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.

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.

Bacteria of the genus Campylobacter are major foodborne pathogens which have become increasingly resistant to clinically important antimicrobial agents (1)....

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.

KBP-7072 is a semi-synthetic aminomethylcycline with broad-spectrum activity against Gram-positive and Gram-negative pathogens including multidrug resistant bacterial strains. The pharmacokinetics (PK) of KBP-7072 after oral and intravenous (IV) administration of single and multiple doses were investigated in animal models including during fed and fasted states and also evaluated the protein binding and excretion characteristics. In Sprague-Dawley (SD) rats, Beagle dogs, and CD-1 mice, KBP-7072 demonstrated a linear PK profile after administration of single oral and IV and multiple oral doses. Oral bioavailability ranged from 12% to 32%. Mean T_max ranged from 0.5 to 4 hours, and mean half-life ranged from approximately 6 to 11 hours. Administration of oral doses in the fed state resulted in a marked reduction in C_max and AUC compared with dosing in fasted animals. The mean bound fractions of KBP-7072 were 77.5%, 69.8%, 64.5%, 69.3%, and 69.2% in mouse, rat, dog, monkey, and human plasma, respectively. Following a single 22.5 mg/kg oral dose of KBP-7072 in SD rats, cumulative excretion in feces was 64% and in urine was 2.5% of the administered dose. The PK results in animal models are consistent with single and multiple ascending dose studies in healthy volunteers and confirm the suitability of KBP-7072 for once daily oral and IV administration in clinical studies.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Pharmacokinetic changes are often seen in patients with severe infections. Administration by continuous infusion has been suggested to optimize antibiotic exposure and pharmacokinetic/pharmacodynamic (PK/PD) target attainment for β-lactams. In an observational study, unbound piperacillin concentrations (n=196) were assessed in 78 critically ill patients following continuous infusion of piperacillin/tazobactam (ratio 8:1). The initial dose of 8, 12 or 16 g (piperacillin component) was determined by individual creatinine clearance (CRCL). Piperacillin concentrations were compared to the EUCAST clinical breakpoint MIC for Pseudomonas aeruginosa (16 mg/L), and the following PK/PD targets were evaluated: 100% fT>1xMIC and 100% fT>4xMIC. A population pharmacokinetic model was developed using NONMEM 7.4.3 consisting of a one-compartment disposition model with linear elimination separated into non-renal and renal (linearly increasing with patient CRCL) clearances. Target attainment was predicted and visualized for all individuals based on the utilized CRCL dosing algorithm. The target of 100% fT>1xMIC was achieved for all patients based on the administered dose, but few patients achieved the target of 100% fT>4xMIC. Probability of target attainment for a simulated cohort of patients showed, that increasing the daily dose by 4 g increments (piperacillin component) did not result in substantially improved target attainment for the 100% fT>4xMIC target. To conclude, in patients with high CRCL combined with high-MIC bacterial infections, even a CI regimen with a daily dose of 24 g may be insufficient to achieve therapeutic concentrations.

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.

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.

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.

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.

Aspergillus niger, the third species responsible for invasive aspergillosis has been considered as a homogeneous species until DNA-based identification uncovered many cryptic species. These species have been recently reclassified into the Aspergillus section Nigri. However little is yet known among the section Nigri about the species distribution and the antifungal susceptibility pattern of each cryptic species. A total of 112 clinical isolates collected from 5 teaching hospitals in France and phenotypically identified as A. niger were analyzed. Identification to the species level was carried out by nucleotide sequence analysis. The Minimum Inhibitory Concentrations (MICs) of itraconazole, voriconazole, posaconazole, isavuconazole and amphotericin B were determined by both the EUCAST and gradient concentration strips methods. Aspergillus tubingensis (n=51, 45.5%) and A. welwitschiae (n=50, 44.6%) were the most common species while A. niger accounted for only 6.3% (n=7). The MICs of azoles drugs were higher for A. tubingensis than for A. welwitschiae. The MIC of amphotericin B was 2 mg/L or less for all isolates. Importantly, MICs determined by EUCAST showed no correlation with those determined by gradient concentration strips methods, these latter being lower than the former (Spearman's rank correlation tests ranging - depending on the antifungal agent - from 0.01 to 0.25; p>0.4). In conclusion, A. niger should be considered as a minority species in the section Nigri. The differences in MICs between species for different azoles underline the importance of accurate identification. Significant divergences in the determination of MIC between EUCAST and gradient concentration strips methods require further investigation.

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.

Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01-0.66 μg/ml) and micafungin levels (<0.01-0.16 μg/ml) were lower than the simultaneous plasma concentrations (0.77-5.07 μg/ml and 1.21-8.70 μg/ml, respectively). In cerebral cortex, anidulafungin and micafungin levels were 0.21-2.34 μg/g and 0.18-2.88 μg/g, respectively. Thus, MIC values of several pathogenic Candida strains exceed concentrations in CSF and in brain.

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.

Many transferable quinolone-resistance mechanisms have been already identified in Gram-negative bacteria. The plasmid-encoded 65 amino-acid long ciprofloxacin-modifying enzyme, namely CrpP, was recently identified in Pseudomonas aeruginosa. We analyzed a collection of 100 clonally-unrelated and multidrug-resistant P. aeruginosa clinical isolates among which 46 (46%) were found positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. Those crpP-like genes were chromosomally located, as part of PAGI-like pathogenicity genomic islands.

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.

Florfenicol belongs to a class of phenicol antimicrobials widely used as feed additives and for the treatment of respiratory infections. In recent years, increasing resistance to florfenicol has been reported in Campylobacter spp., the leading foodborne enteric pathogen causing diarrheal diseases worldwide. Here, we reported the identification of fexA, a novel mobile florfenicol resistance gene in Campylobacter. Of the 100 Campylobacter jejuni strains isolated from poultry in Zhejiang, China, nine of them were shown to be fexA positive, and their whole genome sequences were further determined by integration of Illumina short-read and MinION long-read sequencing. The fexA gene was found in the plasmid of one strain and chromosomes of eight strains, and its location was verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Based on comparative analysis, the fexA gene was located within a region with the tet(L)-fexA-catA-tet(O) gene arrangement, demonstrated to be successfully transferrable among C. jejuni strains. Functional cloning indicated that acquisition of the single fexA gene significantly increased resistance to florfenicol, whereas its inactivation resulted in increased susceptibility to florfenicol in Campylobacter. Taken together, these results indicated that the emerging fexA resistance is horizontally transferable, which might greatly facilitate the adaptation of Campylobacter in food producing environments where florfenicols are frequently used.

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.

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.

CYP450 enzymes are involved in biotransformation of chloroquine (CQ), but the role of the different metabolism profiles of this drug has not been properly investigated in relation to P. vivax recurrences. To investigate the influence of CYPs genotypes associated with CQ-metabolism on early recurrence rates of P. vivax, a case-control study was carried out. Cases included patients presenting an early recurrence (CQ-recurrent), defined as recurrence during the first 28 days after initial infection, plasma concentrations of CQ plus desethylchloroquine (DCQ, the major CQ metabolite) higher than 100 ng/mL. A control (CQ-responsive) with no parasite recurrence over the follow-up was also included. CQ and DCQ plasma levels were measured on Day 28. CQ CYPs (CYP2C8, CYP3A4 and CYP3A5) genotypes were determined by real-time PCR. An ex vivo study was conducted to verify CQ and DCQ efficacy in P. vivax isolates. The frequency of alleles associated with normal and slow metabolism was similar between the cases and controls for CYP2C8 (OR=1.45, 95% CI=0.51-4.14, p=0.570), CYP3A4 (OR=2.38, 95% CI=0.92-6.19, p=0.105) and CYP3A5 (OR=4.17, 95% CI=0.79-22.04, p=1.038) genes. DCQ levels were higher than CQ, regardless of the genotype. Regarding the DCQ/CQ rate, there was no difference between groups or between those patients who had a normal or mutant genotype. DCQ and CQ showed similar efficacy ex vivo. CYPs genotypes had no influence on early recurrence rates. Similar efficacy of CQ and DCQ ex vivo could explain the absence of therapeutic failure, despite presence of alleles associated with slow metabolism.

The off-label use of third generation cephalosporin (3GC) during in ovo vaccination or vaccination of newly hatched chicks, was a common practice worldwide. CMY-2-producing Escherichia coli have been disseminated among broiler production. The objectives of this study were to determine the epidemiological linkage of bla_CMY-2-positive plasmids among broilers both within and outside Japan because grandparent stock and parent stock were imported in Japan. We examined the whole genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002-2014. The predominant 3GC-resistance gene was bla_CMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-I (n=21; 24.1%), IncI (n=12; 13.8%), IncB/O/K/Z (n=28; 32.2%), and IncC (n=22; 25.3%). Those plasmids were subjected to gene clustering and network analyses and plasmid multi-locus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single nucleotide variant (SNV)-based phylogenetic analysis.

MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. ST429 harboring bla_CMY-2-positive IncB/O/K/Z was closely related to isolates from broiler in Germany harboring bla_CMY-2-positive IncB/O/K/Z. pST55-IncI and pST12-IncI1-I and pST3-IncC were prevalent in western Japan. pST12-IncI1-I and pST3-IncC were closely related to those detected in E. coli isolates from chicken in American continent, whereas 26 IncB/O/K/Z were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria in and out of Japan.

Google has made it easier to join the company's Advanced Protection Program, which is designed to stop the most sophisticated hackers from breaking into your Gmail account. Before you needed two security keys to enroll. Now you just need a smartphone.

Six sources confirmed Apple changed its mind on end-to-end encrypted backups two years ago following an FBI complaint and concerns users could lose access to their own data.

The stunning allegation reportedly comes from a forensic analysis Amazon CEO Jeff Bezos commissioned to determine the culprit behind the hack, which resulted in his private photos ending up in the hands of the National Enquirer.

Oil was headed for the longest run of daily gains in more than nine months on signs the worst of the supply glut may be over as production cuts start to take effect.

Oil headed for its first back-to-back weekly gain since February as output cuts from the biggest producers and a nascent recovery in demand began to rebalance a market awash with crude.

BACKGROUND:

The ability of the decades-old Boston and Philadelphia criteria to accurately identify infants at low risk for serious bacterial infections has not been recently reevaluated.

BACKGROUND AND OBJECTIVES:

Antibiotic therapy is often prescribed for suspected community-acquired pneumonia (CAP) in children despite a lack of knowledge of causative pathogen. Our objective in this study was to investigate the association between antibiotic prescription and treatment failure in children with suspected CAP who are discharged from the hospital emergency department (ED).

BACKGROUND:

For children who cannot be discharged from the emergency department, definitive care has become less frequent at most hospitals. It is uncertain whether this is true for common conditions that do not require specialty care. We sought to determine how the likelihood of definitive care has changed for 3 common pediatric conditions: asthma, croup, and gastroenteritis.