Journal of Antimicrobial Chemotherapy

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.

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.

BackgroundThe aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, its potential in the treatment of drug-resistant bloodstream infections (BSIs) has yet to be assessed. MethodsThe resistance gene annotations of 26 493 blood culture isolates were analyzed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for nine E. coli and K. pneumoniae isolates. ResultsGenotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, in comparison to 46.4% of colistin and 2.1% of apramycin resistance. Apramycin activity against methylated ribosomes was > 100-fold higher than other aminoglycosides. Frequencies of resistance were < 10-9 at 8 x MIC. Tentative epidemiological cutoffs (ECOFFs) were determined as 8 g/mL for E. coli and 4 g/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log CFU reduction in the blood and peritoneum. Two doses of 80 mg/kg, resulting in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans, resulted in complete eradication of carbapenem- and aminoglycoside-resistant bacteremias. ConclusionEncouraging coverage and potent in-vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants further consideration of apramycin as a drug candidate for the treatment and prophylaxis of BSI.

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.

ObjectivesSeveral recent studies highlight the high prevalence of resistance to multiple antibiotic classes used in current treatment regimens for neonatal sepsis and new treatment options are urgently needed. We aimed to identify potential new combination antibiotic treatment regimens by investigating the drug-resistance and genetic profiles of the most frequently isolated Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries (LMICs) in the NeoOBS study. Material and methodsGram-negative bacteria isolated from neonates with culture-confirmed sepsis from 13 clinical sites in nine countries, mainly LMICs, were analyzed. Culture-based identification was followed by whole-genome sequencing (WGS). Minimal inhibitory concentrations (MICs) for 8 antibiotics were determined for a representative subset of 108 isolates. ResultsFive bacterial species, Klebsiella pneumoniae (n=135), Acinetobacter baumannii (n=80), Escherichia coli (n=34), Serratia marcescens (n=33) and Enterobacter cloacae complex (ECC) (n=27) accounted for most Gram-negative bacterial isolates received (309/420, 74%). Extended-spectrum {beta}-lactamases (ESBL) genes mostly belonging to CTX-M-15 were found in 107 (79%) K. pneumoniae isolates and 13 (38%) E. coli, as well as in 6 (18%) and 10 (37%) S. marcescens and ECC isolates, respectively. Carbapenem resistance genes were present in 41 (30%) K. pneumoniae, while 73 (91%) of A. baumannii isolates were predicted to be MDR based on carbapenem resistance genes. Apart from A. baumannii, in which two major pandemic lineages predominated, a wide genetic diversity occurred at the intraspecies level with different MDR clones occurring at the different sites. Phenotypic testing showed resistance to the WHO first- and second- line recommended treatment regimens: 74% of K. pneumoniae isolates were resistant to gentamicin and 85% to cefotaxime; E. coli isolates showed resistance to ampicillin, gentamicin and cefotaxime in 90%, 38% and 47%, respectively. For the novel antibiotic regimens involving different combinations of flomoxef, fosfomycin and amikacin, the overall predicted MIC-determined susceptibility for Enterobacterales isolates was 71% (n=77) to flomoxef-amikacin, 76% (n=82) to flomoxef-fosfomycin and 79% (n=85) to fosfomycin-amikacin combinations, compared to 31% and 22% isolates susceptible to ampicillin-gentamicin and cefotaxime, respectively. ESBL-producing Enterobacterales isolates were 100% susceptible both to flomoxef-fosfomycin and flomoxef-amikacin and 92% to fosfomycin-amikacin. ConclusionEnterobacterales carried multiple resistance genes to cephalosporins, carbapenems and aminoglycosides. ESBL-producing K. pneumoniae and E. coli isolates were highly susceptible to the three new antibiotic combination regimens planned to be evaluated in the currently recruiting GARDP-sponsored NeoSep1 trial.

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.

BackgroundCefiderocol is a siderophore-conjugated cephalosporin antibiotic used to treat multi drug resistant Gram negative infections, including metallo-beta-lactamase producing Enterobacterales. Antimicrobial useage is guided by antimicrobial susceptibility testing (AST) which is hampered by differences between EUCAST and CLSI breakpoints, methodological challenges of AST, and lack of information on clinical outcome related to AST. ObjectivesThis study assessed the agreement between AST methods under EUCAST and CLSI breakpoints in a collection of 57 blaNDM producing Enterobacterales isolated from a UK hospital network. MethodsAll isolates, including Klebsiella pneumoniae, Enterobacter hormaechei, Escherichia coli and Citrobacter freundii, were whole-genome sequenced and tested with disk diffusion and MIC gradient test strip, and broth microdilution MICs were determined for a subset. Categorical agreement between methods was calculated using both EUCAST and CLSI breakpoints. Mutations and acquired resistance genes associated with cefiderocol resistance were identified and compared with AST results. ResultsThe disk diffusion method, based on EUCAST interpretation, classified 94.7% of isolates as cefiderocol resistant and 5.3% as susceptible, with 22.8% within the Area of Technical Uncertainty. The CLSI breakpoint classified one isolate as resistant (1.8%) and 5.26% intermediate. Category agreement of broth microdilution and disk diffusion for E. coli using EUCAST guidelines was 38.5%. Mutations associated with cefiderocol resistance were highly prevalent and varied between species. ConclusionsThe discordant EUCAST and CLSI breakpoint values provided have large impacts on the classification of isolates susceptibility to cefiderocol, which will impact global cefiderocol usage and surveillance of resistance, further complicated by poor agreement between AST methods.

BackgroundAntimicrobial resistance (AMR) in E. coli is a global problem associated with substantial morbidity and mortality. AMR-associated genes are typically annotated based on similarity to a variants in a curated reference database with an implicit assumption that uncatalogued genetic variation within these is phenotypically unimportant. In this study we evaluated the potential for discovering new AMR-associated gene families and characterising variation within existing ones to improve genotype-to-susceptibility-phenotype prediction in E. coli. MethodsWe assembled a global dataset of 9001 E. coli sequences of which 8586 had linked antibiotic susceptibility data. Raw reads were assembled using Shovill and AMR genes extracted using the NCBI AMRFinder tool. Mash was used to calculate the similarity between extracted genes using Jaccard distances. We empirically reclustered extracted gene sequences into AMR-associated gene families (70% match) and alleles (ARGs, 100% match). ResultsThe performance of the AMRFinder database for genotype-to-phenotype predictions using strict 100% identity and coverage thresholds did not meet FDA thresholds for any of the eight antibiotics evaluated. Relaxing filters to default settings improved sensitivity with a specificity cost. For all antibiotics, a small number of genes explained most resistance although a proportion could not be explained by known ARGs; this ranged from 75.1% for co-amoxiclav to 3.4% for ciprofloxacin. Only 17,177/36,637 (47%) of ARGs detected had a 100% identity and coverage match in the AMRFinder database. After empirically reclassifying genes at 100% nucleotide sequence identity, we identified 1292 unique ARGs of which 158 (12%) were present [&ge;]10 times, 374 (29%) were present 2-9 times and 760 (59%) only once. Simulated accumulation curves revealed that discovery of new (100%-match) ARGs present more than once in the dataset plateaued relatively quickly whereas new singleton ARGs were discovered even after many thousands of isolates had been included. We identified a strong correlation (Spearman coefficient 0.76 (95% CI 0.72-0.79, p<0.001)) between the number of times an ARG was observed in Oxfordshire and the number of times it was seen internationally, with ARGs that were observed 7 times in Oxfordshire always being found elsewhere. Finally, using the example of blaTEM-1, we demonstrated that uncatalogued variation, including synonymous variation, is associated with potentially important phenotypic differences (e.g. two common, uncatalogued blaTEM-1 alleles with only synonymous mutations compared to the known reference were associated with reduced resistance to co-amoxiclav [aOR 0.57, 95%CI 0.34-0.93, p=0.03] and piperacillin-tazobactam [aOR 0.54, 95%CI 0.32-0.87, p=0.01]). ConclusionsOverall we highlight substantial uncatalogued genetic variation with respect to known ARGs, although a relatively small proportion of these alleles are repeatedly observed in a large international dataset suggesting strong selection pressures. The current approach of using fuzzy matching for ARG detection, ignoring the unknown effects of uncatalogued variation, is unlikely to be acceptable for future clinical deployment. The association of synonymous mutations with potentially important phenotypic differences suggests that relying solely on amino acid-based gene detection to predict resistance is unlikely to be sufficient. Finally, the inability to explain all resistance using existing knowledge highlights the importance of new target gene discovery.

Piperacillin/tazobactam (TZP) is a widely used penicillin/{beta}-lactamase inhibitor combination with broad antimicrobial activity. Recently, Escherichia coli strains resistant to TZP but susceptible to third generation cephalosporins (TZP-R/3GC-S isolates) have been increasingly identified. Here, we investigated resistance mechanisms underlying the TZP-R/3GC-S phenotype in clinical E. coli isolates. A total of 29 TZP-R/3GC-S E. coli isolates were retrieved from urinary cultures and subjected to whole genome sequencing. Resistance to TZP was confirmed by minimum inhibitory concentration determination. {beta}-lactamase activity in the presence and absence of tazobactam was determined to identify hyperproduction of {beta}-lactamase and assess susceptibility to tazobactam inhibition. A previously unrecognized {beta}-lactamase was identified and cloned to determine its resistance profile. Four different resistance mechanisms underlying the TZP-R/3-GC phenotype were identified: 1) In 18 out of 29 isolates (62%) {beta}-lactamase production was increased and in 16 of these either strong alternative promoters or increased gene copy numbers of blaTEM-1 or blaSHV-1 were identified, 2) seven isolates (24%) produced blaOXA-1, 3) three isolates (10%) produced inhibitor-resistant TEM-{beta}-lactamases, and 4) a single isolate (3%) harboured a blaCTX-M gene as the only {beta}-lactamase present. This {beta}-lactamase, CTX-M-255, only differs from CTX-M-27 by a G239S amino acid substitution. In contrast to CTX-M-27, CTX-M-255 conferred resistance to penicillin/{beta}-lactamase inhibitor combinations but remained susceptible to cephalosporins. In conclusion, hyperproduction of blaTEM was the most prevalent mechanism of TZP-resistance underlying the TZP-R/3GC-S phenotype followed by production of blaOXA-1 and inhibitor-resistant TEM-{beta}-lactamases. Furthermore, we identified a previously unrecognized CTX-M-{beta}-lactamase, CTX-M-255 that was resistant to {beta}-lactamase inhibitors.

BackgroundThe rapid development of cefiderocol resistance poses a significant concern, particularly in Enterobacterales that produce New Delhi metallo-{beta}-lactamase (NDM). This study explores the potential of inhibiting the development of cefiderocol resistance by combining cefiderocol with aztreonam. MethodsA resistance induction experiment using 20 clinical isolates was performed to assess the impact of cefiderocol-aztreonam on preventing cefiderocol resistance development at 4x and 10x cefiderocol MIC, with and without aztreonam (2, 4, 8 {micro}g/ml). Additionally, serial passaging with doubling cefiderocol concentrations was performed with and without aztreonam. Whole genome sequencing (WGS) was performed to identify potential genetic factors associated with the phenotype. ResultsAmong the 20 E. cloacae complex isolates, 40% (8/20) exhibited a significant reduction in cefiderocol MIC ([&ge;]4-fold MIC reduction) in the presence of 4 {micro}g/ml aztreonam. Combining cefiderocol with a fixed concentration of 4 {micro}g/ml aztreonam inhibited cefiderocol resistance development in these eight isolates at an inoculum of 107 cfu/ml. Additional resistance induction experiments through serial passaging indicated a delayed emergence of cefiderocol-resistant clones when cefiderocol was combined with aztreonam. WGS analysis revealed a significant positive association between blaCTX-M-15, blaOXA-1, and other co-localized genes with a substantial MIC reduction for cefiderocol-aztreonam compared to cefiderocol alone. ConclusionOur study suggested that cefiderocol resistance development in NDM-producing E. cloacae complex can be delayed or inhibited by combining cefiderocol with aztreonam, even in the presence of multiple {beta}-lactamase genes. A MIC reduction of at least 4-fold emerges as the most reliable predictor for inhibiting resistance development with this dual {beta}-lactam combination.

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.

ObjectivesThe objective of this study was to apply an escalation antibiogram (EA) to community urine data to assess how presumptive resistance (treatment failure or recent microbiological samples) to first-line antibiotics for simple urinary tract infections (UTIs) affects resistance to antibiotics used to treat pyelonephritis. Furthermore, we examined how this varies with age or in instances of recurrent UTI. MethodsWe extracted susceptibility data from Escherichia coli isolates grown from urine samples sent from general practice from a 5-year period (2019-2023) in a region served by three NHS hospital trusts. All female patients over 18 years old were included giving a total of 130,514 isolates. We applied a Bayesian model to estimate antibiotic resistance rates for oral pyelonephritis antibiotics, when presuming resistance to each of the first-line antibiotics used to treat simple UTIs. The model estimates the probability of resistance with 95% credible intervals and was applied to a variety of patient groups based on age and history of recurrent UTIs. The uncertainty in these estimates increases for smaller patient groups. ResultsResistance to first-line UTI antibiotics has a marked effect on the probability of resistance to oral antibiotics used to treat pyelonephritis. In particular amoxicillin-clavulanate should be avoided for pyelonephritis if resistance to pivmecillinam is presumed in UTI because resistant rates may exceed 50%. For patients with presumed resistance to nitrofurantoin or trimethoprim in UTI, the optimal pyelonephritis antibiotic depends on both age group and history of past infections. For example, for patients under 50 with recurrent UTIs, amoxicillin-clavulanate has the lowest estimated resistance rate, but for women over 65 with recurrent UTIs, ciprofloxacin is optimal for pyelonephritis, where there is presumed nitrofurantoin resistance in UTI, but cefalexin is superior if trimethoprim resistance is presumed. ConclusionsEA analysis informed by our Bayesian model is a useful tool to support empiric antibiotic prescribing for pyelonephritis. It provides an estimate of local resistances rates and a comparison of antibiotic options with a measure of the uncertainty in the data.

ObjectivesLinezolid is highly valuable for treating infections caused by Gram-positive bacteria that are resistant to first-line agents. Severe infections require empirical therapy, where treatment is initiated before the pathogen and its susceptibility are identified. This evaluation aims to compare different dosing strategies for empirical dosing of linezolid in an intensive care unit (ICU) setting, with the goal of optimising pathogen eradication while minimising toxicities. MethodsThree dosing strategies were compared, all based on two previously published datasets of prospective clinical studies: (i) standard dosing, (ii) therapeutic drug monitoring (TDM) guided dosing, and (iii) a theoretical model-informed precision dosing (MIPD) guided by a population pharmacokinetic model using a Bayesian approach. Each scenario was combined with an ICU-specific minimal inhibitory concentration (MIC) distribution sampled over ten years and containing over 19,000 pathogens. The pharmacokinetic-pharmacodynamic target for pathogen eradication was defined as the minimum concentration above the MIC, and the toxicity limit was set at minimum concentrations exceeding 8 mg/L. ResultsData from 117 critically ill patients with 2184 TDM samples were available. Within the past 10 years, the rate of resistance to linezolid has increased significantly among intensive care patients, rising from 6.3% in 2015 to 11.4-15% between 2021 and 2024. Using standard dosing, only 35.7% of patients were within the target range. This number increased slightly to 46.3% when dose adaptations with TDM were possible. In contrast, MIPD projected target attainment in 70.9-81.3% of patients. ConclusionsIn the era of increasing antimicrobial resistance, particularly against linezolid, novel dosing strategies such as MIPD become urgently necessary. Our results suggest that empirical linezolid dosing based on standard regimens as well as dose adaptation based on TDM failed to achieve sufficient target concentrations in ICU patients. Dosing guided by MIPD should therefore be further explored and implemented in future clinical practice.

Cefiderocol exhibits excellent in vitro activity against Pseudomonas aeruginosa; however, resistance can emerge. We investigated the molecular mechanisms underlying cefiderocol resistance (MIC >2 mg/L) in 103 clinical strains collected from 61 hospitals (2021-2024). MICs ranged from 4 to >128 mg/L, with 39.8% of strains showing MICs >8 mg/L. Although 37.8% were classified as difficult-to-treat resistant (DTR), acquired {beta}-lactamases were detected in 72.8% of strains, including carbapenemases (39.8%), mainly NDM-1 (29.1%), and Extended Spectrum {beta}-Lactamases (ESBLs) (38.8%). Cloning of 11 {beta}-lactamases into pUCP24, including the acquired cephalosporinase PAC-1 and ESBLs (VEB-1, and VEB-9), resulted in marked increases in cefiderocol MICs (up to 128-fold). Introduction of 6 mutations in the PDC enzyme into a PAO1{Delta}blaPDC-1 background increased MICs up to 4 mg/L and conferred cross-resistance to ceftolozane/tazobactam, notably F121L, G157D, T70I, and E219K. Alterations in siderophore transporters or regulators were identified in 38.8% of strains, most frequently a PirR frameshift (R132fs), consistent with PirR inactivation, which was confirmed in the PAO1 strain to contribute to cefiderocol resistance. Overall, cefiderocol resistance in clinical strains is multifactorial, mainly involving acquired {beta}-lactamases (ESBLs, carbapenemases) and impaired siderophore uptake (PiuA/PiuD, PirA, PiuC), leading to high-level resistance (>8 mg/L). The polyclonal distribution and diversity of mechanisms highlight the need for routine susceptibility testing and surveillance. Detection of NDM producers is critical, as cefiderocol should be used with caution in this context.

Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed animal populations. We conducted whole genome sequencing analysis of 138 representative 3GC-R isolates previously collected from dairy farms in South West England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates: encoding CTX-M-1, -14, -15, -32 and the novel variant, CTX-M-214), blaCMY-2 (6 isolates) and blaDHA-1 (one isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ~220 kb IncHI2 plasmid carrying blaCTX-M-32 was sequenced to closure and designated pMOO-32. It was found experimentally to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure and was found by multiplex PCR to be present on 26 study farms representing a remarkable range of transmission over 1500 square kilometres. However, the plasmid was not found amongst human urinary E. coli we have recently characterised from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two blaCTX-M plasmids circulating amongst eight human and two cattle isolates, and a closely related blaCMY-2 plasmid found in one cattle and one human isolate. However, phylogenetic evidence of recent sharing of 3GC-R strains between farms and humans in the same region was not found. ImportanceThird-generation cephalosporins (3GCs) are critically important antibacterials and 3GC-resistance (3GC-R) threatens human health, particularly in the context of opportunistic pathogens such as Escherichia coli. There is some evidence for zoonotic transmission of 3GC-R E. coli through food, but little work has been done examining possible transmission (e.g. via interaction of people with the local near-farm environment). We characterised acquired 3GC-R E. coli found on dairy farms in a geographically restricted region of the United Kingdom and compared these with E. coli from people living in the same region, collected in parallel. Whilst there is strong evidence for recent farm-to-farm transmission of 3GC-R strains and plasmids - including one epidemic plasmid that has a remarkable capacity to transmit - there was no evidence that 3GC-R found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population.

BackgroundNew therapeutical strategies are urgently needed against multidrug-resistant (MDR) Enterobacterales. Azithromycin is a widely prescribed antibiotic with additional immunomodulatory properties, but traditionally underused for the treatment of enterobacterial infections. We previously identified azithromycin as a potent enhancer of colistin, fosfomycin and tigecycline against Klebsiella pneumoniae ATCC 13883. ObjectivesThe aim of this work was to evaluate the antibacterial in vitro activity of azithromycin-based combinations with last-line antibiotics against an expanded panel of MDR/XDR K. pneumoniae isolates. MethodsTime-kill assays of azithromycin alone and in pair-wise combinations with fosfomycin, colistin and tigecycline were performed against a collection of 12 MDR/XDR K. pneumoniae isolates. Synergistic and bactericidal activities of azithromycin-based combinations were analyzed after 8, 24 and 48 hours of treatment, and compared with antimicrobial combinations frequently used in the clinic for the treatment of MDR Enterobacterales. ResultsSynergistic interactions were detected in 100% (12/12) for azithromycin/fosfomycin, 58.3% (7/12) for azithromycin/colistin and 75% (9/12) for azithromycin/tigecycline of the strains, showing potent killing activities. Clinical combinations currently used in the clinic showed synergy in 41.6% (5/12) for meropenem/ertapenem, 33.33% (4/12) for meropenem/colistin, 75% (9/12) for fosfomycin/colistin and 66.6% (8/12) for fosfomycin/tigecycline of the strains, with lower bactericidal efficacy. ConclusionsNovel azithromycin-based combinations with last-line MDR/XDR K. pneumoniae antibiotics were identified showing in vitro capacity to eradicate MDR/XDR K. pneumoniae. Our results provide an in vitro basis supporting azithromycin used in combinatorial treatment for MDR-related infections.

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.

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.

This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The {beta}-lactam/{beta}-lactamase inhibitor combinations are widely used to circumvent {beta}-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile--KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital--were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a frameshift mutation in KpMVR1s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summaryIllumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institutes database for K. pneumoniae species complex (bigsdb.pasteur.fr/klebsiella/) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statementThis is the first blaKPC-positive K. pneumoniae isolate referred to the UKs national reference laboratory with resistance to three last-resort {beta}-lactam/{beta}-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne blaKPC-2-like allele blaKPC-157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an ISEc68-induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission.

End 2023, the UK Health Security Agency sent an alert about a new hypervirulent Clostridioides difficile PCR ribotype, ribotype 955 (RT955), causing slowly progressing infection clusters in hospitals in the Midlands. Between March 2018 and February 2022, CDI surveillance was performed in southern Serbia with centers providing medical services for approximately 750,000 inhabitants. Using the ECDC recommended protocol, clinical, epidemiological and microbiological data were collected. C. difficile RT955 was identified in 27 (7%) of 383 surveyed patients with CDI. Of 27 patients, 16 (59%) was older than 60 years and 19 (70%) were male. CDI was always associated with previous antibiotic therapy and had a hospital onset in 23 (85%) patients. The clinical presentaiton was milder than reported in UK and at 90 days follow up, no CDI related mortality was found. All sequenced strains belonged to multilocus sequence type (ST) 1 and were highly similar, with 0-1 alleles differences in a cgMLST analysis. The strains differed clearly from the UK RT955 outbreak strain by whole genome sequencing and phenotypic resistance to lincosamides and rifampicin. Interestingly, a high level erythromycin resistance was observed without a genetic resistance determinant. Both the UK and Serbian RT955 strains contained gyrA_p.T82I associated with resistance to fluoroquinolone antimicrobials and carried the PnimBG promoter mutation, suggestive for haem-dependent metronidazole resistance. We conclude that C. difficile RT955 is present in Serbia since 2018, without presentation of large outbreaks. The Serbian RT955 strains differed clearly from a representative UK cluster strain, but shared its haem dependent metronidazole resistance.