Microdilution Reference Method 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.

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.

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 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.

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.

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.

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

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.

Comparison of the Micronaut-AM System and the EUCAST Broth Microdilution Reference Method for MIC Determination of Four Antifungals against Aspergillus fumigatus.

The Antifungal Susceptibility Testing method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) is a reference technique for the determination of the Minimum Inhibitory Concentration (MIC) of antifungals for Aspergillus fumigatus. However, it is time-consuming and requires expertise. Micronaut-AM (M-AM) is a fast, simple, time-saving, and ready-to-use new colorimetric method using an indicator (resazurin) to facilitate the visual reading. The aim of this retrospective study was to evaluate the performance of the M-AM system and compare it with the EUCAST broth microdilution reference method to determine the susceptibility of 77 A. fumigatus clinical strains to amphotericin B, itraconazole, voriconazole, and posaconazole. Overall, the essential agreements within ±2 dilutions were 100%, 62%, 58%, and 30% and the categorical agreements were 100%, 97%, 91%, and 87% for amphotericin B, itraconazole, voriconazole, and posaconazole, respectively. No categorical discrepancy was found for amphotericin B, but several categorical discordances were observed with azole antifungals. However, only 2 of the 16 azole-resistant strains confirmed by the cyp51A sequencing would have been misclassified by M-AM. The use of M-AM is probably suitable for the determination of the MICs of amphotericin B, but further evaluations are needed to confirm its usefulness for the determination of the MICs of azoles for A. fumigatus.

Antimicrobial susceptibility testing of invasive isolates of Streptococcus pneumoniae from Canadian patients: the SAVE study, 2011-2020.

To assess the antimicrobial susceptibility of 14 138 invasive Streptococcus pneumoniae isolates collected in Canada from 2011 to 2020. Antimicrobial susceptibility testing was performed using the CLSI M07 broth microdilution reference method. MICs were interpreted using 2022 CLSI M100 breakpoints. In 2020, 90.1% and 98.6% of invasive pneumococci were penicillin-susceptible when MICs were interpreted using CLSI meningitis or oral and non-meningitis breakpoints, respectively; 96.9% (meningitis breakpoint) and 99.5% (non-meningitis breakpoint) of isolates were ceftriaxone-susceptible, and 99.9% were levofloxacin-susceptible. Numerically small, non-temporal, but statistically significant differences (P < 0.05) in the annual percentage of isolates susceptible to four of the 13 agents tested was observed across the 10-year study: chloramphenicol (4.4% difference), trimethoprim-sulfamethoxazole (3.9%), penicillin (non-meningitis breakpoint, 2.7%) and ceftriaxone (meningitis breakpoint, 2.7%; non-meningitis breakpoint, 1.2%). During the same period, annual differences in percent susceptible values for penicillin (meningitis and oral breakpoints) and all other agents did not achieve statistical significance. The percentage of isolates with an MDR phenotype (resistance to ≥3 antimicrobial classes) in 2011 and 2020 (8.5% and 9.4%) was not significantly different (P = 0.109), although there was a significant interim decrease observed between 2011 and 2015 (P < 0.001) followed by a significant increase between 2016 and 2020 (P < 0.001). Statistically significant associations were observed between resistance rates to most antimicrobial agents included in the MDR analysis (penicillin, clarithromycin, clindamycin, doxycycline, trimethoprim/sulfamethoxazole and chloramphenicol) and patient age, specimen source, geographic location in Canada or concurrent resistance to penicillin or clarithromycin, but not biological sex of patients. Given the large isolate collection studied, statistical significance did not necessarily imply clinical or public health significance in some analyses. Invasive pneumococcal isolates collected in Canada from 2011 to 2020 generally exhibited consistent in vitro susceptibility to commonly tested antimicrobial agents.

Clinical application of Vitek-derived minimum inhibitory concentration values: Proof of concept study.

Minimum inhibitory concentration (MIC) values are useful in guiding appropriate antimicrobial therapy however, routine provision and interpretation of MIC values to guide clinical decision-making is challenging. This proof of concept study aims to demonstrate the clinical utility and application of Vitek®-derived MIC values through categorisation of clinical isolates as wild type. A random selection of clinically relevant Gram negative isolates routinely tested on the Vitek® instrument were included. The Vitek® MIC values, for selected antimicrobials at the lowest calling range of that card, were compared to the broth microdilution reference method. The specified end-point was concordance between the two results if the reference MIC was less than or equal to the EUCAST-defined epidemiological cut-off value (ECOFF) for that drug-bug combination. A total of 525 isolates were included (468 Enterobacterales and 57 Pseudomonas aeruginosa), with an overall concordance rate of 96.4% (508/525). Correct ECOFF categorisation by the Vitek® was highest for ceftazidime and piperacillin (100%, n = 48 and n = 55, respectively) and lowest for cefepime (81.8%, n = 66). Vitek®-derived MIC values can be used to categorise organisms as wild-type if the MIC is reported at the card's lowest calling range (≤) as there is high likelihood that the MIC is at or below the ECOFF. This has important implications for antimicrobial management, assisting in choice of agent and in improving probability of target attainment for desired pharmacodynamic targets which can translate into improved clinical outcomes. Minimum inhibitory concentration data from an automated antimicrobial susceptibility testing instrument can be used to guide clinical decisions.

Performance of discs, sensititre EUMDROXF microplates and MTS gradient strips for the determination of the susceptibility of multidrug-resistant Pseudomonas aeruginosa to cefiderocol

ObjectivesTo evaluate the performance of commercially available tests to determine the susceptibility of multidrug-resistant (MDR) clinical Pseudomonas aeruginosa strains to cefiderocol. MethodsA collection of 150 clinical strains of P. aeruginosa resistant to ceftazidime, (MIC, Minimal Inhibitory Concentration, MIC > 8 mg/L) imipenem (MIC> 4 mg/L) and ceftolozane/tazobactam (MIC> 4/4 mg/L), isolated from 2015 to 2022 was selected. Cefiderocol susceptibility was determined in parallel (a) by disc diffusion using Mast, Oxoid and Liofilchem discs deposited on Mueller-Hinton agar batches from Bio-Rad, BioMérieux, Mast, Becton Dickinson, I2A and Oxoid; (b) by MIC gradient test strips (MTS) (Liofilchem); and (c) by EUMDROXF Sensititre microplates. MICs and inhibition zones were compared with the broth microdilution reference method (BMD) MICs. ResultsThe MIC50 and MIC90 of cefiderocol were 1 mg/L and 8 mg/L by BMD, respectively, including 21.3% (32/150) resistant strains. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ± 30%). Although the Sensititre EUMDROXF microplates overestimated MIC values (categorical agreement [CA] = 86.7% [130/150, 95% CI 80.3–91.2]; EA = 69.3% [104/150, 95% CI 61.6–76.2]; bias = 68.2%), MTS strips underestimated the MIC values for many strains (CA = 86.7%, 130/150, 95% CI 80.3–91.2; EA = 69.3%, 104/150, 95% CI 61.6–76.2; bias = −30.4%), classifying properly only 50% (16/32) of resistant strains. Finally, many cefiderocol-resistant strains were not identified by the disc method, although the CA ranged from 78.0% (117/150, 95% CI 70.7–83.0) to 89.3% (134/150, 95% IC 83.4–93.3) according to Mueller-Hinton agar batches. ConclusionDetermination of cefiderocol susceptibility in MDR P. aeruginosa clinical strains by Sensititre EUMDROXF microplates is an alternative to the reference BMD method. However, MIC values ± 1 dilution apart from the breakpoint (2 mg/L) should be controlled by BMD whereas the use of MTS gradient strips is discouraged. Disc diffusion might be useful for screening, unfortunately many cefiderocol-resistant strains are not detected.

Evaluating the drop test method in measuring colistin susceptibility of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa.

Introduction. Colistin is an antibiotic used to treat Gram-negative bacterial infections, particularly those caused by multidrug-resistant bacteria.Hypothesis/Gap Statement. The broth microdilution (BMD) reference method is the recommended protocol for detecting colistin susceptibility; however, it is laborious and expensive and cannot be performed in all laboratories.Aim. To evaluate the colistin susceptibility in Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa using an alternative method, which has been referred to as the drop test.Methodology. A 16 µg ml-1 colistin solution was deposited on a Mueller-Hinton agar plate previously swabbed with the strain and incubated overnight, and the presence or absence of an inhibition zone was observed.Results. The categorical agreement (CA) of the drop test with respect to BMD was 100 % when 190 Enterobacterales (19 E. coli and 171 K. pneumoniae) were used, and no major errors (MEs) or very major errors (VMEs) were detected. The CA of the drop test with respect to the BMD was 99.2 % for 119 P. aeruginosa isolates, while no ME was detected and only 1 VME (6.7%) was observed.Conclusion. The drop test is an alternative method for antimicrobial susceptibility testing of colistin against K. pneumoniae and E. coli. It is an adequate method for detecting resistant strains of P. aeruginosa, but susceptible isolates should be confirmed using BMD. The drop test is a simpler alternative to the BMD that does not require additional equipment and allows for the testing of numerous isolates in a short period of time.

1692. Ceftibuten-Xeruborbactam: In vitro Potency against Enterobacterales in Comparison with Other Oral Beta-lactams (BL) and Beta-lactamase Inhibitor (BLI) Combinations

Abstract Background The broad spectrum BLI xeruborbactam (XER, formerly, QPX7728) can be delivered IV or orally as the isobutyryloxymethyl prodrug. The prodrug form is in clinical development in combination with ceftibuten (BUT) for oral treatment of resistant gram-negative infections. The objective of this study was to compare the in vitro potency of BUT-XER to that of other investigational oral BL/BLI combinations and tebipenem (TBP) in blinded head-to-head testing against extended beta-lactamase (ESBL) producing and carbapenem-resistant Enterobacterales (CRE). Methods A total of 980 Enterobacterales including 515 ESBL and 460 CRE, including 195 KPC-, 97 OXA-48-like- and 168 metallo beta-lactamase (MBL) producers, were susceptibility tested by reference broth microdilution against BUT-XER (at fixed 4 mg/L) and comparator BLs or BL/BLI combinations (tested at their recommended BLI concentrations). MIC testing was conducted blinded to drug identity using CLSI reference microdilution methods and controlled using approved quality control bacterial strains and ranges. Results BLI inhibitor combinations and TBP had excellent potency against ESBL isolates with MIC90 in the ≤ 0.03-0.06 mg/L range for BUT-avibactam (AVI) and BUT-XER and in 0.125-0.25 mg/L range for TBP, BUT-VNTX-6236 (VNRX) and cefpodoxime (CPD)-ETX1317 (ETX) (Table). Against KPC and OXA-48-producers, BUT-XER and BUT-AVI (MIC90 0.25-0.5 mg/L) were 4-fold more potent than BUT-VNRX and CPD-ETX (MIC90 of 1-2 mg/L); TBP was not active against these strains. BUT-XER and CPD-ETX were the most potent agents against MBL-producing strains; however, anti-MBL activity of CPD-ETX was due to intrinsic antibacterial activity of ETX. TPB, BUT-AVI and BUT-VNTX did not have any relevant activity against MBL-producing strains. Conclusion BUT-XER had the broadest antimicrobial profile, with activity against ESBL and all carbapenemase producing isolates. Its potency against ESBL-producing isolates was comparable to the carbapenem TBP. Its in vitro potency and spectrum combined with the potential of once-daily administration based on PK studies warrants further clinical development. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support.

Clinical, epidemiological, and mycological features of patients with candidemia: Experience in two tertiary referral centers in Iran.

Candidemia is a major cause of morbidity and mortality among patients receiving immunosuppressive therapy and those hospitalized with serious underlying diseases. Here, we investigated the epidemiological, clinical, and mycological features of candidemia in Tehran, Iran. A prospective observational study of all patients diagnosed with candidemia was performed at two referral teaching hospitals in Tehran, Iran, from February to December 2018. Demographic characteristics, underlying diseases, risk factors, clinical symptoms, and laboratory analyses of candidemic patients with positive culture were mined. Candida isolates were molecularly identified by sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2). The antifungal susceptibility testing for fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, caspofungin, micafungin, and anidulafungin against the isolates was performed using CLSI broth microdilution reference method (M27-A3). A total of 89 episodes were identified, with an incidence of 2.1 episodes/1000 admissions. The common underling disease were malignancy (46%), renal failure/dialysis (44%), and hypertension (40%). The overall crude mortality was 47%. C. albicans (44%) was the most frequent causative agent, followed by C. glabrata (21%), C. parapsilosis complex (15%), C. tropicalis (11%), and C. lusitaniae (3.5%). All the isolates were susceptible to amphotericin B. The activity of all four azoles was low against non-albicans Candida species, especially C. tropicalis. The increase in non-albicans Candida species with reduced susceptibility to antifungal drugs might be alarming in high-risk patients. Therefore, accurate knowledge of predisposing factors and epidemiological patterns in candidemia are effective steps for managing and decreasing the mortality rate in candidemia.

Rapid pathogen identification and phenotypic antimicrobial susceptibility directly from urine specimens

Implementing effective antimicrobial therapy close to the onset of infection lowers morbidity and mortality and attenuates the spread of antimicrobial resistance. Current antimicrobial susceptibility testing (AST) methods, however, require several days to determine optimal therapies. We present technology and an automated platform that identify (ID) Urinary Tract Infection pathogens in 45 min and provide phenotypic AST results in less than 5 h from urine specimens without colony isolation. The ID and AST tests count cells fluorescently labeled with specific rRNA probes using non-magnified digital imaging. The ID test detected five pathogens at ≤ 7,000 CFU/mL and had a linear range of ~ 4 orders of magnitude. For contrived specimens, AST tests gave 93.1% categorical agreement with 1.3% Very Major Errors (VME), 0.3% Major Errors (ME), and 6.3% minor Errors (mE) compared to the broth microdilution (BMD) reference method. For clinical specimens, the ID test had 98.6% agreement and the AST test had 92.3% categorical agreement with 4.2% mE, 3.4% ME and 4.0% VME compared to BMD. Data presented demonstrates that direct-from-specimen AST tests can accurately determine antimicrobial susceptibility/resistance for each pathogen in a specimen containing two pathogens. The method is robust to urine matrix effects and off-target commensal and contaminating bacteria.

Activities of Eravacycline, Tedizolid, Norvancomycin, Nemonoxacin, Ceftaroline, and Comparators against 1,871 Staphylococcus and 1,068 Enterococcus Species Isolates from China: Updated Report of the CHINET Study 2019.

To evaluate the in vitro activities of eravacycline, tedizolid, nemonoxacin, norvancomycin, and ceftaroline against Staphylococcus and Enterococcus species isolates were collected as part of the China Antimicrobial Surveillance Network (CHINET) in 2019 to provide susceptibility data for Staphylococcus spp. and Enterococcus spp. for their future development and application in clinical practice. Antimicrobial susceptibility testing was performed using the CLSI broth microdilution reference method. Eravacycline was highly active against Staphylococcus and Enterococcus species isolates, proved by the MIC50/90: 0.06/0.125, 0.06/0.25, 0.06/0.25, 0.06/0.25, 0.125/0.5, 0.125/0.25, and 0.03/0.06 mg/L for Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), S. epidermidis, S. hominis, S. haemolyticus, Enterococcus faecalis, and E. faecium, respectively. S. aureus isolates tested were fully susceptible to tedizolid. Still, nonsusceptible isolates were found for E. faecalis (72/567 [12.7%]) and E. faecium (12/501 [2.4%]). Norvancomycin at 2 mg/L could inhibit 100% of Staphylococcus spp., while 1 mg/L of ceftaroline could inhibit 78.9% of MRSA and 99.9% of methicillin-susceptible S. aureus (MSSA) isolates. Additionally, nemonoxacin was also active against Staphylococcus and Enterococcus species isolates tested (shown by the following MIC90s and ranges, in milligrams per liter: 2 and ≤0.015 to 8 for MRSA, 0.25 and ≤0.015 to 4 for MSSA, 0.5 and ≤0.015 to 8 for S. epidermidis, and 4 and ≤0.015 to >32 for E. faecalis). In conclusion, both eravacycline and tedizolid were highly active against clinical isolates of Staphylococcus spp. and Enterococcus spp. recently collected across China. Nemonoxacin showed potent activity against Staphylococcus spp. and E. faecalis but limited activity against E. faecium. Norvancomycin and ceftaroline displayed highly potent activity against Staphylococcus spp. IMPORTANCE Antimicrobial resistance has become a severe threat to global public health. According to statistics, nearly 700,000 people die from bacterial infections worldwide (J. O'Neill, Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations, 2014; C. Y. Chin, K. A. Tipton, M. Farokhyfar, E. M. Burd, et al., Nat Microbiol 3:563-569, 2018, https://doi.org/10.1038/s41564-018-0151-5). The number of bacterial infections is expected to climb to 10 million by 2050, showing that bacterial resistance has become a significant problem that cannot be ignored. It is crucial to develop new antimicrobial agents to combat antimicrobial-resistant bacteria. In this study, we evaluated the in vitro activities of eravacycline, tedizolid, nemonoxacin, norvancomycin, and ceftaroline against Staphylococcus spp. and Enterococcus species isolates which were collected as part of CHINET in 2019. We believe that this study can provide susceptibility data for Staphylococcus spp. and Enterococcus spp. for their future development and application in clinical practice.

Evaluation of Colistin Susceptibility Results in Multidrug-Resistant Gram-Negative Bacilli by Colistin Broth Disk Elution Method

Objective: Determining colistin susceptibility according to the reference method is a significant problem for clinical microbiology laboratories. There is a need for a fast, easy-to-apply, and accurate method for laboratories with limited opportunities. In our study, we aimed to evaluate the applicability of the colistin broth disk elution (CBDE) test. Methods: A total of 193 Gram-negative bacteria, which were isolated from various clinical specimens in our laboratory, were included in the study. Colistin susceptibility was determined by broth microdilution as the reference method. The CBDE test was performed as previously described. For each bacterial isolate, 0, ½, 1, 2, and 4 colistin discs (10 µg; BD) were added to 5 tubes containing cation-adjusted Mueller-Hinton broth (MHB). The final colistin concentrations in the tubes were 0 (growth control), 0.5, 1, 2, and 4 µg/ml, respectively. The minimum inhibitory concentration values were determined by visual evaluation 16-20 hours after the bacterial inoculation. Results: In the Enterobacterales group, the categorical agreement was 94%, the major error was 5%, and the very major error was 0%. In the non-fermentative bacteria group, the categorical agreement was found to be 94%, the major error 1%, and the very major error 33%, respectively. Conclusion: The CBDE test is a good alternative to broth microdilution in routine microbiology laboratories for the Enterobacterales group since it provides easy-to-apply, cost-effective testing for colistin susceptibility and has a high categorical agreement compared to the reference microdilution method. Keywords: colistin, disc elution, antibiotic resistance, broth microdilution, Enterobacterales

P017 Echinocandin resistance mechanism in Candida tropicalis and Candida glabrata

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjective Candida tropicalis and Candida glabrata account for 41.6% and 7.08% of total Candidaemia cases in India. Echinocandins are the first-line treatment option for these infections. Resistance to Echinocandins is rare with Candida sp. However, in recent years, has been noted across many centers. We determined the mechanism of echinocandin resistance in C. tropicalis and C. glabrata.Methods C. tropicalis and C. glabrata isolated from Candidaemia patients over a period of 3 years (August 2016-July 2019), identified by MALDI-TOF-MS were used in this study. Antifungal susceptibility testing was done following CLSI broth microdilution reference method (M 27A). FKS1 gene was sequenced using species-specific primers for the presence of any mutation. To determine any changes in the cell wall chitin and glucan contents, expression fold changes of chitin synthase (CHS1, CHS2, and CHS3), and glucan synthase genes upon caspofungin treatment were determined using real-time qPCR. These findings were correlated with cell wall chitin and glucan content determined by flowcytometry.ResultsA total of 3558 Candida species were isolated from patients of all age groups at our hospital. C. tropicalis was the predominant agent (34%), while the prevalence of C. glabrata was 6%. A total of 17 (8.5%) C. glabrata and 3 (0.25%) C. tropicalis exhibited reduced susceptibility to echinocandins. All these isolates carried a wild-type FKS genes.In C. tropicalis, inducible expression of Chs1, Chs2 and Chs3 genes were comparable among susceptible and resistant isolates1.8 (0.4-2.8) vs. 2.5 (0.9-6.6), P = .247); [0.7 (0.3-1.8) vs. 0.7 (0.2-1.6), P = .793]; [1.3 (0.14-4.8) vs. 1.1(0.48-1.7), P = .522], respectively. In concordance with gene expression, there was no significant difference in cell wall chitin contents among resistant and susceptible [14.37 (6.5-24.8) vs 16.28 (6.0-24.7), P = .114] C. tropicalis isolates. In contrast in resistant isolates of C. glabrata, caspofungin treatment resulted in significantly higher induction of chitin synthase genes compared to susceptible isolates; Chs1 [2.34 (0.24-9.71) vs. 1.56 (0.55-4.5) (P = .007)], CHS2 [1.59 (0.33-8.0) vs. (2.3 (0.69-6.15), P = .0006], and CHS3 gene [3.8 (0.13-12.73) vs. 1.9 (0.56-7.16), P <.0001]. Flowcytometric data in terms of chitin content, correlated well with expression changes as staining index was significantly higher in resistant compared to susceptible isolates [320 (198-535) vs. 164 (5.34-254.10) P = .0001]. Glucan synthase expression was comparable in susceptible and resistant isolates of C. tropicalis [3.47 (1.57-7.63) vs. 4.41 (0.41-17.51), (P = .518)]. However, glucan synthase gene expression was found significantly increased in resistant C. glabrata isolates compared to susceptible isolates; 3.10 (1.02-16.45) vs. 1.61 (0.13-7.67), P <.001.ConclusionWe evaluated the role of cell wall components in echinocandin resistance in isolates with reduced susceptibility to echinocandins but lacking an FKS1 mutation. While chitin was induced at higher levels in C. glabrata, a similar finding was not observed in C. tropicalis. This warrants further studies to elucidate the role of fungal cell wall polymers in resistance.

P001 Characteristics and dynamics of azole-resistant Aspergillus fumigatus variants emerging over a 28-year period in the Netherlands

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackground Aspergillus fumigatus, a globally distributed opportunistic pathogen, is the main cause of invasive aspergillosis, especially in immunocompromised patients with high mortality. The emergence of azole-resistant A. fumigatus isolates has been a significant concern worldwide and an important clinical problem.ObjectivesWe aim to determine the presence of variants in a large collection of clinical A. fumigatus isolates from the Netherlands, if the number of variants increased over time and if the presence of additional short nucleotide polymorphisms (SNPs) or tandem repeats (TR) variations impacted on the triazole phenotype.MethodsThe Radboud University Medical Center has collected 11 813 clinical A. fumigatus isolates since 1994. The collection includes isolates cultured from patients admitted to our own center, isolates sent from other hospitals for identification and in vitro susceptibility testing, and isolates sent from five university medical centers and five teaching hospitals that contribute to the national Aspergillus resistance surveillance. The genotypes were detected by Cyp51A Sanger sequencing. All isolates were subjected to in vitro susceptibility testing using the EUCAST microdilution reference method. Minimal inhibitory concentrations (MICs) were determined for itraconazole, voriconazole, posaconazole, in all isolates and for isavuconazole in isolates cultured in 2015 and thereafter.ResultsIn total, 1826 A. fumigatus isolates harbored azole-resistant mutations in the Cyp51A-gene with 92 genotypes. Tandem Repeat-associated resistance genotypes accounted for 55.43% of the variants and were involved in 1728 isolates (94.63%). TR34/L98H and TR46/Y121F/T289A resistance mutations remained dominant, and increasingly additional SNPs in the Cyp51A-gene or changes to the gene promoter were observed. The G448S mutation was relatively common and present in various genetic backgrounds. This SNP was most often found in isolates harboring the TR46 resistance mechanism (8 variants) and was also observed in two variants in the TR34 genetic background. TR34 and TR46 resistance mutations are associated with 1170 (64.07%) isolates that exhibited a pan-azole resistance phenotype, 547 (29.96%) a multi-azole resistance phenotype, and 75 (4.11%) resistance to a single azole. TR34/L98H confers high itraconazole resistance, while T289A confers high voriconazole resistance in the TR46 background. Isolates with a G448S point mutation show high MICs for both voriconazole and itraconazole. The TR34/L98H/T289A/G448S isolate showed low itraconazole MICs but high voriconazole resistance, and mutations in the promoter region, TR34/C-86 G/L98H, and (T-66 G)/TR34/L98H variants, showed increased voriconazole and isavuconazole MIC compared with the parent phenotype. TR46/Y121F/M172I/T289A/G448S variant was observed with an increased itraconazole (GM MIC 16 mg/L, 1→16 mg/l) and decreased voriconazole (GM MIC 18.664 mg/l, 4→16 mg/l) compared with the parent MIC of TR46/Y121F/T289A, while TR92/Y121F/M172I/T289A/G448S and TR46/Y121F/ T289A/G448S variants showed the consistent MIC distribution with parent genotype. The variants with more combination mutations showed pan-azole resistance with increased MIC distribution.ConclusionOur survey showed a significant increase in resistance genotypes in clinical A. fumigatus over a period of 28 years. Azoles resistance phenotypes vary from resistant variants in clinical isolates; it is an implication for clinical A. fumigatus infection treatment options and antifungal stewardship.