METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed.
RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage.
CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.
METHODS: Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools.
RESULTS: Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1-2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China.
CONCLUSIONS: The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations.
Methods: Each 24-well plate of Vero cells infected with all four DENV serotypes, singly, was subjected to treatments with various doses of AR-12. Following 48 h of incubation, inhibitory efficacies of AR-12 against the different DENV serotypes were evaluated by conducting a virus yield reduction assay whereby DENV RNA copy numbers present in the collected supernatant were quantified using qRT-PCR. The underlying mechanism(s) possibly involved in the compound's inhibitory activities were then investigated by performing molecular docking on several potential target human and DENV protein domains.
Results: The qRT-PCR data demonstrated that DENV-3 was most potently inhibited by AR-12, followed by DENV-1, DENV-2 and DENV-4. Our molecular docking findings suggested that AR-12 possibly exerted its inhibitory effects by interfering with the chaperone activities of heat shock proteins.
Conclusions: These results serve as vital information for the design of future studies involving in vitro mechanistic studies and animal models, aiming to decipher the potential of AR-12 as a potential therapeutic option for DENV infection.
PATIENTS AND METHODS: A direct observational study was conducted in which plasma levels of drug and amino acids (tryptophan, tyrosine and phenylalanine) were monitored during quinine treatment of malaria patients with Plasmodium falciparum infections.
RESULTS: Consistent with competition for uptake from plasma into cells, plasma tryptophan and tyrosine levels increased ≥2-fold during quinine therapy. Plasma quinine levels in individual plasma samples were significantly and positively correlated with tryptophan and tyrosine in the same samples. Control studies indicated no effect on phenylalanine. Chloroquine treatment of Plasmodium vivax-infected patients did not affect plasma tryptophan or tyrosine. During quinine treatment, plasma tryptophan was significantly lower (and quinine significantly higher) in patients experiencing adverse drug reactions.
CONCLUSIONS: Plasma quinine levels during therapy are related to patient tryptophan and tyrosine levels, and these interactions can determine patient responses to quinine. The study also highlights the potential for extrapolating insights directly from the yeast model to human malaria patients.
METHODS: A 'meta-model' with 4894 concentrations from 1631 neonates was built using NONMEM, and Monte Carlo simulations were performed to design an optimal intermittent infusion, aiming to reach a target AUC0-24 of 400 mg·h/L at steady-state in at least 80% of neonates.
RESULTS: A two-compartment model best fitted the data. Current weight, postmenstrual age (PMA) and serum creatinine were the significant covariates for CL. After model validation, simulations showed that a loading dose (25 mg/kg) and a maintenance dose (15 mg/kg q12h if <35 weeks PMA and 15 mg/kg q8h if ≥35 weeks PMA) achieved the AUC0-24 target earlier than a standard 'Blue Book' dosage regimen in >89% of the treated patients.
CONCLUSIONS: The results of a population meta-analysis of vancomycin data have been used to develop a new dosing regimen for neonatal use and to assist in the design of the model-based, multinational European trial, NeoVanc.
Methods: This was a single-centre, retrospective cohort study evaluating posaconazole suspension pre-emptive therapy in LTx recipients between January 2009 and December 2015.
Results: Forty-two LTx recipients were prescribed posaconazole suspension pre-emptively. Aspergillus fumigatus was the most commonly isolated fungal organism. Of the patients receiving posaconazole suspension as the initial antifungal post-LTx, 93% had eradication of colonization at 6 months after commencing therapy. In contrast, only 61% had eradication of fungal colonization when posaconazole suspension was administered following initial therapy with voriconazole. Posaconazole suspension appeared to be well tolerated, although one case was curtailed following concern about abnormal liver function and another due to nausea/vomiting. TDM was performed in 37 patients. The initial median (IQR) trough plasma concentration ( C min ) following 400 mg twice-daily posaconazole suspension was 0.78 (0.46-1.19) mg/L. Doses beyond 800 mg daily did not appear to result in a higher median C min.
Conclusions: Early initiation of posaconazole suspension pre-emptive therapy in LTx recipients appears to be well tolerated and may potentially afford favourable clinical outcomes.
Methods: A single-centre, retrospective study evaluating posaconazole Cmin in LTx recipients receiving posaconazole suspension or MR tablets between January 2014 and December 2016.
Results: Forty-seven LTx patients received posaconazole suspension, and 78 received the MR tablet formulation; a total of 421 and 617 Cmin measurements were made, respectively. Posaconazole was concurrently administered with proton pump inhibitor in ≥ 90% of patients. The median (IQR) of initial posaconazole Cmin following 300 mg daily of posaconazole tablet was significantly higher than that of 800 mg daily of posaconazole suspension [1.65 (0.97-2.13) mg/L versus 0.81 (0.48-1.15) mg/L, P
METHODS: Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR.
RESULTS: Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific.
CONCLUSIONS: RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates.
Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.
Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.
Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.
Methods: We searched PubMed, Embase and the Cochrane register of trials systematically for studies that examined treatment options for patients with MDR- and XDR-AB infections until April 2016. Network meta-analysis (NMA) was performed to estimate the risk ratio (RR) and 95% CI from both direct and indirect evidence. Primary outcomes were clinical cure and microbiological cure. Secondary outcomes were all-cause mortality and nephrotoxic and non-nephrotoxic adverse events.
Results: A total of 29 studies with 2529 patients (median age 60 years; 65% male; median APACHE II score 19.0) were included. Although there were no statistically significant differences between treatment options, triple therapy with colistin, sulbactam and tigecycline had the highest clinical cure rate. Colistin in combination with sulbactam was associated with a significantly higher microbiological cure rate compared with colistin in combination with tigecycline (RR 1.23; 95% CI 1.03-1.47) and colistin monotherapy (RR 1.21; 95% CI 1.06-1.38). No significant differences in all-cause mortality were noted between treatment options. Tigecycline-based therapy also appeared less effective for achieving a microbiological cure and is not appropriate for treating bloodstream MDR- and XDR-AB infections.
Conclusions: Combination therapy of colistin with sulbactam demonstrates superiority in terms of microbiological cure with a safety profile similar to that of colistin monotherapy. Thus, our findings support the use of this combination as a treatment for MDR- and XDR-AB infections.
METHODS: In total, 7541 organisms causing documented infections were consecutively collected in 66 centres in 33 countries, excluding the USA. Susceptibility testing was performed by broth microdilution. Isolates displaying linezolid MIC results of ≥4 mg/L were molecularly characterized.
RESULTS: Linezolid inhibited all Staphylococcus aureus at ≤2 mg/L, with MIC50 results of 1 mg/L, regardless of methicillin resistance. A similar linezolid MIC50 result (i.e. 0.5 mg/L) was observed against CoNS, with the vast majority of isolates (99.4%) also inhibited at ≤2 mg/L. Six CoNS that exhibited elevated linezolid MIC values were found to contain alterations in the 23S rRNA and/or L3 ribosomal protein. Linezolid exhibited consistent modal MIC and MIC50 results (1 mg/L) against enterococci, regardless of species or vancomycin resistance. Three Enterococcus faecalis from Galway and Dublin (Ireland) and Kelantan (Malaysia) showed MIC results of 4 to 8 mg/L and carried optrA. All Streptococcus pneumoniae, viridans-group streptococci and β-haemolytic streptococci were inhibited by linezolid at ≤2, ≤2 and ≤1 mg/L, respectively, with equivalent MIC90 results (1 mg/L for all groups).
CONCLUSIONS: These results document the continued long-term and stable in vitro potency of linezolid and reveal a limited number of isolates with decreased susceptibility to linezolid (i.e. MIC ≥4 mg/L). The latter isolates primarily showed mutations in the 23S rRNA gene and/or L3 protein, but cfr was not detected. Moreover, this study shows that isolates carrying the newly described ABC transporter optrA are not restricted to China.