factors of the hosts and causative EFM isolates were assessed to determine associations with the 30-day mortality of EFM BSI patients via multivariable logistic regression analyses.

Results: The vanA gene was detected in 35.2% (179/509) of EFM isolates; 131 EFM isolates exhibited typical VanA phenotypes (group vanA-VanA), while the remaining 48 EFM isolates exhibited atypical phenotypes (group vanA-Atypical), including VanD (n = 43) and vancomycin-variable phenotypes (n = 5). A multivariable logistic regression indicated that vanA-positivity of causative pathogens was independently associated with the increased 30-day mortality rate in the patients with an EFM BSI; however, there was no significant difference in the survival rates between the patients of the vanA-VanA and vanA-Atypical groups (log-rank test, P = 0.904).

Conclusions: A high 30-day mortality rate was observed in patients with vanA-positive EFM BSIs, and vanA-positivity of causative EFM was an independent risk factor for early mortality irrespective of the susceptibility phenotypes to glycopeptides; thus, intensified antimicrobial stewardship is needed to improve clinical outcome of patients with vanA-positive EFM BSI.

As resistance to artemisinins (current frontline drugs in malaria treatment) emerges in south East Asia, there is an urgent need to identify the genetic determinants and understand the molecular mechanisms underpinning such resistance. Such insights could lead to prospective interventions to contain resistance and prevent the eventual spread to other malaria endemic regions. Artemisinin reduced susceptibility in South East Asia (SEA) has been primarily linked to mutations in P. falciparum Kelch-13, which is currently widely recognised as a molecular marker of artemisinin resistance. However, 2 mutations in a ubiquitin hydrolase, UBP-1, have been previously associated with artemisinin reduced susceptibility in a rodent model of malaria and some cases of UBP-1 mutation variants associating with artemisinin treatment failure have been reported in Africa and SEA. In this study, we have employed CRISPR-Cas9 genome editing and pre-emptive drug pressures to test these artemisinin susceptibility associated mutations in UBP-1 in P. berghei sensitive lines in vivo. Using these approaches, we have shown that the V2721F UBP-1 mutation results in reduced artemisinin susceptibility, while the V2752F mutation results in resistance to chloroquine and moderately impacts tolerance to artemisinins. Genetic reversal of the V2752F mutation restored chloroquine sensitivity in these mutant lines while simultaneous introduction of both mutations could not be achieved and appears to be lethal. Interestingly, these mutations carry a detrimental growth defect, which would possibly explain their lack of expansion in natural infection settings. Our work has provided independent experimental evidence on the role of UBP-1 in modulating parasite responses to artemisinin and chloroquine under in vivo conditions.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and heart failure in Latin American countries. The treatment of Chagas disease is directed at eliminating the parasite, decreasing the probability of cardiomyopathy, and disrupting the disease transmission cycle. Benznidazole (BZ) and nifurtimox (NFX) are recognized as effective drugs for the treatment of Chagas disease by the World Health Organization, but both have high toxicity and limited efficacy, especially in the chronic disease phase. At low doses, aspirin (ASA) has been reported to protect against T. cruzi infection. We evaluated the effectiveness of BZ in combination with ASA at low doses during the acute disease phase and evaluated cardiovascular aspects and cardiac lesions in the chronic phase. ASA treatment prevented the cardiovascular dysfunction (hypertension and tachycardia) and typical cardiac lesions. Moreover, BZ+ASA-treated mice had a smaller cardiac fibrotic area than that in BZ-treated mice. These results were associated with an increase in the number of eosinophils and reticulocytes and level of nitric oxide in the plasma and cardiac tissue of ASA-treated mice relative to respective controls. These effects of ASA and BZ+ASA in chronically infected mice were inhibited by pretreatment with the LXA₄ receptor antagonist, Boc-2, indicating that the protective effects of ASA are mediated by ASA-triggered lipoxin. These results emphasize the importance of exploring new drug combinations for treatments of acute phase of Chagas disease that are beneficial for chronic patients.

Two non-amidated host defence peptides named Pin2[G] and FA1 were evaluated against three types of pathogenic bacteria; two isolated from diabetic foot ulcer patients, Staphylococcus aureus UPD13 and Pseudomonas aeruginosa UPD3, and another from a commercial collection, Salmonella enterica serovar Typhimurium (ATCC 14028). In vitro experiments showed that the antimicrobial performance of the synthetic peptides, Pin2[G] and FA1, was modest, although FA1 was more effective than Pin2[G]. In contrast Pin2[G] had superior in vivo anti-infective activity to FA1 in rabbit wound infections by the diabetic foot ulcer pathogens S. aureus UPD13 and P. aeruginosa UPD3. Indeed, Pin2[G] reduced bacterial colony counts of both S. aureus UPD13 and P. aeruginosa UPD3 by >100,000-fold after 48-72 h on skin wounds of infected rabbits, while in similar infected wounds, FA1 had no major effects at 72-96 h of treatment. Ceftriaxone was equally effective vs. Pseudomonas but less effective vs. S. aureus infections. Additionally, the two peptides were evaluated in mice against intragastrically inoculated S. enterica ser. Typhimurium (ATCC 14028). Only Pin2[G], at 0.56 mg/kg, was effective in reducing systemic (liver) infection by >67-fold, equivalent to the effect of treatment with levofloxacin. Pin2[G] showed superior immunomodulatory activity in increasing chemokine production by a human bronchial cell line and suppressing poly(IC)-induced pro-inflammatory IL6 production. These data showed that the in vitro antimicrobial activity of these peptides was not correlated with their in vivo anti-infective activity, and suggest that other factors such as immunomodulatory activity were more important.

Methicillin-resistant Staphylococcus aureus (MRSA) has grown to become a major burden on healthcare systems. The cumulation of limited therapeutic options and worsened patient outcomes with persistent MRSA bacteremia has driven research in optimizing its initial management. The guidelines published by the Infectious Disease of America currently recommend combination therapy for refractory MRSA bacteremia, but the utility of combining antibiotics from the start of therapy is under investigation. The alternative strategy of early use of a β-lactam antibiotics in combination with vancomycin upon initial MRSA bacteremia detection has shown promise. While this concept has gained international attention, providers should give this strategy serious consideration prior to implementation. The objective of this review is to examine retrospective and prospective evidence for early combination with vancomycin and β-lactam antibiotics, as well as explore potential consequences of combination therapy.

Manogepix (APX001A) is the active moiety of the novel drug candidate fosmanogepix (APX001). We previously reported the broad-spectrum activity of manogepix but also observed a correlation between increased manogepix and fluconazole MICs. Here we extended this study and included isolates with acquired fluconazole resistance.

Isolates (n=835) were identified using CHROMagar, MALDI-TOF and, when needed, ITS-sequencing. EUCAST E.Def 7.3.1 susceptibility testing included manogepix, amphotericin B, anidulafungin, micafungin, fluconazole and voriconazole. Manogepix wildtype-upper-limit (WT-UL) values were established following EUCAST-principles for ECOFF setting allowing wildtype/non-wildtype classification. Drug-specific MIC correlations were investigated using Pearson's correlation.

Manogepix modal MICs were low (range 0.004-0.06 mg/L against 16/20 included species). Exceptions were C. krusei and C. inconspicua, and to a lesser extent C. kefyr and Pichia kluyveri. The activity was independent of Fks echinocandin hot-spot alterations (n=17). Adopting the WT-UL established for C. albicans, C. dubliniensis, C. glabrata, C. parapsilosis and C. tropicalis, 14/724 (1.9%) isolates were non-wildtype for manogepix. Twelve of these (85.7%) were also non-wildtype for fluconazole. A statistically significant correlation was observed between manogepix and fluconazole MICs for C. albicans, C. dubliniensis, C. glabrata, C. parapsilosis and C. tropicalis (Pearson r=0.401-0.575), but not between manogepix and micafungin or amphotericin B MICs for any species except C. tropicalis (r=0.519 for manogepix versus micafungin).

Broad-spectrum activity was confirmed for manogepix against contemporary yeast. However, a 1-4 two-fold-dilution increase in manogepix MICs is observed in a subset of isolates with acquired fluconazole resistance. Further studies on the potential underlying mechanism and implication for optimal dosing are warranted.

Treatment of exacerbations of chronic Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) is highly challenging due to hypermutability, biofilm formation and an increased risk of resistance emergence. We evaluated the impact of ciprofloxacin and meropenem as monotherapy and in combination in the dynamic in vitro CDC biofilm reactor (CBR). Two hypermutable P. aeruginosa strains, PAOmutS (MIC_{ciprofloxacin} 0.25 mg/L, MIC_meropenem 2 mg/L) and CW44 (MIC_{ciprofloxacin} 0.5 mg/L, MIC_meropenem 4 mg/L), were investigated for 120h. Concentration-time profiles achievable in epithelial lining fluid (ELF) following FDA-approved doses were simulated in the CBR. Treatments were ciprofloxacin 0.4g every 8h as 1h-infusions (80% ELF penetration), meropenem 6 g/day as continuous infusion (CI; 30% and 60% ELF penetration) and their combinations. Counts of total and less-susceptible planktonic and biofilm bacteria and MICs were determined. Antibiotic concentrations were quantified by UHPLC-PDA. For both strains, all monotherapies failed with substantial regrowth and resistance of planktonic (≥8log₁₀ CFU/mL) and biofilm (>8log₁₀ CFU/cm²) bacteria at 120h (MIC_{ciprofloxacin} up to 8 mg/L, MIC_meropenem up to 64 mg/L). Both combination treatments demonstrated synergistic bacterial killing of planktonic and biofilm bacteria of both strains from ~48h onwards and suppressed regrowth to ≤4log₁₀ CFU/mL and ≤6log₁₀ CFU/cm² at 120h. Overall, both combination treatments suppressed amplification of resistance of planktonic bacteria for both strains, and biofilm bacteria for CW44. The combination with meropenem at 60% ELF penetration also suppressed amplification of resistance of biofilm bacteria for PAOmutS. Thus, combination treatment demonstrated synergistic bacterial killing and resistance suppression against difficult-to-treat hypermutable P. aeruginosa strains.

Ebola Virus (EBOV) is among the most devastating pathogens causing fatal hemorrhagic fever in humans. The 2013–2016 epidemics resulted in over 11000 deaths, while another outbreak is currently ongoing. Since there is no FDA-approved drug so far to fight EBOV infection, there is an urgent need to focus on drug discovery. Considering the tight correlation between the high EBOV virulence and its ability to suppress the type-I Interferon (IFN-I) system, identifying molecules targeting viral protein VP24, one of the main virulence determinants blocking IFN response, is a promising novel anti-EBOV therapy approach. Hence, in the effort of finding novel EBOV inhibitors, a screening of a small set of flavonoids was performed, showing that Quercetin and Wogonin can suppress the VP24 effect on IFN-I signaling inhibition. The mechanism of action of the most active compound, Quercetin, showing an IC₅₀ value of 7.4 μM, was characterized to significantly restore the IFN-I signaling cascade, blocked by VP24, by directly interfering with the VP24 binding to karyopherin-α and thus restoring P-STAT1 nuclear transport and IFN genes transcription. Quercetin significantly blocked viral infection, specifically targeting EBOV VP24 anti-IFN-I function. Overall, Quercetin is the first identified inhibitor of the EBOV VP24 anti-IFN function, representing a molecule interacting with a viral binding site that is very promising for further drug development aiming to block EBOV infection at the early steps.