Reference Broth Microdilution Methods Research Articles

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.

Assessment of Lefamulin 20 µg Disk versus Broth Microdilution When Tested against Common Respiratory Pathogens

ObjectiveTo evaluate the performance of the disk diffusion test with lefamulin 20 µg compared with the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method. Methods572 clinical stains, including 240 Staphylococcus aureus, 211 Streptococcus pneumoniae and 121 Haemophilus influenzae, isolated from 71 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2020. Broth microdilution method and disk diffusion methods were performed according to CLSI. Categorical agreement (CA), major error (ME), and very major error (VME) were calculated. ResultsLefamulin showed potent activity against S. aureus, S. pneumoniae, and H. influenzae. Using the broth microdilution method, lefamulin inhibited 97.1% of S. aureus isolates at 0.25 mg/L; seven isolates were not susceptible. For S. pneumoniae and H. influenzae, the percentage of susceptibility to lefamulin was 100% and no non-susceptible strains were found in this study. Compared with the reference broth microdilution method, the CA of the lefamulin 20 µg disk testing was 99.8% (571/572), with 14.3% (1/7) VME and no ME. In our study, VME was determined in S. aureus. For S. pneumoniae and H. influenzae, the VME was not determined due to the lack of lefamulin non-susceptible strains. ConclusionThe lefamulin 20 µg disk diffusion testing showed excellent CA and ME with the reference broth microdilution method for S. aureus, S. pneumoniae, and H. influenzae. The VME exceeding CLSI recommendations may be a bias due to fewer lefamulin non-susceptible isolates. Our results suggest that lefamulin non-susceptible isolates detected by disk diffusion should be confirmed by the reference BMD.

Commercially available tests for determining cefiderocol susceptibility display variable performance in the Achromobacter genus

BackgroundCefiderocol is a siderophore-conjugated cephalosporin increasingly used in the management of Achromobacter infections. Testing for cefiderocol susceptibility is challenging with distinct recommendations depending on the pathogens.ObjectivesWe evaluated the performance of commercial tests for testing cefiderocol susceptibility in the Achromobacter genus and reviewed the literature.MethodsDiffusion (disks, MIC gradient test strips [MTS], Liofilchem) and broth microdilution (BMD) methods (ComASP™, Liofilchem; UMIC®, Bruker) were compared with the BMD reference method according to the EUCAST guidelines on 143 Achromobacter strains from 14 species with MIC50/90 of ≤ 0.015/0.5 mg/L. A literature search was conducted regardless of method or species.ResultsNone of the methods tested fulfilled an acceptable essential agreement (EA). MTS displayed the lowest EA (30.8%) after UMIC® (49%) and ComASP™ (76.9%). All methods achieved an acceptable bias, with MICs either underestimated using MTS (-1.3%) and ComASP™ (-14.2%) or overestimated with UMIC® (+ 9.1%). Inhibition zone diameters ranged from 6 to 38 mm (IZD50/90=33/30 mm). UMIC® and ComASP™ failed to categorize one or the two cefiderocol-resistant strains of this study as resistant unlike the diffusion-based methods. The literature review highlighted distinct performance of the available methods according to pathogens and testing conditions.ConclusionsThe use of MTS is discouraged for Achromobacter spp. Disk diffusion can be used to screen for susceptible strains by setting a threshold diameter of 30 mm. UMIC® and ComASP™ should not be used as the sole method but have to be systematically associated with disk diffusion to detect the yet rarely described cefiderocol-resistant Achromobacter sp. strains.

Erythromycin-metal complexes: One-step synthesis, molecular docking analysis and antibacterial proficiency against pathogenic strains

The study focused on the extraction of free erythromycin from commercially manufactured tablets and the use of metal salts to synthesize erythromycin-metal complexes, specifically involving silver (Ag), nickel (Ni), cobalt (Co), and copper (Cu). The synthesis was confirmed through various methods, including elemental analysis, thermogravimetric analysis, Fourier-transform infrared (FTIR), and UV–visible spectroscopy. The microbiological investigation involved Salmonella typhi, Escherichia coli, Staphylococcus aureus, Bacillus cereus, Candida albicans, and Microsporum canis as test organisms. The NCCLS broth microdilution reference method was used to determine the minimum fungicidal concentration and minimum inhibitory concentration of the complexes. The synthesized complexes were highly effective against a variety of fungi and bacteria, with compound Ery-Cu having MIC as low as 1.56 mg/mL, Ery-Cu and Ery-Ni with MBCs of 6.25 mg/mL and Ery-Cu having MFC of 6.25 mg/mL. Dose-dependent inhibitory effects were found upon examination of the antimicrobial susceptibility of specific complexes (Cu, Ni, Co and Ag) at varying concentrations of 100, 50, 25 and 12.5 mm/mL. Antibiotic susceptibility testing revealed efficacy against the tested pathogens. The study suggests that the synthesis of erythromycin-metal complexes, coupled with their antibacterial effectiveness against a diverse spectrum of bacteria and fungi, as they showed promising inhibitory properties when tested against a range of test species (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Candida albicans, and Microsporum canis), could lead to the development of innovative antibacterial agents. Molecular docking simulations were used to examine the interactions between metal complexes with proteins filamentous temperature-sensitive protein Z and lanosterol 14α-demethylase. The study highlights the need for further exploration in pharmaceutical research.

Rapid detection of imipenem/relebactam susceptibility/resistance in Pseudomonas aeruginosa

ObjectivesImipenem-relebactam (IPR) has been reported to exhibit a good activity against non-metallo-ß-lactamase carbapenem-resistant Pseudomonas aeruginosa (CRPA), and the rapid detection of susceptibility/resistance to this new therapeutic alternative may be crucial. Therefore, the Rapid IPR Pseudomonas NP test was developed to quickly identify IPR susceptibility/resistance among multidrug-resistant P. aeruginosa. MethodsThe principle of the Rapid IPR Pseudomonas NP test is based on visually detecting glucose metabolization by observing (or not) a color change from yellow to red or orange of the red phenol pH indicator in the presence of imipenem at 2 mg/L and relebactam at 4 mg/L A total of 80 clinical Pseudomonas aeruginosa isolates were analyzed, among which 42 isolates were IPR resistant according to EUCAST guidelines (MICs, susceptible ≤2 mg/L, resistant >2 mg/L). Results obtained with the Rapid IPR Pseudomonas NP test were compared with the reference broth microdilution (BMD). ResultsThe sensitivity, specificity and accuracy of the test were found to be 100 %, 89.5 % and 95 %, respectively, using the BMD reference method as a comparator. Moreover, five out of the IPR-susceptible isolates (n = 38) exhibiting an MIC of IPR close to the breakpoint (MIC = 1 mg/L, n = 2; MIC = 2 mg/L, n = 3) yielded to a major error result, namely a positive result with the rapid IPR Pseudomonas NP test (resistance). By contrast, all IPR-resistant isolates (n = 42) were all correctly categorized. ConclusionsThe Rapid IPR Pseudomonas NP test is sensitive, specific, and easy to perform and interpret. Therefore, it is suitable for implementation in routine clinical laboratories.

Cefiderocol susceptibility of Achromobacter spp.: study of an accurately identified collection of 230 strains

BackgroundAchromobacter spp. are opportunistic pathogens, mostly infecting immunocompromised patients and patients with cystic fibrosis (CF) and considered as difficult-to-treat pathogens due to both intrinsic resistance and the possibility of acquired antimicrobial resistance. Species identification remains challenging leading to imprecise descriptions of resistance in each taxon. Cefiderocol is a broad-spectrum siderophore cephalosporin increasingly used in the management of Achromobacter infections for which susceptibility data remain scarce. We aimed to describe the susceptibility to cefiderocol of a collection of Achromobacter strains encompassing different species and isolation sources from CF or non-CF (NCF) patients.MethodsWe studied 230 Achromobacter strains (67 from CF, 163 from NCF patients) identified by nrdA gene-based analysis, with available susceptibility data for piperacillin–tazobactam, meropenem and trimethoprim–sulfamethoxazole. Minimal inhibitory concentrations (MICs) of cefiderocol were determined using the broth microdilution reference method according to EUCAST guidelines.ResultsStrains belonged to 15 species. A. xylosoxidans represented the main species (71.3%). MICs ranged from ≤ 0.015 to 16 mg/L with MIC50/90 of ≤ 0.015/0.5 mg/L overall and 0.125/2 mg/L against 27 (11.7%) meropenem-non-susceptible strains. Cefiderocol MICs were not related to CF/NCF origin or species although A. xylosoxidans MICs were statistically lower than those of other species considered as a whole. Considering the EUCAST non-species related breakpoint (2 mg/L), 228 strains (99.1%) were susceptible to cefiderocol. The two cefiderocol-resistant strains (A. xylosoxidans from CF patients) represented 3.7% of meropenem-non-susceptible strains and 12.5% of MDR strains.ConclusionsCefiderocol exhibited excellent in vitro activity against a large collection of accurately identified Achromobacter strains, irrespective of species and origin.

Evaluation of commercial, customized microdilution plates for Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis antimicrobial susceptibility testing and determination of antimicrobial resistance prevalence in France.

Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring.

Comparison of Colistin Susceptibility via Two Different Methods in Gram-Negative Extensive Drug-Resistance Isolates from ICU Patients.

To compare the susceptibility of colistin by two methods in extensive drug-resistant (XDR) Gram-negative isolates from ICU patients. Cross-sectional comparative analysis. Place and Duration of the Study: Department of Microbiology, Combined Military Hospital Karachi, Pakistan, from August 2022 to February2023. A total of 100 clinical specimens received from the intensive care unit yielded growth of extensively drug-resistant gram-negative bacteria, which were evaluated for polymyxin E susceptibility. The agar dilution method was compared with the reference broth microdilution (BMD) method. Minimum inhibitory concentration (MIC) was noted for both methods. Comparison of the MIC method by agar dilution showed a 90% correlation with the reference method of broth microdilution. With MICs within the acceptable range of the clinical and laboratory standards institute (CLSI) recommendations, 89 isolates were susceptible to colistin, whereas only 11 remained resistant. Polymyxin E's MIC 50 and MIC 90 were determined to be 1 and 2 µg/ml, respectively, with 97% susceptibility. Agar dilution susceptibility method can be used for screening purposes for the susceptibility testing of polymyxin E. This method is reliable and can easily identify the heteroresistance. Extensively drug-resistant, Broth microdilution, Multidrug-resistant, Agar dilution, Minimum inhibitory concentration, Colony forming unit.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4μg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 μg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Antimicrobial photodynamic effect of the photosensitizer riboflavin, alone and in combination with colistin, against pandrug-resistant Pseudomonas aeruginosa clinical isolates

IntroductionDevelopment of multi-, extensively-, and pandrug-resistant (MDR, XDR, and PDR) strains of Pseudomonas aeruginosa remains a major problem in medical care. The present study evaluated the effect of antimicrobial photodynamic therapy (aPDT) as a monotherapy and in combination with colistin against P. aeruginosa isolates. MethodsTwo P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Minimum inhibitory concentration (MIC) of colistin was determined by the colistin broth disk elution (CBDE) and the reference broth microdilution (rBMD) methods. aPDT was performed using the photosensitizer (Ps) riboflavin at several concentrations and a light-emitting diode (LED) emitting blue light for different irradiation times with or without colistin at 1/2 × MIC concentration. ResultsBoth PA1 and PA2 isolates were identified as colistin-resistant P. aeruginosa with a MIC ≥4 μg/mL by the CBDE and MICs of 512 μg/mL and 256 μg/mL, respectively, by the rBMD. In aPDT, neither riboflavin nor LED light alone had antibacterial effects. The values of colony forming units per milliliter (CFU/mL) in both isolates were significantly reduced by LED + Ps treatments in a time-dependent manner (LED irradiation time) and dose-dependent manner (Ps concentration). In comparison with control, treatment with Ps (50 μM) + LED (120 s) and Ps (100 μM) + LED (120 s) resulted in 0.27 log10 CFU/mL and 0.43 log10 CFU/mL reductions in PA1, and 0.28 log10 CFU/mL and 0.34 log10 CFU/mL reductions in PA2, respectively, (P < 0.01). The best results were obtained after the combination of aPDT followed by colistin, which increased bacterial reduction, resulting in a 0.41−0.7 log10 CFU/mL reduction for PA1 and 0.35−0.83 log10 CFU/mL reduction for PA2 (P = 0.001). ConclusionsThis study suggests the potential implications of aPDT in combination with antibiotics, such as colistin for treatment of difficult-to-treat P. aeruginosa infections.

Lessons learned from an external quality assurance program in applying CLSI interpretive criteria for reporting piperacillin/tazobactam susceptibility

We evaluated the performance of automated susceptibility testing for piperacillin/tazobactam (PTZ) MICs against the reference microbroth dilution method. The Minimum Inhibitory Concentration of PTZ against a clinical isolate of Klebsiella pneumoniae was determined by reference broth micro-dilution method in 10 replicates which yielded a modal MIC of 16 mg/L (susceptible dose-dependent). Out of 434 laboratories who obtained MIC of 16 mg/L correctly, only 301 interpreted the result as susceptible dose dependent as per 2022 revised CLSI criteria. Educating the clinical laboratories in validating AST methods as per latest CLSI guidelines is of utmost important.

Evaluation of different linezolid susceptibility testing methods and detection of linezolid resistance gene (cfr) in staphylococcal isolates

Linezolid is an effective oxazolidinone antibiotic against multi resistant Gram-positive organisms. Linezolid resistance is an emerging problem and some controversy exists about the reliability of different phenotypic methods of linezolid susceptibility testing. Fifty isolates each of methicillin resistant S. aureus (MRSA) and Staphylococcus haemolyticus were tested for linezolid susceptibility using Kirby-Bauer disc diffusion, E-test, automated system VITEK2, Broth micro-dilution (reference method) and PCR for the cfr gene. Six resistant isolates were identified, three each in MRSA and S. haemolyticus, all carrying the cfr gene. E-test and VITEK2 were found to be more accurate than disc diffusion test.

2755. Activity of Aztreonam-avibactam and Carbapenem-resistant Enterobacterales (CRE) Isolates Collected in a Six-year Period (2017–2022)

Abstract Background Beta-lactam/beta-lactamase inhibitor (BL/BLI) combinations and cefiderocol (FDC) have been recommended for the treatment of carbapenem-resistant Enterobacterales (CRE), but extensive comparisons of these agents are limited. We tested the activity of aztreonam-avibactam (ATM-AVI), ceftazidime-avibactam (CAZ-AVI), meropenem-vaborbactam (MEV), imipenem-relebactam (IR), all BL/BLIs, and FDC against &amp;gt; 500 CRE isolates collected in US hospitals during a 6-year period (2017–2022). Methods A total of 54,576 Enterobacterales (ENT) isolates collected during 2017–2021 were susceptibility (S) tested by reference broth microdilution methods. CRE isolates displayed meropenem and/or imipenem MIC results at &amp;gt; 1 mg/L and were submitted to whole genome sequencing for analysis of resistance mechanisms. Results The CRE isolates (n=511; 0.9% overall) were mainly KPC producers without metallo-beta-lactamases (n=346; 67.7%). Genes encoding MBLs and OXA-48–like enzymes alone were detected among 50 and 12 isolates, respectively. Another 12 isolates carried genes encoding NMC-A and SME. ATM-AVI inhibited 98.4% of the CRE isolates when applying ATM breakpoints for comparison. FDC, CAZ-AVI, MEV, and IR inhibited 94.7%, 89.2%, 86.7%, and 81.9% of the CRE isolates, respectively. Against class A serine-carbapenemase–producing isolates without MBLs, ATM-AVI, CAZ-AVI, FDC, and MEV demonstrated activity &amp;gt; 98.6%. IR inhibited 92.7% of these isolates. Except for ATM-AVI and FDC, other BL/BLI had limited activity against MBLs, ranging from 2.0% S for IR to 16% for MEV against MBL isolates. ATM-AVI inhibited 98.0% of the MBL isolates while FDC inhibited 74.0%. ATM-AVI, CAZ-AVI, IR, FDC, and MEV inhibited 94.5%, 96.7%, 90.1%, 89.0%, and 87.9% of the carbapenemase-negative isolates (n=91), respectively. Among comparators, tigecycline was the most active, inhibiting 94.7% of the CRE isolates; 84.7% of these isolates displayed a colistin intermediate MIC value of ≤ 2 mg/L. Conclusion The new BL/BLIs and FDC displayed good activity against a large collection of CRE isolates that were mainly KPC producers from US hospitals. Against MBLs, ATM-AVI was the most active agent, followed by FDC. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Joshua Maher, PhD, AbbVie: Grant/Research Support|Affinity Biosensors: Grant/Research Support|AimMax Therapeutics, Inc: Grant/Research Support|Alterity Therapeutics: Grant/Research Support|Amicrobe, Inc: Grant/Research Support|Arietis Pharma: Grant/Research Support|Armata Pharmaceuticals, Inc: Grant/Research Support|Astrellas Pharma, Inc.: Grant/Research Support|Basilea Pharmaceutica AG: Grant/Research Support|Becton Dickinson And Company: Grant/Research Support|bioMerieux, Inc: Grant/Research Support|Boost Biomes: Grant/Research Support|Diamond V: Grant/Research Support|Fedora Pharmaceuticals, Inc: Grant/Research Support|Iterum Therapeutics plc: Grant/Research Support|Johnson &amp; Johnson: Grant/Research Support|Kaleido Biosciences, Inc.: Grant/Research Support|Meiji Seika Pharma Co. Ltd.: Grant/Research Support|National Institutes of Health: Grant/Research Support|Pfizer Inc.: Grant/Research Support|Roche Holding AG: Grant/Research Support|Shionogi Inc.: Grant/Research Support|Summmit Therapeutics, Inc.: Grant/Research Support|Zoetis Inc: Grant/Research Support Katie Simpson, BS, AbbVie: Grant/Research Support Cory Hubler, BS, AbbVie: Grant/Research Support|Shionogi: Grant/Research Support Helio S. Sader, MD, PhD, FIDSA, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

865. Same Shift Susceptibility Testing of Cefepime-Taniborbactam and Cefiderocol Using The Selux Next-Generation Phenotyping System

Abstract Background Robust, efficient, and reliable antimicrobial susceptibility testing (AST) systems are an integral component of effective antibiotic treatment. Multi-drug resistant (MDR) bacteria continue to emerge at alarming rates, requiring continuous innovation and development of new antibiotics. Among the new promising treatments for severe bacterial infections caused by carbapenem-resistant gram-negative bacteria, Cefiderocol (FDC) and Cefepime-Taniborbactam (FTB) have emerged as two frontrunners. FDC is the only FDA-approved siderophore cephalosporin, and FTB pairs the fourth-generation cephalosporin Cefepime, with Taniborbactam, a novel beta-lactamase inhibitor. AST results are paramount when directing therapy for patients with MDR infections, yet there is an average 3-year delay between the approval of new antibiotics and 510k submissions for new agents on automated AST systems. This is typified by FDC, for which there are currently no FDA-cleared automated AST systems. Here we assess the accuracy of FDC and FTB AST results using the Selux Next-Generation Phenotyping System. Methods For both FTB and FDC susceptibility, AST was performed using the Selux AST system and compared to CLSI broth microdilution (BMD) reference method in either cation-adjusted Muller Hinton broth (CAMHB) or its Iron depleted counterpart (ID-CAMHB). FDC, was tested in ID-CAMHB across 44 total CDC AR-Bank carbapenem-resistant K. pneumoniae, A. baumannii, and P. aeruginosa species. 34 CDC AR-Bank Strains predominantly resistant (30/34) to Cefepime were used to assess FTB. For FDC Essential Agreement (EA) and Categorical Agreement (CA) relative to ID-CAMHB were determined according to FDA breakpoints. FTB breakpoints were based on FDA breakpoints for Cefepime. For both agents, a comparison of BMD and the Selux system was accessed by EA and CA. Results The Selux AST system produced MICs to FDC with an average accuracy of 98% EA and 98% CA in ID-CAMHB (Table 1). In FTB, the Selux AST system produced MICs with an average accuracy of 94% EA and 100% CA in CAMHB (Table 2). Conclusion These data demonstrate the ability of the Selux NGP system to provide accurate results for FDC and FTB. This is the first known demonstration of an automated AST system for these important new drugs. Disclosures David Jimenez, B.S., Selux Diagnostics: Employee Kristin Baker, PhD, Selux Diagnostics: Employee Kelly Flentie, PhD, Selux Diagnostics: Employee Kristen Roberts, BS, Selux Diagnostics: Stocks/Bonds Gabriel Michaels, n/a, Selux Diagnostics: Employee Eric Stern, PhD, Selux Diagnostics: Board Member|Selux Diagnostics: Ownership Interest

In vitro manogepix susceptibility testing of South African Emergomyces africanus, Emergomyces pasteurianus, and Blastomyces emzantsi clinical isolates.

We performed in vitro antifungal susceptibility testing of manogepix against the yeast phase of 78 Emergomyces africanus, 2 Emergomyces pasteurianus, and 5 Blastomyces emzantsi isolates using a reference broth microdilution method following Clinical and Laboratory Standards Institute recommendations. All three pathogens had low minimum inhibitory concentrations ranging from <0.0005 to 0.008 mg/L. Manogepix should be investigated in animal models and potentially in future human clinical trials for endemic mycoses.

In-vitro Susceptibility Testing Methods for Ceftazidime-avibactam against Carbapenem-resistant Enterobacterales: Comparison with Reference Broth Microdilution Method.

β-lactam antibiotics, mainly cephalosporins, and carbapenems, have been the mainstay of treatment for infections caused by Enterobacterales. However, their role in treating clinical infections has become limited because of the increase in resistance. There is a need to have cost-effective and rapid methods for antimicrobial susceptibility testing methods for newer antibiotics like ceftazidime-avibactam against carbapenem-resistant Enterobacterales (CRE), which can be applied in routine clinical microbiology laboratories. With this aim, the present study was conducted to compare the disk diffusion and gradient diffusion, i.e., the E-test method with the reference broth microdilution (BMD) method for in-vitro testing of ceftazidime-avibactam against CRE. A total of 111 CRE isolates from various clinical samples were included. Conventional PCR (Polymerase Chain Reaction) was done for the detection of genes encoding carbapenemases and to see their expression, modified carbapenem inactivation method (mCIM) along with EDTA (Ethylenediaminetetraacetic acid) carbapenem inactivation method (eCIM) was done. 42.3% (47/111) isolates were resistant to ceftazidime-avibactam by the standard broth microdilution method; however, 45.9% (51/111) were resistant by both disk diffusion and E-test. In 5.4% of isolates (similar in both methods), microbroth dilution method results did not match with E-strip and disk diffusion. Very major errors (VME) by both disk diffusion and E-test were found in 2.1% (1/47), and major errors (ME) were found in 7.8% (5/64) isolates (similar isolates in both methods). The overall categorical agreement (CA) rate was 94.6% for both E-test and disk diffusion, and the essential agreement (EA) rate was 90.1% (100/111) for E-test. 98% (109/111) of CRE harbored carbapenemase genes either singly (30.3%) or in combination with others (69.7%). In conclusion, for CRE, E-test and the disk diffusion method for ceftazidimeavibactam depicted an acceptable performance as an alternative to the reference broth microdilution method.

Optimization of the EUCAST reference broth microdilution method for echinocandin susceptibility testing of Aspergillus fumigatus.

Because of the high inoculum (105 cfu/mL) used in the EUCAST susceptibility testing of Aspergillus spp., determination of the minimal effective concentration (MEC) of echinocandins is challenging as the morphological differences are subtle. The MECs of 10 WT and 4 non-WT Aspergillus fumigatus isolates were determined with the EUCAST E.Def 9.4. Plates were inoculated with increasing inocula (102-105 cfu/mL) and after 24 and 48 h of incubation, MECs were determined macroscopically (magnifying mirror) and microscopically (inverted microscope) by two observers, spectrophotometrically (OD at 405 nm) and colorimetrically (absorbance at 450/630 nm after 2 h incubation with 400 mg/L XTT/6.25 μM menadione). The interobserver (between observers)/intermethod (compared with the microscopic method) essential agreement (EA, ±1 2-fold dilution) and categorical agreement (CA) were determined for each inoculum. Echinocandin-induced microscopic hyphal alterations or macroscopic changes in turbidity were subtle with a 105 cfu/mL inoculum compared with the lower inocula of 103 and 102 cfu/mL, where more distinct changes in turbidity and formation of characteristic rosettes were obvious at the MEC after 48 h. A 105 cfu/mL inoculum resulted in wider MEC distributions (3-6 dilutions) and lower interobserver EA (69%), macroscopic-microscopic EA (26%) and CA (71%) compared with a 103 cfu/mL inoculum (2-3 dilutions, 100%, 100% and 100%, respectively). Spectrophotometric readings using a 103 cfu/mL inoculum showed good EA (57-93%) and excellent CA (86%-100%), while the XTT assay demonstrated excellent EA (93%) and CA (100%). A 48 h incubation using a 103 cfu/mL inoculum improved echinocandin MEC determination for A. fumigatus with the EUCAST method, while the colorimetric assay could allow automation.

Performance of the Disc Diffusion Method, MTS Gradient Tests and Two Commercially Available Microdilution Tests for the Determination of Cefiderocol Susceptibility in Acinetobacter spp.

Cefiderocol is a siderophore-conjugated cephalosporin with potent activity against multidrug-resistant Gram-negative pathogens including Acinetobacter baumannii. The aim of this study was to evaluate cefiderocol testing methods on a relevant collection of 97 Acinetobacter spp. isolates. Commercialized broth microdilution methods (ComASP®, Liofilchem and UMIC®, Bruker), MIC test strips (Liofilchem) and disc diffusion using discs of three different brands (Mast Diagnostic, Liofilchem and Oxoid-Thermo Fisher Scientific) were compared with the broth microdilution reference method. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ±30%) but both BMD methods achieved acceptable categorical agreement rates (CA = 95.9% [93/97, 95% CI 89.9-98.4] and CA = 93.8% [91/97, 95% CI 87.2-97.1] for ComASP® and UMIC®, respectively) and bias < 30% (-7.2% and -25.2% for ComASP® and UMIC®, respectively). The use of MIC gradient testing is strongly discouraged due to misclassification of 55% (n = 23/42) of resistant strains. Finally, the disc diffusion method could be used to rapidly screen for susceptible strains by setting a critical diameter of 22 mm.