MIC Distributions Research Articles

Macrolide resistance is pervasive in oral streptococci in the Belgian general population: a cross-sectional survey.

Background. Commensal streptococci are common inhabitants of the oral microbiome and regulate its structure and function in beneficial ways for human health. They can, however, also be opportunistic pathogens and act as a reservoir of resistance genes that can be passed on to other bacteria, including pathogens. Little is known about the prevalence of these commensals in parents and their children and their antimicrobial susceptibilities in the Belgian general population.Gap Statement. The macrolide susceptibility of commensal oral Streptococci in Belgium is unknown.Methods. We assessed the prevalence and azithromycin susceptibility of commensal streptococcal species in the parents (n=38) and children (n=50) of 35 families in Belgium.Results. The most frequently detected taxonomic grouping was Streptococcus mitis/oralis, which was detected in 78/181 (43.1%) of the children's isolates and 66/128 (51.6%) of the parents' isolates. Of the 311 isolates collected in this study, 282 isolates (90.7%) had an azithromycin MIC value greater than the breakpoint of 0.25 mg l-1 and 146 isolates (46.9%) had azithromycin MICs greater than 2 mg l-1. There was no difference in the azithromycin MIC distribution of all streptococcal isolates between children and parents. All individuals were colonized by streptococci with azithromycin MICs greater than 0.25 mg l-1, and 87.5% of individuals had streptococci with MICs greater than 2 mg l-1.Interpretation. The most prevalent species identified in both age groups was S. mitis/oralis. All individuals harboured streptococci with macrolide resistance. This highlights the need for additional antimicrobial stewardship initiatives to reduce the consumption of macrolides in the general population.

Assessing the Efficacy of Chlorhexidine in Combating Most Important Causative Agents of Fungal Keratitis: An in Vitro Comparative Study With Seven Antifungal Agents

Purpose Fungal keratitis (FK) is a difficult condition to treat, particularly in underdeveloped nations. The study aimed to compare the in vitro activity of chlorhexidine (CHX) and seven antifungal agents against a collection of fungi recovered from FK patients. Methods Seventy-three fungi were collected from patients with FK included in study. The antifungal agents tested were fluconazole, voriconazole, posaconazole, miconazole, natamycin, amphotericin B, and caspofungin. The concentration range for CHX was 1–1024 μg/mL. Assessments of antifungal susceptibility were conducted using the EUCAST broth microdilution reference method. Results The findings demonstrated the beneficial in vitro inhibition of filamentous and yeast fungi by CHX. CHX demonstrated efficacy against Fusarium spp., Aspergillus spp., Candida spp., S. apiospermum and dematiceous fungi at concentrations of 4–64, 32–64, 4–32, 8, and 4–16 µg/mL respectively. The median MICs and MIC distributions of CHX showed no significant differences among the evaluated spp. (p > 0.05). The most effective antifungal drug was posaconazole, which was followed by voriconazole, caspofungin, and amphotericin B. Conclusion In situations where access to a range of antifungal medications and microbiological facilities is limited, CHX should be further investigated as a potential treatment for FK. It might be able to treat the condition as an inexpensive topical treatment.

Wild-type MIC distributions and epidemiological cutoff values for 5-flucytosine and Candida species as determined by EUCAST broth microdilution.

EUCAST has established clinical breakpoints and epidemiological cutoff values (ECOFFs) for Candida spp. However, limited data are available for 5-flucytosine (5-FC). We assessed the in vitro susceptibility of 5-FC against a large collection of clinical Candida species using EUCAST methodology and determined the associated ECOFFs. A total of 5622 Candida isolates were collected from patients across the Netherlands between 2008 and 2024. 5-FC MICs were determined using the EUCAST microbroth dilution reference method. Furthermore, MICs were extracted from the EUCAST website. The MICs from this study and those extracted were used to determine ECOFFs and local ECOFFs (L-ECOFFs). 5-FC exhibited potent in vitro activity against C. albicans, N. glabratus and C. parapsilosis, while decreased susceptibility was observed for C. tropicalis, Pichia species, K. marxianus, Y. lipolytica, and C. auris. The ECOFFs (mg/L) and the percentages of WT isolates for 5-FC were: C. albicans: 0.5 (97.2%), N. glabratus: 0.5 (96.6%), C. parapsilosis: 0.5 (99.5%) and P. kudriavzevii: 8 (99.4%). The L-ECOFF (mg/L) and the percentages of WT isolates for 5-FC were: C. dubliniensis: 0.25 (96.8%), C. tropicalis: 0.25 (67.2%), K. marxianus: 0.25 (48.0%), C. lusitaniae: 0.25 (86.5%), M. guillermondii: 0.125 (95.9%) and P. norvegiensis: 8 (94.2%). 5-FC remains a valuable drug to manage difficult-to-treat invasive Candida infections. In vitro susceptibility cannot be predicted based on species identification for most Candida species, but requires MIC-testing. ECOFFs will help to interpret the MICs to support treatment decisions.

Assessment of pharmacokinetics-pharmacodynamics to support omadacycline dosing regimens for the treatment of patients with acute bacterial skin and skin structure infections.

Pharmacokinetic-pharmacodynamic (PK-PD) relationships for efficacy were evaluated using data from omadacycline-treated patients with acute bacterial skin and skin structure infections (ABSSSI) enrolled in two phase 3 studies. Patients received omadacycline 100 mg intravenously (IV) every 12 hours for two doses, followed by 100 mg IV every 24 hours (q24h), with the option to switch to 300 mg oral (PO) q24h after 3 days or 450 mg PO q24h for two doses, followed by 300 mg PO q24h for a total duration of 7-14 days. Clinical response was evaluated at 48-72 hours [early clinical response (ECR)], end of treatment (EOT), and 7-14 days after EOT. Using a population pharmacokinetic (PK) model and PK data from patients with Staphylococcus aureus at baseline, omadacycline free-drug plasma area under the concentration-time curve (AUC) values were determined, and the relationships between free-drug plasma AUC:MIC ratio and dichotomous efficacy endpoints were evaluated. Using these relationships, the population PK model, simulation, and an omadacycline MIC distribution for S. aureus, mean percent probabilities of response were evaluated. Statistically significant PK--PD relationships were identified for ECR (P = 0.016 and 0.013 for optimized two- and three-group free-drug plasma AUC:MIC ratios, respectively). At an MIC value of 0.5 µg/mL, percent probabilities of model-predicted success for ECR based on the univariable PK-PD relationships using continuous and two-group free-drug plasma AUC:MIC ratio variables were 91.9 and 95.6%, respectively, for the IV-to-PO dosing regimen and 89.3 and 88.4%, respectively, for the PO-only dosing regimen. These data support for omadacycline IV-to-PO and PO-only dosing regimens for ABSSSI and an omadacycline susceptibility breakpoint of 0.5 µg/mL for S. aureus.

Pharmacokinetics and pharmacodynamics of two in-feed chlortetracycline regimens provided to beef cattle.

Plasma chlortetracycline (CTC) concentration data were subjected to Monte Carlo simulation of area under the concentration curve (AUC) values related to bovine respiratory disease pathogen MIC distributions to evaluate target attainment rates. Crossbred Hereford heifers were randomly assigned into two treatment groups. Treatment group (A) received chlortetracycline (CTC) at a target dose of 22 mg/kg of bodyweight daily for 5 consecutive days (n = 8) and group (B) received CTC at 350 mg/head per day (1.5 ± 0.2 mg/kg based on actual bodyweights) for seven consecutive days (n = 8). Non-compartmental analysis was used to calculate plasma-free drug CTC area under the concentration curves. The mean observed (±SD) free drug AUC values were 4.18 (±1.72) μg × h/mL and 0.30 (±0.06) μg × h/mL for treatment groups A and B, respectively. The probability of target attainment for AUC24/MIC values of 25 and 12.5 was modeled using Monte Carlo simulations. Treatment group A achieved >90% target attainment (AUC24/MIC of 25) at an MIC of 0.06 μg/mL, whereas treatment group B displayed only 12.6% target attainment (AUC24/MIC of 12.5) at the lowest MIC evaluated (0.015 μg/mL). Both in-feed CTC regimens failed to obtain a reasonable target attainment rate in light of expected MIC distributions of potential pathogens.

Twenty Years in EUCAST Anti-Fungal Susceptibility Testing: Progress & Remaining Challenges.

Since its inception in 2002, the EUCAST Antifungal Susceptibility Testing Subcommittee (AFST) has developed and refined susceptibility testing methods for yeast, moulds and dermatophytes, and established epidemiological cut-off values and breakpoints for antifungals. For yeast, three challenges have been addressed. Interpretation of trailing growth in fluconazole susceptibility testing, which has been proven without impact on efficacy if below the 50% endpoint. Variability in rezafungin MIC testing due to laboratory conditions, which has been solved by the addition of Tween 20 to the growth medium in E.Def 7.4. And third, interpretation of MICs for rare yeast with no breakpoints, where recommendations have been established for MIC-based clinical advice. For moulds, refinements include the validation of spectrophotometer reading for A. fumigatus to facilitate objective MIC determination, and for dermatophytes the establishment of a microdilution method with automated reading and a selective medium to minimise the risk of contaminations. Recent initiatives involve development and validation of agar-based screening assays for detection of potential azole and echinocandin resistance in A. fumigatus and Aspergillus species, respectively, and of terbinafine resistance in Trichophyton species. Moreover, the development of a EUCAST guidance document for molecular resistance testing represents an advancement, particularly for identifying target gene alterations associated with resistance. In summary, EUCAST AFST continues to play a pivotal role in standardizing AFST and facilitating accurate interpretation of susceptibility data for clinical decision-making. Adoption of EUCAST breakpoints for commercial test methods, however, requires thorough validation to ensure concordance with EUCAST reference testing species-specific MIC distributions.

Adequacy of the Dosing and Infusion Time of Ceftazidime/Avibactam for the Treatment of Gram-Negative Bacterial Infections: A PK/PD Simulation Study.

Recent studies suggested the potential benefits of extended infusion times to optimize the treatment efficacy of ceftazidime/avibactam, which indicated that the current pharmacokinetic/pharmacodynamic (PK/PD) target may not be sufficient, especially for severe infections. The purpose of this study is to assess the adequacy of dosing strategies and infusion durations of ceftazidime/avibactam when applying higher PK/PD targets. This study utilized published PK parameters to conduct Monte Carlo simulations. Different dosages including the recommended regimen based on renal function were simulated and evaluated by the probability of target attainment (PTA) and cumulative fraction of response (CFR). Different PK/PD targets were set for ceftazidime and avibactam. MIC distributions from various sources were used to calculate the CFR. Multiple PK/PD targets have been set in this study, All recommended dosage could easily achieve the target of 50%fT ≥ MIC (ceftazidime) and 50%fT ≥ CT=1.0 mg/L (avibactam). However, for severe infection patients with normal renal function and augmented renal clearance at the recommended dosage (2000 mg/500 mg, every 8 hours), the infusion duration needs to be extended to 3 hours and 4 hours to achieve the targets of 100%fT ≥ MIC and 100%fT ≥ CT=1.0 mg/L. Only continuous infusion at higher dosages achieved 100%fT ≥ 4×MIC and 100%fT ≥ CT=4.0 mg/L targets to all currently recommended regimens. According to the varying MIC distributions, higher concentrations are needed for Pseudomonas aeruginosa, with the attainment rates vary across different regions. The current recommended dosing regimen of ceftazidime/avibactam is insufficient for severe infection patients, and continuous infusion is suggested.

Optimizing Antibiotic Therapy for Stenotrophomonas maltophilia Infections in Critically Ill Patients: A Pharmacokinetic/Pharmacodynamic Approach.

Stenotrophomonas maltophilia is an opportunistic, multidrug-resistant non-fermentative Gram-negative bacillus, posing a significant challenge in clinical treatment due to its numerous intrinsic and acquired resistance mechanisms. This study aimed to evaluate the adequacy of antibiotics used for the treatment of S. maltophilia infections in critically ill patients using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The antibiotics studied included cotrimoxazole, levofloxacin, minocycline, tigecycline, cefiderocol, and the new combination aztreonam/avibactam, which is not yet approved. By Monte Carlo simulations, the probability of target attainment (PTA), the PK/PD breakpoints, and the cumulative fraction of response (CFR) were estimated. PK parameters and MIC distributions were sourced from the literature, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the SENTRY Antimicrobial Surveillance Program collection. Cefiderocol 2 g q8h, minocycline 200 mg q12h, tigecycline 100 mg q12h, and aztreonam/avibactam 1500/500 mg q6h were the best options to treat empirically infections due to S. maltophilia. Cotrimoxazole provided a higher probability of treatment success for the U.S. isolates than for European isolates. For all antibiotics, discrepancies between the PK/PD breakpoints and the clinical breakpoints defined by EUCAST (or the ECOFF) and CLSI were detected.

Population pharmacokinetic/pharmacodynamic target attainment analysis of IV fosfomycin for the treatment of MDR Gram-negative bacterial infections.

IV fosfomycin is used against MDR Gram-negative bacilli (GNB) but has dose-limiting side effects, especially in patients with impaired kidney function. To determine the optimal dosage of IV fosfomycin for patients with varying degrees of kidney function. Adult patients receiving IV fosfomycin for treatment of GNB were eligible. Five serial blood samples were collected after at least three doses of fosfomycin; plasma was assayed by LC-MS/MS and modelled by population pharmacokinetic analysis. The PTA for AUC24/MIC of 98.9 for Escherichia coli and Klebsiella pneumoniae, and 40.8 for Pseudomonas aeruginosa were computed by Monte Carlo simulations. Cumulative fractions of response (CFR) were analysed for each pathogen using EUCAST MIC distributions. A total of 24 patients were included. Creatinine clearance (CLCR) and gender significantly influenced fosfomycin clearance. The kidney function-adjusted dosing regimens are proposed by using the lowest dose that can achieve ≥90% PTA for AUC24/MIC of 98.9 at an MIC of ≤32 mg/L (EUCAST v.13 susceptibility breakpoint for Enterobacterales). For patients with normal kidney function (CLCR 91-120 mL/min), a dosage of 15 g/day is suggested. This regimen achieved 97.1% CFR against E. coli, whereas CFR was 72.9% for K. pneumoniae and 76.7% for P. aeruginosa. A fosfomycin dosage of 15 g/day with adjustment according to kidney function provided high PTA and CFR when treating E. coli. This dosage is lower than that used in current practice and may improve tolerability. Higher dosages may be needed for P. aeruginosa; however, safety data are limited.

45 years of tetracycline post exposure prophylaxis for STIs and the risk of tetracycline resistance: a systematic review and meta-analysis

There is considerable interest in the use of doxycycline post exposure prophylaxis (PEP) to reduce the incidence of bacterial sexually transmitted infections (STIs). An important concern is that this could select for tetracycline resistance in these STIs and other species. We searched PubMed and Google Scholar, (1948–2023) for randomized controlled trials comparing tetracycline PEP with non-tetracycline controls. The primary outcome was antimicrobial resistance (AMR) to tetracyclines in all bacterial species with available data. Our search yielded 140 studies, of which three met the inclusion criteria. Tetracycline PEP was associated with an increasedprevalence of tetracycline resistance in Neisseria gonorrhoeae, but this effect was not statistically significant (Pooled OR 2.3, 95% CI 0.9-3.4). PEP had a marked effect on the N. gonorrhoeae tetracycline MIC distribution in the one study where this was assessed. Prophylactic efficacy was 100% at low MICs and 0% at high MICs. In the one study where this was assessed, PEP resulted in a significant increase in tetracycline resistance in commensal Neisseria species compared to the control group (OR 2.9, 95% CI 1.5-5.5) but no significant effect on the prevalence of tetracycline resistance in Staphylococcus aureus. The available evidence suggests that PEP with tetracyclines could be associated with selecting tetracycline resistance in N. gonorrhoeae and commensal Neisseria species.

The MIC distribution of dalbavancin differs between different coagulase-negative staphylococci.

CoNS constitute a significant part of the human microbiota of skin and mucous membranes. They can cause nosocomial infections, and have shown decreased susceptibility to several antibiotics. The few remaining treatment options include (lipo)glycopeptides such as dalbavancin. However, there is a lack of knowledge concerning whether susceptibility to lipoglycopeptides varies between different species of CoNS. To determine the susceptibility to dalbavancin in different species of CoNS. We investigated 480 bacterial isolates from 10 CoNS species: Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus pettenkoferi, Staphylococcus hominis, Staphylococcus sciuri and Staphylococcus simulans. The isolates were randomly selected from different sources of infection, including blood isolates, as well as deep and superficial infections. Antibiotic susceptibility was tested with the gradient test method. There was a statistically significant difference (ANOVA; P < 0.0001) in the MIC distribution for dalbavancin between different CoNS species. S. sciuri was the least susceptible species, with 90% of the isolates having an MIC value for dalbavancin above the EUCAST breakpoint of 0.125 mg/L. The lowest MIC90 values were seen for S. capitis, S. simulans and S. caprae (all 0.032 mg/L). This study demonstrated a difference in dalbavancin susceptibility between different CoNS species, suggesting that species-specific breakpoints for CoNS should be further investigated.

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45–55%) compared to the Norwegian isolates (10–13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Target attainment of intravenous lefamulin for treatment of acute bacterial skin and skin structure infections.

Lefamulin is a pleuromutilin antibiotic approved for the treatment of community-acquired bacterial pneumonia (CABP). Its spectrum of activity, good penetration into soft tissues and low rates of cross-resistance also make lefamulin a potentially valuable option for treatment of acute bacterial skin and skin structure infections (ABSSSIs). A Phase 2 trial of lefamulin for ABSSSI indicated similar efficacy of 100 and 150 mg q12h IV dosing regimens. In the present study, the potential of lefamulin for this indication was further evaluated from a translational pharmacokinetic/pharmacodynamic perspective. PTA was determined for various dosages using Monte Carlo simulations of a population pharmacokinetic model of lefamulin in ABSSSI patients and preclinical exposure targets associated with bacteriostasis and a 1-log reduction in bacterial count. Overall target attainment against MSSA and MRSA was calculated using lefamulin MIC distributions. Overall attainment of the bacteriostasis target was 94% against MSSA and 84% against MRSA for the IV dosage approved for CABP (150 mg q12h). Using the same target, for the 100 mg q12h regimen, overall target attainment dropped to 68% against MSSA and 50% against MRSA. Using the 1-log reduction target, overall target attainment for both regimens was <40%. Lefamulin at the currently approved IV dosage covers most Staphylococcus aureus isolates when targeting drug exposure associated with bacteriostasis, suggesting potential of lefamulin for the treatment of ABSSSIs. Lefamulin may not be appropriate in ABSSSI when rapid bactericidal activity is warranted.

Vibrio species: development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints.

Most human infections caused by Vibrio spp. do not warrant antimicrobial treatment but in severe cases, targeted antimicrobial treatment can be lifesaving. For Vibrio spp., standardized antimicrobial susceptibility testing (AST) guidelines with EUCAST methodology are lacking. In this study, we aimed to produce data suitable for EUCAST to establish clinical MIC breakpoints and zone diameter correlates for Vibrio spp. An intercontinental collection (N = 524) comprising five important Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. parahaemolyticus and V. vulnificus) was organized. All isolates were subjected to broth microdilution (BMD) against 11 antimicrobial agents according to ISO 20776-1 using unsupplemented Mueller-Hinton broth on freeze-dried Sensititre panels (Thermo Scientific, UK), and most isolates (n = 371) were also tested with disc diffusion according to EUCAST methodology for non-fastidious organisms. Aggregated results were used to generate MIC and zone diameter distributions and to prepare graphs of MIC-zone diameter correlation. Based on these results, the EUCAST Steering Committee determined clinical susceptible (S) and resistant (R) MIC (mg/L) breakpoints (S≤/R>) for the five Vibrio spp. for piperacillin/tazobactam (1/1), cefotaxime (0.25/0.25), ceftazidime (1/1), meropenem (0.5/0.5), ciprofloxacin (0.25/0.25), levofloxacin (0.25/0.25), azithromycin (4/4), doxycycline (0.5/0.5) and trimethoprim/sulfamethoxazole (0.25/0.25). The corresponding zone diameter breakpoints were identified. We demonstrated the validity of using standard BMD and EUCAST disc diffusion methodology for AST of five Vibrio spp., and generated suitable data to allow EUCAST to determine clinical MIC and zone diameter breakpoints for five pathogenic Vibrio spp., including both non-toxigenic and toxigenic V. cholerae.

In vitro activity of cefiderocol against a global collection of carbapenem-resistant Pseudomonas aeruginosa with a high level of carbapenemase diversity.

To determine the in vitro activity of cefiderocol in a global collection of carbapenem-resistant Pseudomonas aeruginosa including >200 carbapenemase-producing isolates. Isolates (n = 806) from the ERACE-PA Surveillance Program were assessed. Broth microdilution MICs were determined for cefiderocol (iron-depleted CAMHB) and comparators (CAMHB). Susceptibility was interpreted by CLSI and EUCAST breakpoints and reported as percent of isolates. The MIC distribution of cefiderocol in the entire cohort and by carbapenemase status was assessed. In the entire cohort, cefiderocol was the most active agent (CLSI 98% susceptible; EUCAST 95% susceptible; MIC50/90, 0.25/2 mg/L). Amikacin (urinary only breakpoint) was the second most active, with 70% of isolates testing as susceptible. The percentage of isolates susceptible to all other agents was low (<50%) including meropenem/vaborbactam, imipenem/relebactam, piperacillin/tazobactam and levofloxacin. Cefiderocol maintained significant activity against the most commonly encountered carbapenemases including VIM- (CLSI 97% susceptible; EUCAST 92% susceptible) and GES (CLSI 100% susceptible; EUCAST 97% susceptible)-harbouring isolates. The cefiderocol MIC distribution was similar regardless of carbapenemase status, with MIC50/90 values of 0.5/4 mg/L, 0.5/2 mg/L and 0.25/1 mg/L for MBL, serine carbapenemase and molecular carbapenemase-negative isolates, respectively. Cefiderocol displayed potent in vitro activity in this global cohort of carbapenem-resistant P. aeruginosa including >200 carbapenemase-harbouring isolates. Cefiderocol was highly active against MBL-producing isolates, where treatment options are limited. These data can help guide empirical therapy guidelines based on local prevalence of carbapenemase-producing P. aeruginosa or in response to rapid molecular diagnostics.

829. 5-Flucytosine Longitudinal Antifungal Susceptibility Testing of Cryptococcus neoformans: A Sub-study of the EnACT Trial Testing Oral Amphotericin

Abstract Background The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B for treatment of cryptococcal meningitis. Oral LNC amphotericin B applies nanotechnology, which hopes to improve intracellular drug delivery to affected tissues while reducing extracellular concentrations and thereby adverse events. When flucytosine (5-FC) is used as monotherapy, it can induce stable, highly resistant mutants of Cryptococcus. Since LNC amphotericin is a novel drug delivery mechanism, we assessed whether resistance to 5-FC develops in this context. Results from the ACTA and AMBITION Clinical Trials 10-week mortality by antifungal regimen for cryptococcal meningitis from the clinical trial by Molloy et al. (ACTA) in top section and Jarvis et al. (AMBITION) in bottom section. Mean survival for each group is represented by diamonds and 95% confidence interval represented by error bars. Encapsulation of Amphotericin B in a Lipid nanocrystal Particle Lipid nanocrystal particle encapsulates amphotericin B molecule and delivers the drug to antigen presenting cells. Methods The trial enrolled subjects with HIV who were diagnosed with cryptococcal meningitis and randomized to receive 5-FC and either standard IV amphotericin or LNC amphotericin. Subjects had a lumbar puncture (LP) performed on screening and days 3, 7, and 14. We used broth microdilution methods, standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to assess the MIC of isolates in each arm of the trial. We tested cryptococcal isolates from the cerebrospinal fluid (CSF) on the day of screening and then on the last positive CSF to contain cryptococcal growth. Statistical analysis included chi-square to test for a difference in MIC between the control and LNC amphotericin groups; and linear regression to compare MIC with early fungicidal activity (EFA), which is a surrogate endpoint. Intervention and Control Gropus for EnACT Phase 2 The EnACT trial phase 2 was completed in 4 cohorts to determine the ideal combination of IV and LNC amphotericin. The MIC analysis was completed in the 4th cohort. In the 4th cohort, participants randomized to the intervention group started LNC amphotericin B from the day of enrollment. Broth Microdilution for 5-Flucytosine Susceptibility Testing A 96-well plate was prepared with serial dilutions of 5-FC ranging from 256 µg/mL to 0.5 µg/mL in columns 1-10. Columns 11 and 12 were drug-free controls. The first and last CSF isolate from each unique participant was inoculated into rows B-D and E-G, respectively. The first row is a negative control and the last row is a positive control. Results The MIC50 was 4 µg/mL and MIC90 was 8 µg/mL for both the control and LNC amphotericin B groups. There was no evidence of 5-FC resistance in any Cryptococcus isolate tested after 2 weeks of therapy. 73% (n=8) of participants in the control group and 73% (n=19) in the LNC amphotericin group had no change or a decrease in MIC from the first to last Cryptococcus isolate. 27% (n=3) in the control group and 19% (n=5) in the LNC amphotericin group had a 2-fold increase in MIC. 9% (n=2) in the LNC amphotericin group had a 4-fold increase in MIC. There was no association with MIC and EFA. 5-FC CSF levels were above the MIC50. Distribution of MIC for flucytosine The MIC for each CSF isolate in the control and oral amphotericin B arms of the trial with the percents of each MIC level in each trial arm. Chi-squared statistic testing for a difference in MIC between each trial arm. Early Fungicidal Activity Compared to MIC for Each Trial Arm The EFA is compared to MIC in the control and oral amphotericin groups. Linear regression comparing EFA and control group shows that there is no correlation between MIC and EFA. Change in MIC During Treatment with Flucytosine and Amphotericin B A) Percent of participants in each trial group who had an increase in MIC over the treatment course. B) Individual participants are represented by a line with the MIC represented by a dot on the end of each line. Conclusion There is no evidence of baseline resistance to 5-FC or incident resistance during therapy in individuals presenting with cryptococcal meningitis in Uganda. LNC amphotericin B can safely be used in combination with 5-FC. Disclosures All Authors: No reported disclosures

2108. In-vitro Activity of Five Novel Anti-Fungal Compounds Against Thirty-Two Strains of Talaromyces marneffei

Abstract Background Talaromyces marneffei (Tm) is a thermally dimorphic fungus causing invasive mycosis in human immunodeficiency virus (HIV) infected patients in Southeast Asia and is a leading cause of AIDS-related deaths in this region with mortality on anti-fungal therapy as high as 30%. The limited options, routes of administration, and toxicity of antifungals with established efficacy against Tm calls for assessment of efficacy for additional antifungals against Tm. Here we evaluate the in-vitro efficacy of 5 novel anti-fungal agents against 32 clinical strains of Tm. The agents tested include ibrexafungerp, two manogepix compounds (APX001A and APX2039), olorofim, and oteseconazole. Methods 32 Tm isolates were cultured from clinical samples from patients in Vietnam and identified as Tm by standard morphological characteristics. Inocula were prepared from these clinical samples in conjunction with CLSI standards and plated onto 96-well microplates. Concentrations for each drug ranged from 0.008 µg/mL to 4 µg/mL. AlamarBlueTM, a resazurin-based cell viability indicator dye, was added to the drug-inoculum solution. Use of this reagent enabled utilization of a microplate fluorescence intensity(FI) reader to objectively identify the designated MIC endpoint of 50% inhibition of growth. Methods (Figure 1). MIC endpoint determination using AlamarBlue indicator dye method. Clinical Tm isolates were thawed and plated on Yeast Peptone Dextrose (YPD) agar and grown for 3-5 days at 37°C. Isolates were then sub-cultured on fresh YPD agar and allowed to grow for 3 days prior to inoculum preparation. Inocula were prepared according to CLSI standards. Following suspension in solution, inocula were diluted in PBS until an optical density (OD) of 0.5 to 0.6 was achieved. Inocula were further diluted in RPMI + MOPS and added to the drug-dilution microplates to achieve a final concentration of 1000-5000 cells/mL. After incubation at 37°C for 24 hours, AlamarBlue was added to all wells. Following incubation for an additional 48 hours, fluorescence intensity (FI) was measured using a microplate reader to determine the MIC endpoint of 50% inhibition of fungal growth. Results Of the drugs tested, olorofim showed the highest activity against the 32 isolates, with MICs consistently ≤ 0.008 µg/mL. This was followed by oteseconazole with MICs between ≤ 0.008 and 0.031 µg/mL APX001A with MICs between 0.031 and 0.25 µg/mL and APX2039 with MICs between 0.25 and 4 µg/mL. Ibrexafungerp had the broadest MIC distribution with MICs between 0.063 and 4 µg/mL. Figure 2/Table 1. MIC Endpoint Distribution. Figure 2 shows a scatter plot of MIC endpoints for each drug. Error bars demonstrate the geometric mean and 95% confidence intervals. Table 1 illustrates the MIC endpoint distribution in a graded heat map format. In addition, the absolute values of the geometric mean (GM), mode, MIC50, and MIC90 are displayed at the right side of the table. Both Figure 2 and Table 1 illustrate high efficacy for olorofim and oteseconazole, with low MIC endpoints consistent across the 32 isolates. The manogepix compounds(APX001A and APX2039) demonstrate surprisingly contrasted performance against Tm, despite the similar chemical structure and identical mechanism of action of the drugs. APX001A had significantly lower MIC endpoints compared to APX2039. Ibrexafungerp had the broadest range of MIC endpoints from 0.063 to 4µg/mL and the highest MIC50 and MIC90 of all the drugs. However, a significant proportion of isolates were highly susceptible to the drug. Conclusion In-vitro activity of the 5 drugs tested against the 32 Tm strains was variable, but all should be considered potential candidates for treating Tm infection. To further evaluate the potential role of each drug in treating Tm infection, the next step should be to evaluate each in in-vivo models. Disclosures All Authors: No reported disclosures

Individual meropenem epithelial lining fluid and plasma PK/PD target attainment.

It is unclear whether plasma is a reliable surrogate for target attainment in the epithelial lining fluid (ELF). The objective of this study was to characterize meropenem target attainment in plasma and ELF using prospective samples. The first 24-hour T>MIC was evaluated vs 1xMIC and 4xMIC targets at the patient (i.e., fixed MIC of 2 mg/L) and population [i.e., cumulative fraction of response (CFR) according to EUCAST MIC distributions] levels for both plasma and ELF. Among 65 patients receiving ≥24 hours of treatment, 40% of patients failed to achieve >50% T>4xMIC in plasma and ELF, and 30% of patients who achieved >50% T>4xMIC in plasma had <50% T>4xMIC in ELF. At 1xMIC and 4xMIC targets, 3% and 25% of patients with >95% T>MIC in plasma had <50% T>MIC in ELF, respectively. Those with a CRCL >115 mL/min were less likely to achieve >50%T>4xMIC in ELF (P < 0.025). In the population, CFR for Escherichia coli at 1xMIC and 4xMIC was >97%. For Pseudomonas aeruginosa, CFR was ≥90% in plasma and ranged 80%-85% in ELF at 1xMIC when a loading dose was applied. CFR was reduced in plasma (range: 75%-81%) and ELF (range: 44%-60%) in the absence of a loading dose at 1xMIC. At 4xMIC, CFR for P. aeruginosa was 60%-86% with a loading dose and 18%-62% without a loading dose. We found that plasma overestimated ELF target attainment inup to 30% of meropenem-treated patients, CRCL >115 mL/min decreased target attainment in ELF, and loading doses increased CFR in the population.

In vitro activity of new combinations of β-lactam and β-lactamase inhibitors against the Mycobacterium tuberculosis complex.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a β-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as β-lactams in combination with β-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians.

Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (n = 21; blaKPC) and non-CP-CRE (n = 19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.