Journal of Antimicrobial Chemotherapy

IntroductionApramycin is an aminoglycoside antimicrobial that has been used in veterinary medicine since the 1980s but not licensed for human medicine. Because it is not impacted by common aminoglycoside resistance mechanisms, there is interest in repurposing the drug for use in humans, as a treatment of multidrug resistant Gram negative bacterial infections. Gap StatementThe prevalence and factors associated with apramycin resistance in bacteria isolated from humans has received limited study but is important foundational information for considering repurposing apramycin for use in humans. AimTo systematically review and analyze data pertaining to apramycin resistance in bacteria isolated from humans and to identify knowledge gaps, to inform work evaluating the potential for re-purposing of apramycin for clinical use in humans. MethodologyA systematic review was performed to evaluate apramycin resistance in bacteria isolated from humans. ResultsA total of 1626 references were identified during the search, with 34 studies were deemed eligible for inclusion. Pooled estimates for apramycin resistance were 6% (95% CI 1-12%) for E. coli, 1% (0-3%) for Acinetobacter spp, 2% (0-5%) for Enterobacter spp, 7% (2-15%) for Klebsiella spp, 4% (0-13%) for Pseudomonas and 0% (0-0%) for Salmonella spp. Multivariable mixed-effects meta-regression identified no effect of year (P=0.36), bacterial species (all P>0.19), geographic region (all P>0.13) or enrollment of known carbapenem-resistant isolates (P=0.44). The only significant variable was datasets that used known gentamicin-resistant isolates (P=0.003). ConclusionDespite nearly 50 years of apramycin use in animals, apramycin resistance was identified in bacteria of human origin but was rare, supporting the potential value of re-purposing this drug for use in humans and suggesting that there is limited spillover of resistance from veterinary and agricultural use of apramycin.

Two new antibiotics, zoliflodacin and gepotidacin, were recently approved for the treatment of urogenital gonorrhea. While combination therapy could, in principle, delay the emergence and spread of resistance, doing so depends on the absence of antagonism between the co-administered drugs. Using in vitro checkerboard testing, we observed no evidence of antagonism for all pairwise combinations of zoliflodacin, gepotidacin, and ciprofloxacin, including in strains with elevated ciprofloxacin MICs.

Objective: To consolidate publicly available United States National Antimicrobial Resistance Monitoring System (NARMS) human isolate data spanning 2004-2021 and comprehensively quantify trends in fluoroquinolone resistance, tetracycline resistance, ceftriaxone resistance, and early-stage azithromycin resistance across four NARMS-monitored enteric bacterial pathogens: nontyphoidal Salmonella, Campylobacter jejuni, Campylobacter coli, and Shigella spp. Methods: Annual resistance percentages were compiled from publicly available CDC, FDA, and USDA NARMS Human Isolates Surveillance Reports and Integrated Reports. Multi-year reference-period averages were assigned to their period midpoint years. Ordinary least-squares linear regression was applied to each pathogen-drug series. Slope estimates, 95% confidence intervals, Pearson correlation coefficients, and p-values were computed. A sensitivity analysis was conducted excluding reference-period midpoint years. Results: Ciprofloxacin decreased susceptibility in Salmonella rose from 2.4% to 11% (slope 0.62 pp/year, 95% CI 0.39-0.85; p less than 0.001). Ciprofloxacin resistance in C. jejuni rose from 21% to 34% (slope 1.68 pp/year, 95% CI 1.10-2.25; p=0.001) and in C. coli from 28% to 45% (slope 2.08 pp/year, 95% CI 0.97-3.19; p=0.007). Shigella showed a 5.8-fold increase (underpowered, n=3). Tetracycline resistance in C. jejuni and C. coli rose significantly. Salmonella multidrug resistance declined (slope -0.34 pp/year; p=0.001) while ceftriaxone resistance rose (slope 0.10 pp/year; p=0.028). Azithromycin-resistant Salmonella isolates detected in 2017 equalled the cumulative total from 2011-2016. Discussion: Fluoroquinolone and tetracycline resistance rose significantly across NARMS-monitored Campylobacter species, while Salmonella showed diverging trends: declining classical multidrug resistance but rising fluoroquinolone and ceftriaxone resistance. The emerging azithromycin resistance signal threatens remaining oral treatment options. These trends support sustained investment in harmonized antimicrobial resistance surveillance and stewardship globally.

Trimethoprim is recommended as a first-line treatment of urinary tract infections (UTIs) in the UK. In 2018, 31.4% of Escherichia coli isolated from UTIs in England were trimethoprim resistant, leading to overreliance on other first and second-line antibiotics. Here, we assessed whether prior selection with trimethoprim results in collateral effects to other antibiotics recommended for the treatment of UTIs. As collateral effects, we considered changes in susceptibility, mutation-selection window and population establishment probability. We selected 10 trimethoprim-resistant derivatives from three clinical isolates of uropathogenic Escherichia coli. We found that mutations conferring trimethoprim resistance did not have any collateral effects to fosfomycin. In contrast, resistance to trimethoprim resulted in decreased susceptibility (collateral resistance) to nitrofurantoin, below the clinical breakpoint, and narrowed the mutation-selection window thereby reducing the maximum concentration for selection of nitrofurantoin resistance mutations. Our analyses demonstrate that multiple collateral responses should be accounted for when predicting and optimising antibiotic use, limiting future AMR emergence.

Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been proposed to be effective at treating COVID-19 patients. We, and others, have previously reported on the capacity of CQ to reduce HIV-1 replication in vitro. We tested CQ administration in post-partum mothers on influencing HIV-1 viral loads in human milk as a means of lowering mother to child transmission. A Phase I/II, randomized, placebo-controlled study to evaluate chloroquine administration to reduce HIV-1 RNA levels in human milk: the CHARGE study. Thirty HIV-1 positive pregnant Rwandese women (CQ n = 20; placebo n = 10) were enrolled in a 16-week study, with the treatment group receiving a 200 mg oral dose of CQ daily. Base-line plasma viral load (pVL) measurements and CD4 counts were determined prior to delivery, and pVL, breast milk VL (bmVL) and CQ levels measured during treatment. For women receiving treatment, CQ concentration was higher in breast milk compared to plasma (over 2.5-fold), with a positive correlation between the levels in the two compartments (P < 0.003). A link between high CQ concentrations in plasma and high CD4 counts (P < 0.001) was observed. Surprisingly, we found a significant increase in pVL after CQ treatment in over half of the mothers (n=11; P < 0.001) and with no alteration to bmVL measurements. No specific amino acid alterations in the gp120 envelope sequences could be associated with CQ administration. CQ usage is associated with a significant increase to pVL in early breastfeeding mothers from Rwanda which cautions against the use of CQ in such individuals. Our results highlight a discrepancy between CQ effects on modulating HIV-1 replication in vitro versus in vivo and indicate caution when prescribing CQ to postpartum HIV-1 untreated mothers. This discrepancy should be taken into consideration when testing CQ or HCQ treatment in COVID-19 clinical trials, especially relating to the post-partum setting.

BackgroundWe showed that exposure of an AcrB D408A mutant to efflux inhibitors applied evolutionary pressure to select bacteria with the wild type acrB sequence. This suggested that reversion to wild type can differentiate between efflux inhibitors. Thus, we hypothesized that this experiment could identify inhibitors of the primary RND pump, AcrB or its homologues in other species. ObjectivesTo construct three mutants, Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A and expose the mutants to substrates and non-substrates of AcrB and/or MexB and determine the rate of reversion to wildtype acrB/mexB sequence. MethodsMutant Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A were constructed with site-directed mutagenesis of the relevant nucleotide in the acrB/mexB gene. Mutants were exposed on agar to substrates and the mutation frequency and mutation rate determined. The MIC of antibiotics and the presence/absence of the D408A substitution was determined for mutants. ResultsExposure to the AcrB substrates chlorpromazine and minocycline reverted the D408A genotypes to wild type in a species-dependent manner. Exposure to a non-AcrB substrate, spectinomycin, did not select wild type acrB. Chlorpromazine selected for wild type acrB K. pneumoniae as it had for S. Typhimurium, whereas minocycline selected for wild type E. coli acrB. None of the antibiotics selected wild type mexB, including the tested MexB substrates. ConclusionsEvolutionary paths depend upon the genetic background of the species and availability of alternative routes/genetic pathways that can confer resistance/decreased susceptibility to antibiotics.

BackgroundSurveillance and prediction of antibiotic resistance in Escherichia coli relies on curated databases of genes and mutations. Such databases currently lack quantitative data estimating the effect on MIC caused by the acquisition of any given element for a particular antibiotic-species combination. MethodsUsing a collection of 2875 E. coli isolates with linked whole genome sequencing and MIC data, we used multivariable interval regression models to estimate the change in MIC for specific antibiotics associated with the acquisition of genes and mutations in the AMRFinder database with and without an adjustment for population structure. We then tested the ability of these models to predict MIC and binary resistance/susceptibility using leave-one-out cross validation. FindingsWe provide quantitative estimates (with confidence intervals) of the change in MIC associated with the acquisition of genes/mutations in the NCBI-AMRFinder database. Whilst the majority of genes and mutations (89/111 (80.2%) were associated with an increased MIC, a much smaller number (27/111, 24.3%) were found to be putatively independently resistance conferring (i.e. associated with an MIC above the EUCAST breakpoint) when acquired in isolation. We found evidence of differential effects of acquired genes and mutations between different generations of cephalosporin antibiotics and demonstrated that sub-breakpoint variation in MIC can be linked to genetic mechanisms of resistance. 20,697/24,858 (83.3%, range 52.9-97.7 across all antibiotics) of MICs were correctly exactly predicted and 23,677/24,858 (95.2%, range 87.3-97.7) to within +/-1 doubling dilution. InterpretationQuantitative estimates of the independent effect on MIC of the acquisition of antibiotic resistance genes add to the interpretability and utility of existing databases. Using these estimates to predict antibiotic resistance phenotype demonstrates performance that is comparable to or better than approaches utilising machine learning models and crucially more readily interpretable. The methods outlined here could be readily applied to other antibiotic/pathogen combinations. FundingThis work was funded by the NIHR and the MRC. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed from inception to 05/04/2024 using the terms ((Escherichia coli OR E. coli) AND ((MIC) OR (minimum inhibitory concentration))) AND (predict*) AND (whole genome sequencing). Of the 56 articles identified by these search terms, eight were of direct relevance to this study. These studies generally focused on single antibiotics (3 studies), had relatively small datasets (6 studies {inverted exclamation}1000 isolates) or used machine learning approaches on pan-genomes to predict binary (i.e. susceptible/resistant) phenotypes (2 studies). Only one study attempted to predict ciprofloxacin MICs in 704 E. coli isolates using a machine learning approach with known resistance conferring genes/mutations as features. To our knowledge, there are no studies estimating the independent effect (as opposed to the total effect of all elements present) of the acquisition of specific antibiotic resistance genes (ARGs) or resistance-associated mutations on MICs of different antibiotics in E. coli more generally. What this study addsIn this study we estimate the change in MIC for particular antibiotics associated with the acquisition of specific ARGs or resistance-associated mutations, adjusting for the presence of other relevant genes and population structure. In doing so we provide an approach to greatly enhance the information provided by existing ARG databases and approaches based on predicting binary susceptible/resistant phenotypes, for example by demonstrating differential effects of ARGs on resistance to antibiotics of the same class, enriching our understanding of the relationship between genotype and phenotype in a way that is easily interpretable. Using more "parsimonious" models for prediction, we demonstrate high overall accuracy comparable to or better, and crucially more readily interpretable, than recent machine learning models. We also demonstrate a genetic basis behind sub-breakpoint variation in MIC for some antibiotics, demonstrating the value of non-dichotomised phenotypes for identifying wildtype isolates (i.e. those carrying no ARGs) with greater confidence. Implications of all available evidenceWhole genome sequencing data can be used to predict MICs for most commonly used antibiotics for managing E. coli infections with accuracy approaching that of conventional phenotyping techniques, though very major error rates remain too high for deployment in routine clinical practice. Further studies focusing on genotypes with high phenotypic heterogeneity should investigate the phenotypic replicability, genetic heritability and clinical outcomes associated with these isolates.

Long-acting cabotegravir and rilpivirine combination (LA-CAB/RPV) is approved for HIV treatment whilst long-acting cabotegravir alone (LA-CAB) is approved for HIV prevention, both in adults. However, individuals who become pregnant might prefer to discontinue it due to lack of definitive data on safety. The aim of this study was to characterise the tail-phase maternal and fetal pharmacokinetics of LA-CAB/RPV following discontinuation at steady-state early in pregnancy. A virtual population of non-pregnant women (n = 100 per scenario) initiated intramuscular injections of LA-CAB/RPV at the approved dosage and continued maintenance dose (400/600 mg once monthly or 600/900 mg once every two months) until steady state. We simulated discontinuation at steady state after only one injection during pregnancy. Tail-phase pharmacokinetics of CAB and RPV from LA injections were characterised during gestation and until 6 months postpartum. Pharmacokinetic tails of LA-CAB/RPV were driven by the residual drug in the muscle depot which stabilised at steady state and reduced steadily upon dosing discontinuation. Upon discontinuation of the monthly dosing, predicted median (IQR) maternal plasma concentrations for LA-CAB were 415 (386-448) ng/mL at delivery and 125 (115-139) ng/mL 6 months postpartum. For LA RPV, these were 11.6 (11.0-12.6) ng/mL and 7.84 (7.30-8.49) ng/mL at delivery and 6 months postpartum, respectively. Pharmacokinetic tails of LA-CAB/RPV extend to several months postpartum, with levels falling below established minimum effective concentration in most women after gestation week 33. Potential strategies to minimise potential risks associated with LA-CAB/RPV discontinuation in this population are needed.

Using mixed culture selection, we show how reduced envelope permeability, reduced target-site affinity, and increased {beta}-lactamase production layer to confer aztreonam/{beta}-lactamase inhibitor resistance in Escherichia coli. We report a clinical isolate producing CTX-M-15 and CMY-4, lacking OmpF, and carrying a PBP3 mutation. It is resistant to aztreonam plus the inhibitors avibactam, relebactam and vaborbactam. Mobilisation of blaSHV-12 into this isolate generated a derivative additionally resistant to aztreonam plus the bicyclic boronate inhibitors 2 and taniborbactam.

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

Cefepime is widely used in the intensive care unit (ICU) for the treatment of complicated Gram-negative infections. Cefepime is a candidate for therapeutic drug monitoring (TDM) and model-informed precision dosing (MIPD), especially in critically ill patients, notably because of its concentration-dependent neurological toxicity. In this study, we aimed to evaluate the impact of the implementation of cefepime MIPD on PK/PD targets attainment in intensive care. We performed a monocentric, retrospective, before-after study, including all adult patients hospitalized in our ICU and for whom at least two cefepime plasma through concentrations (Cmin) were measured on separate days. The main endpoint was a Cmin between 4xMIC (or 10mg/L if the MIC was unavailable) and 20mg/L. We modelled the odds of target attainment via a mixed-effect logistic model and the rate of target attainment with a spline of time. A total of 281 patients were included, of whom 121 were in the MIPD group and 160 in the control group. Median age, weight and follow-up duration were 65 years, 76 kg and 3 days respectively. The two most common infections were pneumonia (n=239) and peritonitis (n=16). A total of 728 cefepime through concentrations were collected. MIPD was non-significantly associated with higher odds of being in the therapeutic range (aOR 1.40 [0.88 - 2.22]) and significantly associated with lower odds of being over-exposed (aOR 0.58 [0.35 - 0.96]). HR of target attainment was 1.1 [0.7 - 1.9] at day 1 and 1.6 [0.9 - 2.9] at day 7. In summary, we demonstrated that cefepime MIPD in the ICU reduces the risk of over-exposure, and may be beneficial on the odds and on the rate of PK/PD targets attainment. What is already known about this subjectO_LICefepime is a candidate for TDM/MIPD programs, with established PK/PD targets for both antimicrobial efficacy and neurological toxicity (100% f T >4xMIC and Cmin>20mg/L, respectively). C_LIO_LIPrevious randomized controlled trials (RCT) of beta-lactams model-informed precision dosing (MIPD) versus standard dosing approaches failed to demonstrate a superiority of MIPD in clinical endpoints attainment, and provided conflicting results on pharmacokinetic target attainment. C_LI What this study addsO_LICefepime MIPD in the intensive care units reduces the odds of being over-exposed, and thus, possibly, the risk of elicit dose-dependent neurological toxicity. C_LIO_LIFurther RCT evaluating MIPD efficacy should focus on evaluating target attainment rate instead of probability alone. C_LI

A tedizolid-resistant isolate of MRSA was selected by serial passage. Whole genome sequencing revealed only a single nucleotide variant in rpoB. Cross-resistance to linezolid, chloramphenicol, and quinupristin-dalfopristin was observed but susceptibility to other drugs including rifampin was unchanged. Models of the RNA-polymerase-ribosomal complex revealed that the mutated residue was unlikely to interact directly with the oxazolidinone binding site. This is the first time that rpoB mutation has been associated with resistance to the PhLOPSa antimicrobials.

SynopsisO_ST_ABSBackgroundC_ST_ABSMeropenem, gentamicin and ciprofloxacin have been used as empiric broad-spectrum combination therapy in different combinations. Recent restrictions on the use of quinolones jeopardises the rational of administering this combination to increase the spectrum of coverage for this particular case. A mechanistic understanding of pharmacodynamic interaction for these combinations is lacking but can provide insight in the necessity of using the different moieties. ObjectivesTo study pharmacodynamic drug-drug interaction between meropenem, gentamicin and ciprofloxacin against Escherichia coli. MethodsStatic time kill curve experiments were conducted with Escherichia coli (NCTC(R) 12241) at 0.25 - 16 x MIC for a duration of 24 hours with samples being collected at 0, 2, 4, 6, 8, and 24 hour. Meropenem, gentamicin and ciprofloxacin were tested alone, in two- and three-way combinations. Bacterial load time series data were enumerated on Meuller Hinton plates and Colony Forming Unit data was modelled using nonlinear mixed-effects models in nlmixr. ResultsMeropenem, gentamicin and ciprofloxacin two- and three-way combinations prevented regrowth, but did not when these moieties were studied alone. Gentamicin and meropenem were synergistic by decreasing ciprofloxacin IC50 and the combination effects of meropenem and gentamicin and the addition of meropenem on top of a gentamicin and ciprofloxacin combination were indifferent. ConclusionsOur findings emphasize the added value of a quinolone in the drug combination. In light of the recent move towards reduced use of quinolones, a quinolone free combination still prevented regrowth, it just did not display further synergy on IC50 and was indifferent in initial killing.

BackgroundThe combination of aztreonam (ATM) and avibactam (AVI) is an attractive option to treat infections caused by extended spectrum {beta}-lactamase plus NDM-1-producing Enterobacteriaceae. Since ATM activity was shown to be severely impacted by an increase in the inoculum size in vitro, we wondered whether ATM-AVI activity could be impaired in high-inoculum infections. MethodsWe analyzed the impact of the inoculum size on ATM-AVI activity in vitro and in a murine model of peritonitis due to susceptible E. coli CFT073-pTOPO and its isogenic derivatives producing NDM-1 (E. coli CFT073-NDM1) and CTX-M-15 plus NDM-1 (E. coli CFT073-CTXM15-NDM1). The impact of the inoculum size on bacterial morphology was studied by microscopic examination. ResultsIn vitro, at standard (105) inoculum, E. coli CFT073-CTXM15-NDM1 was resistant to ATM but susceptible to the ATM-AVI combination. At high (107) inoculum, MICs of ATM alone and of the ATM-AVI combination reached > 512 and 64 mg/L respectively, against all tested strains. ATM led to bacterial filamentation when active against the bacteria, i.e., in monotherapy or in combination with AVI against susceptible E. coli CFT073-pTOPO, and only in combination with AVI against E. coli CFT073-CTXM15-NDM1. In vivo, increase in the inoculum led to a drastic decrease in the activity of ATM alone against E. coli CFT073-pTOPO, and of ATM-AVI against E. coli CFT073-CTXM15-NDM1. ConclusionOur results suggest a high in vivo impact of the inoculum increase on the activity of ATM alone against ATM-susceptible E. coli, and of ATM-AVI against CTX-M-15 plus NDM-1 producing E. coli. Clinicians must be aware of the risk of failures when using AZT-AVI in high inoculum infections.

ObjectivesTo investigate an outbreak of Enterococcus faecium in a hospital haematology ward and uncover the mechanism of a vancomycin resistance phenotype-genotype disparity in an isolate from this outbreak. MethodsWhole genome shotgun sequencing was used for the phylogenetic analysis of E. faecium isolates (n = 39) and to identify the carriage of antibiotic resistance genes. A long-read sequencing approach was adopted to identify structural variations in the vancomycin resistance region of a vancomycin-variable E. faecium (VVE) and to uncover the resistance reversion mechanism in this isolate. RT-qPCR and RT-PCR were used to determine differences in the expression of vanRS and vanHAX among strains. ResultsThe E. faecium strains isolated in the hospital haematology ward were extensively drug resistant and highly diverse. The notable expansion of ST262 among patients was the likely driver of a VRE outbreak. A VVE isolate was identified that could rapidly revert to a vancomycin-resistant state in the presence of vancomycin. Disruption of the vanR gene in this isolate by an IS6-family element impaired its response to vancomycin. However, when the isolate was evolved to vancomycin resistance, it could constitutively express the vanHAX genes at levels up to 36,000-fold greater than the parent isolate via co-transcription with a ribosomal RNA operon. ConclusionIn this study, we report a VVE isolate that was isolated during a VRE outbreak. This strain was capable of rapidly reverting to a resistant phenotype through a novel mechanism involving integration of vanHAX downstream of a ribosomal RNA operon. During VRE outbreaks, attention should be paid to contemporaneous vancomycin-susceptible strains as these may carry silent vancomycin resistance genes that can be activated through genomic rearrangements upon exposure to vancomycin.

BackgroundClinical bedaquiline resistance predominantly involves mutations in mmpR5 (Rv0678). However, mmpR5 resistance-associated variants (RAVs) have a variable relationship with phenotypic M. tuberculosis resistance. We performed a systematic review to (1) assess the maximal sensitivity of sequencing bedaquiline resistance-associated genes and (2) evaluate the association between RAVs and phenotypic resistance, using traditional and machine-based learning techniques. MethodsWe screened public databases for articles published until October 2022. Eligible studies performed sequencing of at least mmpR5 and atpE on clinically-sourced M. tuberculosis isolates and measured bedaquiline minimum inhibitory concentrations (MICs). We performed genetic analysis for identification of phenotypic resistance and determined the association of RAVs with resistance. Machine-based learning methods were employed to define test characteristics of optimised sets of RAVs, and mmpR5 mutations were mapped to the protein structure to highlight mechanisms of resistance. ResultsEighteen eligible studies were identified, comprising 975 M. tuberculosis isolates containing [&ge;]1 potential RAV (mutation in mmpR5, atpE, atpB or pepQ), with 201 (20.6%) demonstrating phenotypic bedaquiline resistance. 84/285 (29.5%) resistant isolates had no candidate gene mutation. Sensitivity and positive predictive value of taking an any mutation approach was 69% and 14% respectively. Thirteen mutations, all in mmpR5, had a significant association with a resistant MIC (adjusted p<0.05). Gradient-boosted machine classifier models for predicting intermediate/resistant and resistant phenotypes both had receiver operator characteristic c-statistics of 0.73. Frameshift mutations clustered in the alpha 1 helix DNA binding domain, and substitutions in the alpha 2 and 3 helix hinge region and in the alpha 4 helix binding domain. DiscussionSequencing candidate genes is insufficiently sensitive to diagnose clinical bedaquiline resistance, but where identified a limited number of mutations should be assumed to be associated with resistance. Genomic tools are most likely to be effective in combination with rapid phenotypic diagnostics.

BackgroundIn high dose, cefepime causes neurotoxicity in patients with kidney injury; however, the relationship between exposure and observed neurotoxicity is not clear, and no animal model presently recapitulates the human condition. ObjectivesThis study sought to describe plasma and tissue pharmacokinetics and pharmacodynamics (PK/PD) of cefepime in rats experiencing neurotoxicity. MethodsMale Sprague-Dawley rats (n=21) received escalating cefepime total daily doses ranging from 531-1593 mg/kg body weight/day administered as a short infusion (0.5 mL/min) every 24h for 5 days. Cefepime was quantified in plasma, cerebral cortex and hippocampus via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple PK/PD models of cefepime transit between plasma and brain compartments (i.e. cerebral cortex and hippocampus) and neurotoxic response were explored using Monolix 2021R1 (LixoftPK). ResultsExposure estimation of cerebral cortex demonstrated a median (IQR) AUC0 -24 and Cmax 0 -24 of 181.8 (85.2-661.3) mg {middle dot} 24 h/liter and 13.9 (1.0-30.1) mg/L, respectively. The median cerebral cortex/blood percentage of penetration was 1.7%. Exposure estimation of hippocampus demonstrated a median (IQR) AUC0 -24 and Cmax 0 -24 of 291.4 (126.6-1091.6) mg {middle dot} 24 h/liter and 8.8 (3.4-33.4) mg/L, respectively. The median hippocampus/blood percentage of penetration was 4.5%. Rats that reached a cefepime Cmax of {square}17 mg/L in the hippocampus exhibited signs of neurotoxicity. A hippocampal cefepime concentration of 4.1 {micro}g/100 mg brain tissue best described seizure stages >1 for cefepime-induced neurotoxicty. ConclusionsA cefepime plasma AUC0 -24 of 28,000 mg*24h/L and hippocampal concentrations of 4.1 {micro}g/100 mg brain tissue may be a threshold for cefepime-induced neurotoxicity. This model provides a methodology for future interrogation of the relationship between plasma concentrations, brain tissue concentrations, and neurotoxicity.

IntroductionBuruli ulcer (BU) is a neglected tropical disease primarily affecting skin and sometimes bone. Standard therapy consists of rifampicin (RIF, once daily) plus clarithromycin (CLA, twice daily) over 8 weeks. Adding amoxicillin-clavulanate (AMX/CLV) may shorten treatment, but predicting treatment success before clinical trial implementation is challenging. AimsTo assess the probability of bacterial eradication following treatment with novel investigational BU regimens over different intervals using a mechanism-based modelling and simulation approach. MethodsIn vitro time-kill assays with RIF, CLA, and AMX/CLV alone and in combination were performed with a range of clinical isolates of Mycobacterium ulcerans. Bactericidal activity was characterized using a bacterial growth dynamics model, including an Emax function to describe the drug effect. Subsequently, clinical trial simulations were performed to evaluate drug disposition and skin penetration in a cohort of virtual subjects, taking into account interindividual variability in pharmacokinetics and pharmacodynamics (n=70/arm). Several regimens, including standard therapy and AMX/CLV-containing combinations with higher RIF doses were assessed. The probability of eradication at 4-8 weeks was assessed across strains with different susceptibility and assuming varying bacterial load at start of treatment. ResultsBeta-lactam containing combinations resulted in higher potency and maximum killing rates relative to the currently recommended regimens. Consequently, regimens containing AMX/CLV with higher RIF doses (20 mg/kg q.d. or 10 mg/kg b.i.d.) outperformed standard therapy, achieving 100% eradication within 4 weeks for baseline loads up to 1,000 CFU/mL across most isolates, except one from China. At higher loads (10,000 CFU/mL), 6 weeks were required. ConclusionsThe use of mechanism-based modelling and clinical trial simulations provides a robust translational framework for the evaluation of novel therapies for neglected diseases, such as BU. Irrespective of differences in bacterial susceptibility, adding AMX/CLV or using RIF-AMX/CLV dual therapy may reduce BU treatment from 8 to 4 weeks.

The ANDROMEDA software, based on machine learning, conformal prediction and a new physiologically-based pharmacokinetic model, was used to predict and characterize the human clinical pharmacokinetics of 30 selected modern small antibiotic compounds (investigational and marketed drugs). A majority of clinical pharmacokinetic data was missing. ANDROMEDA successfully filled this gap. Most antibiotics were predicted and measured to have limited permeability, good metabolic stability and multiple elimination pathways. According to predictions, most of the antibiotics are mainly eliminated renally and biliary and every other antibiotic is mainly eliminated via the renal route. Mean prediction errors for steady state volume of distribution, unbound fraction in plasma, renal and total clearance, oral clearance, fraction absorbed, fraction excreted renally, oral bioavailability and half-life were 1.3- to 2.3-fold. The overall median and maximum prediction errors were 1.5- and 4.8-fold, respectively, and 92 % of predictions had <3-fold error. Results are consistent with those obtained in previous validation studies and are better than with the best laboratory-based prediction methods, which validates ANDROMEDA for predictions of human clinical pharmacokinetics of modern antibiotic drugs, which to a great extent demonstrate pharmacokinetic characteristics challenging for laboratory methods (metabolic stability, limited permeability, efflux and multiple elimination pathways). Advantages with ANDROMEDA include that results are produced without the use of animals and cells and that predictions and decision-making can be done already at the design stage.

In this study we explored phenotypic resistance using traditional Kirby-Bauer methods in human and animal derived Campylobacter isolates that were concurrently resistant to both azithromycin and ciprofloxacin to an expanded panel of antimicrobials, including clindamycin, fosfomycin, ampicillin sulbactam and tigecycline. Out of 236 Campylobacter isolates, over 85% of C. jejuni and C. coli were resistant to clindamycin, over 60% were resistant to ampicillin sulbactam and over 30% to fosfomycin. Less than 2% of isolates were resistant to tigecycline and there was no observed resistance to Imipenem.