Broth Microdilution Panels Research Articles

Comparison of two commercial broth microdilution panels for multidrug-resistant Gram-negative bacteria: Thermo Scientific™ Sensititre DKMGN vs. Beckman Coulter MicroScan NMDRM1.

Antimicrobial susceptibility testing (AST) using broth microdilution (BMD) is usually the reference method to obtain accurate minimum inhibitory concentrations and optimally manage infections with resistant organisms. Several commercial dry BMD are available for AST in clinical laboratories. Two commercial BMD panels for testing of multidrug-resistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. A total of 207 isolates were tested: three ATCC strains and one NCTC strain, six quality control strains from the Belgian National Antimicrobial Committee, and 197 clinical isolates, including carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 breakpoints version 13.1 were used to assign susceptibility categories. Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multi-resistant organisms showed CA and EA under 90%, that is, meropenem, imipenem, and colistin for Enterobacterales and meropenem and colistin for P. aeruginosa. For Enterobacterales, the NMDRM1 panel showed a significantly higher resistance rate for meropenem, imipenem, amikacin, and colistin. For carbapenems, the minimal inhibitory concentrations (MICs) were underestimated by the DKMGN panel, as already pointed out by a warning on the EUCAST website. To better assess carbapenem susceptibility in carbapenem-resistant organisms, the DKMGN panel now requires the use of a higher inoculum in the insert kit. However, for a given isolate whose susceptibility to carbapenems is not known, there is a risk of underestimating the MIC values. Our results show that colistin testing remains a challenge, highlighting the urgent need for the development of more accurate commercial methods. The use of a single commercial method cannot guarantee good precision in the determination of the MIC value for colistin.

Resistance to Antifungals in Non-albicans Candida Species Isolates in the Southeast Region

Background: Antimicrobial resistance is a growing problem in Candida spp., leading to treatment challenges and increased morbidity and mortality. The World Health Organization (WHO) fungal priority pathogens list classifies C. glabrata, C. tropicalis, and C. parapsilosis as high priority and leading causes of candidemia with high fluconazole resistance. In the US, these organisms are the most frequently isolated non-albicans Candida species. In 2016, the Antibiotic Resistance Laboratory Network (ARLN) was created to monitor resistance threats, including in Candida spp. This study describes the proportion of resistance in C. glabrata, C. parapsilosis, and C. tropicalis isolates sent to the Southeast ARLN from 2017 to 2023. Methods: This study evaluated C. glabrata, C. parapsilosis, and C. tropicalis submitted to the Southeast ARLN from Alabama, Florida, Georgia, Louisiana, Mississippi, and Tennessee from February 2017- September 2023. Species identification was confirmed by Bruker Biotyper matrix assisted laser desorption-ionization time of flight (MALDI-TOF). Antifungal susceptibility testing (AFST) was performed using TREK frozen broth microdilution panels. Minimum inhibitory concentration values from the clinical instrument were used to determine susceptibility based on Clinical and Laboratory Standards Institute (CLSI) standard interpretations from the 2020 CLSI M60 guidelines. Data were extracted from the laboratory information management system. Analyses were conducted using SAS v9.4. Results: AFST testing was performed on 660 C. glabrata, 500 C. parapsilosis, and 233 C. tropicalis isolates from within the Southeast region. The predominant specimen sources by species were blood 25.30% C. glabrata; other/not specified 27.80% C. parapsilosis; and lower respiratory 36.91% C. tropicalis. Resistance to fluconazole is as follows: C. glabrata, 12.88%; C. parapsilosis, 3.41%; C. tropicalis, 36.64%. Resistance to voriconazole is as follows: C. parapsilosis, 1.00%; C. tropicalis 30.04%. Resistance to at least one echinocandin (Anidulafungin, Capsofungin, Micafungin) is as follows: C. glabrata, 1.67%; C. parapsilosis, 0.60%; C. tropicalis, 0.43%. Overall, there was a decreasing trend in resistance to fluconazole, and voriconazole in all three species between 2017 and 2023. Conclusions: Antifungal resistance in non-albicans Candida species represents an emerging public health threat, however, within the Southeast region, ARLN data has shown a decreasing trend of azole resistance. This may be due in part to changes in reporting requirements and submission criteria from within the region. Nevertheless, C. tropicalis showed high resistance to azoles within the Southeast region. These Candida species should be monitored to inform clinical decision making and identify resistance patterns in other US regions due to their increase in resistance worldwide.

Comparison of Minimum Inhibitory Concentrations of Selected Antimicrobials for Non-Aureus Staphylococci, Enterococci, Lactococci, and Streptococci Isolated from Milk Samples of Cows with Clinical Mastitis.

The objective of this study was to compare the minimum inhibitory concentrations of antimicrobials included in a commercial broth microdilution panel among Gram-positive pathogens that caused non-severe clinical mastitis on three Michigan dairy farms. Duplicate quarter milk samples were collected from eligible quarters of cows enrolled in a randomized clinical trial, cultured in a university laboratory, and identified using MALDI-TOF. Etiologies were grouped by genus as Enterococcus species (n = 11), Lactococcus species (n = 44), non-aureus Staphylococcus species (n = 39), or Streptococcus species (n = 25). Minimum inhibitory concentrations (MICs) were determined using the mastitis panel of a commercially available broth microdilution test. In vitro susceptibility was determined using approved guidelines and included breakpoints for mastitis pathogens, or when not available, breakpoints from other species. Most isolates were inhibited at or below breakpoints that demonstrated in vitro susceptibility. The proportions of susceptible isolates varied among pathogens for pirlimycin, penicillin, and tetracycline. The greatest proportion of resistance was observed for pirlimycin, tetracycline, and sulfadimethoxine. Survival analysis was performed to evaluate differences in MICs among pathogen groups. MIC values varied among pathogens for ceftiofur, cephalothin, erythromycin, penicillin, pirlimycin, and tetracycline. However, nearly all isolates were susceptible to ceftiofur and cephalothin, indicating that pathogen differences in MIC are not likely clinically relevant, as these are the two most commonly administered mastitis treatments in the United States. While differences in vitro susceptibility were observed for some antimicrobials, susceptibility was high to cephalosporin-based IMM treatments that are most commonly used and did not vary among pathogens.

Antimicrobial Susceptibility Patterns Among a Large, Nationwide Cohort of Chryseobacterium Species Clinical Isolates

Abstract Background Chryseobacterium species are ubiquitous in nature that can be found in soil, water, sinks, and medical devices. They have been implicated in various infections including ventilator-associated pneumonia, catheter-related bloodstream infections (CRBSI), skin and soft tissue infections, and meningitis. C. indologenes has been shown to possess an extended-spectrum beta-lactamase (ESBL) and metallo-β-lactamase (MBL) that render it resistant to most antibiotics. Antimicrobial susceptibility testing (AST) data are required to help clinicians choose effective empiric antibiotics and to investigate potential resistance mechanisms. Methods Chryseobacterium spp. identification was determined prior to submission by client laboratories or performed in our laboratory by MALDI-TOF (Bruker Biotyper) or 16S rRNA gene sequencing. AST was performed on Chryseobacterium species isolates received from across the United States between April 2004 and December 2018. AST was performed using custom-made broth microdilution panels, and minimal inhibitory concentrations (MICs) were interpreted using CLSI M100 MIC breakpoints for non-Enterobacterales organisms. Isolates from a range of clinical specimens including blood, respiratory, wound, tissue, bone, and body fluids were included. Quality control was performed using Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Results were included only if the QC values were within range. The MIC50, MIC90, and MIC ranges for antimicrobials for frequently tested Chryseobacterium species were determined. Results AST was completed for 245 Chryseobacterium species clinical isolates, and the most frequently tested species were C. gleum/ indologenes (n = 78), C. hominis (n = 25), C. taklimakanense (n = 14), C. anthropi/ haifense (n = 12), C. arthrospharae (n = 12), C. bernardetii/jejuense (n = 3), C. pallidum (n = 3), and C. solincola/treverense (n = 3). 84 isolates were unable to be identified to species level, and were grouped as “Chryseobacterium spp.” Regarding susceptibility, across all Chryseobacterium species, the antibiotics with the most activity included minocycline (97.8%), trimethoprim-sulfamethoxazole (TMP-SMX; 95.5%), levofloxacin (92.3%), and ciprofloxacin (82.8%). Comparing the β-lactams, piperacillin-tazobactam had the most activity (59.7%), whereas ceftazidime (42.2%), cefepime (44.3%), meropenem (41.2%), and imipenem (46.3%) had similar low activity. The highest resistance rates were found for tobramycin (96%), aztreonam (94.6%), and gentamicin (62.7%). Among the Chryseobacterium isolates, certain species were found to be largely pan-susceptible, including C. anthropic/haifense, C. hominis, and C. taklimakanense. Conclusions Chryseobacterium species display significant resistance against β-lactam antibiotics, which are often used for empiric therapy. Minocycline, TMP-SMX, and fluoroquinolones have the most activity against Chryseobacterium species clinical isolates.

2747. Aztreonam-Avibactam Susceptibility Testing Program for Metallo-β-Lactamase-Producing Enterobacterales collected through the Antimicrobial Resistance Laboratory Network, March 2019 to April 2023

Abstract Background Metallo-β-lactamase-producing Enterobacterales (MBL-E) are a major public health concern as they cause infections with few effective treatment options. Aztreonam-avibactam (AZA) is a β-lactam/β-lactamase inhibitor combination agent in the drug development pipeline that is active against MBL-E. Though clinical studies are ongoing, the effective components can be administered by combining two FDA-approved drugs: aztreonam and ceftazidime-avibactam. In 2019, CDC initiated a program in the Antimicrobial Resistance Laboratory Network (AR Lab Network), the Expanded Antimicrobial Susceptibility Testing Program for Hard-to-Treat Infections, to provide antimicrobial susceptibility testing (AST) of MBL-E for AZA. We provide a summary of the program’s results. Methods Seven AR Lab Network laboratories validated the use of a digital dispenser to create custom broth microdilution panels for AZA AST. Testing requests must be for clinical decision-making purposes. Isolates must be an Enterobacterales and have laboratory results positive for an MBL gene (i.e., blaNDM, blaVIM, or blaIMP), or be not susceptible to all β-lactams tested. AZA testing results for confirmed MBL-E were reported back to submitters within 3 working days from receipt of the isolate and shared with CDC. Results From March 2019 through April 2023, AZA AST was performed for 250 isolates: 103 Escherichia coli, 86 Klebsiella pneumoniae, 46 Enterobacter cloacae complex, 8 K. oxytoca, 2 Providencia rettgeri, 1 K. aerogenes, 1 Morganella morganii, 1 Proteus mirabilis, 1 E. hormaechei, and 1 Citrobacter amalonaticus. Carbapenemase genes detected were: 206 blaNDM, 24 blaNDM/blaOXA-48-like, 14 blaKPC/blaNDM, 2 blaIMP, 2 blaKPC/blaIMP, and 2 blaNDM/blaIMP. All isolates were resistant to ceftazidime-avibactam; 227/250 (90.8%) were not susceptible to aztreonam (≥8 µg/mL). For isolates not susceptible to aztreonam, the addition of avibactam resulted in a median 128-fold MIC reduction of aztreonam. The MIC range of AZA was ≤0.03/4 - >64/4 µg/mL. Overall, the MIC50 and MIC90 of AZA were 0.5/4 µg/mL and 8/4 µg/mL, respectively; the MIC50 and MIC90 for E. coli were higher than other species, 4/4 µg/mL and 16/4 µg/mL. Conclusion Our data suggest that AZA has potent in vitro activity against MBL-E collected in the United States. Disclosures All Authors: No reported disclosures

Antimicrobial Resistance Laboratory Network's multisite evaluation of the ThermoFisher Sensititre GN7F broth microdilution panel for antimicrobial susceptibility testing.

In 2017, the Centers for Disease Control and Prevention (CDC) established the Antimicrobial Resistance Laboratory Network to improve domestic detection of multidrug-resistant organisms. CDC and four laboratories evaluated a commercial broth microdilution panel. Antimicrobial susceptibility testing using the Sensititre GN7F (ThermoFisher Scientific, Lenexa, KS) was evaluated by testing 100 CDC and Food and Drug Administration AR Isolate Bank isolates [40 Enterobacterales (ENT), 30 Pseudomonas aeruginosa (PSA), and 30 Acinetobacter baumannii (ACB)]. We assessed multiple amounts of transfer volume (TV) between the inoculum and tubed 11-mL cation-adjusted Mueller-Hinton broth: 1 µL [tribe Proteeae (P-tribe) only] and 10, 30, and 50 µL, resulting in respective CFU per milliter of 1 × 104, 1 × 105, 3 × 105, and 5 × 105. Four TV combinations were analyzed: standard (STD) [1 µL (P-tribe) and 10 µL], enhanced standard (E-STD) [1 µL (P-tribe) and 30 µL], 30 µL, and 50 µL. Essential agreement (EA), categorical agreement, major error (ME), and very major error (VME) were analyzed by organism then TVs. For ENT, the average EA across laboratories was <90% for 7 of 15 β-lactams using STD and E-STD TVs. As TVs increased, EA increased (>90%), and VMEs decreased. For PSA, EA improved as TVs increased; however, MEs also increased. For ACB, increased TVs provided slight EA improvements; all TVs yielded multiple VMEs and MEs. For ENT and ACB, Minimum inhibitory concentrations (MICs) trended downward using a 1 or 10 µL TV; there were no obvious MIC trends by TV for PSA. The public health and clinical consequences of missing resistance warrant increased TV of 30 µL for the GN7F, particularly for P-tribe, despite being considered "off-label" use.

A-273 Multicenter Assessment of the Accuracy of MIC Results for Piperacillin/Tazobactam with MicroScan Dried Gram-Negative MIC Panels using CLSI Breakpoints

Abstract Background MIC data from MicroScan Dried Gram-negative MIC (MSDGN) Panels with piperacillin/tazobactam were evaluated with CLSI-M100-ED32 breakpoints for Acinetobacter, Enterobacterales, Other-Non-Enterobacterales, and proposed CLSI breakpoints for Pseudomonas aeruginosa from a multicenter clinical study. MIC results were compared to results obtained with CLSI frozen broth microdilution panels. Materials/Methods The study included a total of 683 clinical isolates tested using Prompt and turbidity during efficacy and challenge phases. MSDGN panels were evaluated at three clinical sites, comparing MIC values obtained using MSDGN panels to MICs utilizing CLSI reference panel. MSDGN panels were incubated at 35 ± 1°C and read on WalkAway System, autoSCAN-4 instrument, and read visually at 16–20 h. Frozen reference panels were prepared according to CLSI methodology, incubated for 16–20 h for Enterobacterales, Other Non-Enterobacterales, and Pseudomonas aeruginosa and for 20–24 h for Acinetobacter and read visually. CLSI breakpoints(mg/L) used for interpretation of MIC results were: ≤16/4 S, 32/4–64/4 I, ≥128/4 R for Acinetobacter and Other Non Enterobacterales, ≤8/4 S, 16/4 SDD, ≥32/4 R for Enterobacterales, and ≤16/4 S, 32/4 I, &amp;gt;64/4 R for Pseudomonas aeruginosa. Results When compared to frozen reference panel results, essential and categorical agreements for all isolates tested are as follows with these recommendations: Due to elevated major error rates for S. marcescens and WalkAway reads with Prompt, results should be confirmed visually prior to reporting. Due to performance with P. rettgeri and S. liquefaciens complex, do not report drug, therapy, or MIC. Conclusion Piperacillin/tazobactam MIC results for gram-negative clinical isolates obtained with the MSDGN panel correlate with MICs obtained using frozen reference panels with updated CLSI interpretive criteria.

A Multicenter Evaluation of Overall Susceptibility and Antimicrobial Resistance Among Streptococcus pneumoniae Isolates.

S. pneumoniae ranks as the fourth-most lethal pathogen globally in terms of fatalities associated with or attributable to resistance. In this study, the Antimicrobial Testing Leadership and Surveillance (ATLAS) analysis from India aims to study the overall antimicrobial susceptibility (AMS) among pneumococcal isolates collected between 2018 and 2021. Non-duplicate clinically significant S. pneumoniae isolates were collected between 2018 and 2021. In vitro activity of antibiotics was assessed against S. pneumoniae. Susceptibility was confirmed at an International Health Management Associates (IHMA) laboratory using supplied broth microdilution panels (Omron Microscan Systems, Inc., Omron Corp., Kyoto, Japan), according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for all antibiotics. Of the total 86 non-duplicate isolates of Streptococcus pneumoniae collected from the tertiary care centers, the proportion of isolates increased from 8.14% (n=7) in 2018 to 43.02% (n=37) in 2020. Most isolates (n = 18; 48.65%) were collected from the age group of 31-60 years in the year 2020. Erythromycin revealed a decrease in susceptibility from the year 2018 (71.43%) to 2020 (16.22%). A decreased susceptibility of 90% was recorded for levofloxacin in the year 2021. Meropenem revealed a decrease in susceptibility from the year 2018 (85.71%) to 2020 (35.14%). Penicillin susceptibility decreased from 37.5% in 2019 to 27.03% in the year 2020. Clindamycin indicated a 100% susceptibility in the year 2018 which then decreased to 71.88% in 2019 and 56.76% in 2020. Linezolid and vancomycin were found to have uniform susceptibility of 100% throughout the years from 2018 to 2021. An increase in resistance to penicillin and macrolides among S. pneumoniae isolates was observed in the Indian population. Addressing the elevating rates of S. pneumoniae resistance may require pneumococcal conjugate vaccines (PCVs) with expanded serotype coverage and targeted antimicrobial stewardship efforts.

345. Performance Evaluation of the LifeScale AST Rapid Automated Antimicrobial Susceptibility System

Abstract Background Reducing the time for antimicrobial susceptibility test (AST) results directly from positive blood cultures is essential for managing patients with suspected sepsis/bacteremia caused by the most predominate Gram-negative bacteria. The aim of this study was to evaluate the LifeScale AST system using a custom LifeScale 96-well broth microdilution panel. The system employs a microfluidic sensor that detects and measures the mass of individual organisms at high throughput with MIC results available within 4-6 hours. Methods Fresh blood cultures detected as positive by a continuous monitoring blood culture system and confirmed by Gram stain for Gram-negative rods were enrolled in the study. If a mixed Gram stain was observed, the sample was ineligible for the study. Testing was performed within 12 hours of the blood culture being flagged as positive. LifeScale performance was evaluated by comparing MICs and interpretive results to the MicroScan Walkaway 96. The following metrics were assessed: Essential Agreement (EA), Category Agreement (CA), Very Major Discrepancy (VM), Major Discrepancy (M), and Minor Discrepancy (Min). Results A total of 65 samples were tested that met criteria for LifeScale (intended) claimed species: E. coli (N = 45), Klebsiella pneumoniae (N = 9), Pseudomonas aeruginosa (N = 8), Klebsiella aerogenes (N = 2), and one isolate each of Acinetobacter lwoffi, Acinetobacter species, and Klebsiella oxytoca. Three organisms, 2 E. coli, one P. aeruginosa, were excluded due to insufficient growth or amplitude error. The average time to results was 4.31 hours. Following resolution of discrepant AST results, there was 97.92% final agreement between LifeScale and MicroScan Walkaway 96, 36% categorical agreement, no very major discrepancies, and 0.16% major discrepancies. Conclusion The LifeScale system provided reliable AST results for Gram-negative organisms directly from positive blood cultures with minimal hands-on time (approximately less than 8 minutes), allowing for more rapid antimicrobial management compared to standard methods. This is the first AST system that detects and measures the mass of each organism compared to traditional growth-based AST methods. Results for additional organisms will be provided once development is complete. Disclosures All Authors: No reported disclosures.

668. Presence of the Narrow-Spectrum OXA-1 Beta-Lactamase Enzyme is not Associated with Elevated Ceftolozane-Tazobactam MIC Values among ESBL-Producing Escherichia coli Clinical Isolates (CANWARD, 2011-2018)

Abstract Background Beta-lactam-beta-lactamase inhibitor combinations have been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli as an antimicrobial stewardship initiative. However, in a recent randomized trial evaluating patients with bacteremia, presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Enterobacterales was associated with higher piperacillin-tazobactam MICs, and in turn excess mortality among patients treated with piperacillin-tazobactam relative to meropenem. The purpose of this study was to determine whether the in vitro activity of ceftolozane-tazobactam versus ESBL-producing E. coli is similarly compromised by the presence of OXA-1. Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2011 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection. Results In total, 485 ESBL-producing E. coli identified as part of the CANWARD study were included. The majority of isolates (91.3%; 443/485) harbored a CTX-M ESBL enzyme. OXA-1 was present in 39.6% (192/485) of isolates. OXA-1 was detected in 62.5% (187/299) of isolates with a CTX-M-15 ESBL enzyme versus only 2.7% (5/186) of isolates with other ESBL enzyme types. Ceftolozane-tazobactam MIC50 and MIC90 values were identical (0.25 µg/mL and 1 µg/mL, respectively) for the isolate subsets with and without OXA-1. Overall, 97.4% and 96.9% of isolates with and without OXA-1 remained susceptible (CLSI breakpoint) to ceftolozane-tazobactam. Conclusion The presence of OXA-1 among ESBL-producing E. coli clinical isolates was not associated with an elevation in MIC values for ceftolozane-tazobactam. These data support further evaluation of ceftolozane-tazobactam as an alternative to carbapenems for the treatment of infections caused by ESBL-producing E. coli, as is being done with the MERINO-3 trial. Disclosures George Zhanel, PhD, Merck: Grant/Research Support.

230. Multicenter Assessment of the Accuracy of MIC Results for Colistin with MicroScan Dried Gram Negative MIC Panels using CLSI Breakpoints

Abstract Background A multicenter study was performed to evaluate the accuracy of MIC results for colistin on a MicroScan Dried Gram Negative MIC (MSDGN) Panel when compared to results obtained with frozen broth microdilution panels prepared according to CLSI/EUCAST methodology. Note: Colistin is not available in all countries. Methods MSDGN panels were evaluated at three clinical sites (including Beckman Coulter) by comparing MIC values obtained using the MSDGN panels to MICs obtained utilizing a CLSI/EUCAST broth microdilution reference panel. The study included 407 clinical isolates tested using the turbidity and Prompt® methods of inoculation. MSDGN panels were incubated in the WalkAway System (35 ± 1°C) and read on the WalkAway System, the autoSCAN-4 instrument, and read visually at 16-20 hours. Frozen reference panels were prepared and inoculated according to CLSI/EUCAST Joint Working Group Recommendations for MIC determination of Colistin (Polymyxin E) and incubated for 16-20 hours and read visually. CLSI M100 ED32 breakpoints (µg/mL) used for interpretation of MIC results were: Enterobacterales ≤ 2 I, ≥ 4 R; P. aeruginosa ≤ 2 I, ≥ 4 R. Results Essential, categorical agreement, and categorical errors were calculated using MIC results from WalkAway reads for MSDGN panels in Table 1. All other read methods yielded similar results. Categorical agreement was not calculated for Acinetobacter spp. as MIC only is reported. Table 1- Results Conclusion This multicenter study showed that colistin MIC results for Gram Negative bacteria obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels with CLSI interpretive criteria. © 2022 Beckman Coulter. All rights reserved. All other trademarks are the property of their respective owners. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the U.S. and other countries. Disclosures Stefan Riedel, M.D., PhD., D(ABMM), FCAP, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Maria Traczewski, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Denise Beasley, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Regina Brookman, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Jose Diaz, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Zabrina Lockett, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter Jennifer Chau, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter.

Antifungal susceptibility profiles and drug resistance mechanisms of clinical Candida duobushaemulonii isolates from China.

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 μg/ml. One isolate showed a high MIC value of ≥64 μg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment.

Validation of Three MicroScan® Antimicrobial Susceptibility Testing Plates Designed for Low-Resource Settings.

Easy and robust antimicrobial susceptibility testing (AST) methods are essential in clinical bacteriology laboratories (CBL) in low-resource settings (LRS). We evaluated the Beckman Coulter MicroScan lyophilized broth microdilution panel designed to support Médecins Sans Frontières (MSF) CBL activity in difficult settings, in particular with the Mini-Lab. We evaluated the custom-designed MSF MicroScan Gram-pos microplate (MICPOS1) for Staphylococcus and Enterococcus species, MSF MicroScan Gram-neg microplate (MICNEG1) for Gram-negative bacilli, and MSF MicroScan Fastidious microplate (MICFAST1) for Streptococci and Haemophilus species using 387 isolates from routine CBLs from LRS against the reference methods. Results showed that, for all selected antibiotics on the three panels, the proportion of the category agreement was above 90% and the proportion of major and very major errors was below 3%, as per ISO standards. The use of the Prompt inoculation system was found to increase the MIC and the major error rate for some antibiotics when testing Staphylococci. The readability of the manufacturer’s user manual was considered challenging for low-skilled staff. The inoculations and readings of the panels were estimated as easy to use. In conclusion, the three MSF MicroScan MIC panels performed well against clinical isolates from LRS and provided a convenient, robust, and standardized AST method for use in CBL in LRS.

Antimicrobial activity of ceftazidime-avibactam and comparators against levofloxacin-resistant Escherichia coli collected from four geographic regions, 2012\u20132018

BackgroundIncreases in resistance to fluoroquinolones have been correlated with the use of levofloxacin in the treatment of infections caused by Escherichia coli. The analysis presents the in vitro activity of ceftazidime-avibactam and comparator agents against 10,840 levofloxacin-resistant E. coli isolates collected from four geographic regions (Africa/Middle East, Europe, Asia/South Pacific, Latin America) between 2012 and 2018.MethodsNon-duplicate clinical isolates of E. coli were collected from participating centres and shipped to IHMA, Inc., (Schaumburg, IL, USA). Susceptibility testing was performed with frozen broth microdilution panels manufactured by IHMA, according to CLSI guidelines. Levofloxacin-resistance was defined at a minimum inhibitory concentration of ≥ 2 mg/L. Isolates collected between 2012 and 2015 were tested for extended-spectrum β-lactamase (ESBL) activity by determining susceptibility to cefotaxime, cefotaxime-clavulanate, ceftazidime, and ceftazidime-clavulanate as recommended by CLSI guidelines. Isolates collected between 2016 and 2018 were identified as ESBL-positive by genotype using multiplex polymerase chain reaction assays.ResultsA total of 74.8% of levofloxacin-resistant E. coli isolates in the analysis were from three culture sources: urinary tract infections (N = 3229; 29.8%), skin and skin structure infections (N = 2564; 23.7%) and intra-abdominal infections (N = 2313; 21.3%). Susceptibility rates to ceftazidime-avibactam were consistently high in all regions against both ESBL-positive (97.0% in Asia/South Pacific to 99.7% in Africa/Middle East and Latin America) and ESBL-negative isolates (99.4% in Asia/South Pacific to 100% in Latin America). Susceptibility was also high in each region among ESBL-positive and ESBL-negative isolates to colistin (≥ 98.5%), imipenem (≥ 96.5%), meropenem (≥ 96.5%) and tigecycline (≥ 94.1%).ConclusionsAntimicrobial susceptibility to ceftazidime-avibactam among levofloxacin-resistant E. coli isolates, including ESBL-positive isolates, collected from four geographical regions between 2012 and 2018 was consistently high. Susceptibility to the comparator agents colistin, tigecycline, imipenem and meropenem was also high.

P43 In vitro activity of cefiderocol and comparators against Gram-negative pathogens: ARTEMIS study in the UK

ObjectivesCefiderocol (CFDC) is a novel siderophore cephalosporin approved in Europe for the treatment of infections caused by aerobic Gram-negative (GN) bacteria in adults with limited treatment options. The aim of the ARTEMIS study was to evaluate the in vitro activity of CFDC and comparators against recent clinical isolates collected across five countries in Europe. Here we report susceptibility data from isolates collected in the UK.MethodsFrom January to December 2020, GN clinical isolates were collected from hospitalized patients from all infection sites (excluding the urinary tract). Duplicate isolates of the same species from a single patient were excluded. As a prespecified target, each laboratory collected 75 isolates, with: 20 Klebsiella spp., 20 other Enterobacterales, 20 Pseudomonas aeruginosa and 15 Acinetobacter baumannii isolates expected to be included. CFDC susceptibility testing was conducted using disc diffusion (with 30 μg discs) on Mueller–Hinton agar and Sensititre™ broth microdilution (BMD) panels [EUMDROXF; centrally tested at International Health Management Associates (IHMA)]. Susceptibility by disc diffusion was reported using zone diameter breakpoints (BPs) of ≥22 mm (or ≥17 mm for A. baumannii isolates, corresponding to MIC values below the pharmacokinetic/pharmacodynamic BPs of ≤2 mg/L). Comparator susceptibility was determined using custom research use only Sensititre™ BMD panels (CMP2SHIH) according to the EUCAST method for BMD. Antimicrobial susceptibility was interpreted according to EUCAST clinical BPs (v.11 2021).ResultsIn total, 517 isolates were collected from nine UK hospitals, of which: 308 (59.6%) were Enterobacterales [including 147 (28.4%) Klebsiella spp.], 148 (28.6%) were P. aeruginosa and 33 (6.4%) were A. baumannii. The most common sites of infection were bloodstream (n = 245; 47.4%), respiratory tract (n = 158; 30.6%) and skin (n = 59; 11.4%). A high percentage of Enterobacterales (90.2%), P. aeruginosa (96.6%) and A. baumannii (96.9%) isolates were susceptible to CFDC by disc diffusion. By central laboratory testing (MIC), 99.0% of Enterobacterales, 99.3% of P. aeruginosa and 93.9% of A. baumannii isolates were susceptible to CFDC. High susceptibility rates (>85%) were also observed for all comparator agents (Table 1). A total of 32/517 (6.2%) isolates were carbapenem resistant, the majority of which (22/32, 68.8%) were susceptible to CFDC by disc diffusion.Table 1.CFDC susceptibility based on disc zone diameters; CFDC and comparator agent susceptibility based on MIC from BMD (EUCAST clinical BPs v.11, 2021)Susceptibility, n/N (%)CFDC (disc)No. isolates in ATUCFDC (MIC)MEMaC/TCZAMVBI/RCSTEnterobacterales277/307 (90.2)40/307 (13.0)285/288 (99.0)295/308 (95.8)264/308 (85.7)298/307 (97.1)303/308 (98.4)286/306 (93.5)265/307 (86.3) P. aeruginosa 143/148 (96.6) 8/148 (5.4)144/145 (99.3)135/148 (91.2)140/148 (94.6)141/148 (95.3)137/148 (92.6)133/147 (90.5)142/148 (95.9) A. baumannii 31/32 (96.9)N/A31/33 (93.9)30/33 (90.9)N/AN/AN/A30/33 (90.9)30/33 (90.9)ATU, area of technical uncertainty; CFDC, cefiderocol; CST, colistin; C/T, ceftolozane/tazobactam; CZA, ceftazidime/avibactam; I/R, imipenem/relebactam; MEM, meropenem; MVB, meropenem/vaborbactam.aIsolates were defined as MEM-susceptible with a BP of ≤8 mg/L [relating to high-dose extended-infusion (2 g, 3 h infusion) MEM], based on EUCAST recommendations.ConclusionsAmong clinical GN isolates collected from UK hospitals in 2020, a high percentage (98.6%), including carbapenem-resistant isolates, were susceptible to CFDC by BMD. These data support the use of CFDC in patients with GN infections and limited treatment options. The differences identified between EUCAST disc diffusion and BMD using Sensititre™ panels for CFDC highlight that disc diffusion underestimates Enterobacterales susceptibility to CFDC, which is mainly a result of the area of technical uncertainty (where isolates with MIC of 2 mg/L have a zone diameter of <22 mm and are characterized as resistant). This requires further investigation to explore whether the EUCAST zone diameter BP is optimal for CFDC disc testing.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)

1261. Antimicrobial Susceptibilities of Carbapenem-Resistant Klebsiella pneumoniae (CRKP) Isolates from a Multi-state Network of Long-term Acute Care Hospitals (LTACHs)

Abstract Background CRKP infection is common among LTACH patients. However, CRKP antimicrobial susceptibility testing (AST) with newer antibiotics has not been described in this population. In this study, we performed AST on CRKP in LTACHs. Methods CRKP clinical cultures were collected from 21 Kindred Healthcare LTACHs (12 southern California, 6 Texas, 2 Florida, 1 Kentucky) from 8/1/14-7/25/15. AST was performed using a custom SensititreTM broth microdilution panel (ThermoFisher Scientific, Waltham, MA). 2021 Clinical &amp; Laboratory Standards Institute interpretive criteria were used for all agents except plazomicin (PLZ), for which US Food and Drug Administration breakpoints were used. Results 459 CRKP clinical isolates were collected, including 254 (55.5%) respiratory, 155 (33.8%) urine, 39 (8.5%) blood, and 10 (2.2%) wound cultures. Most (419, 91.0%) were from southern California. 151 (32.9%) were colistin-resistant. 42 (9.2%) isolates were non-susceptible to meropenem-vaborbactam (MVB), 16 (8.9%) to imipenem-relebactam (IPR), 4 (0.9%) to ceftazidime-avibactam (CZA), and 3 (0.7%) to PLZ. Cross-resistance patterns are shown in Table 1. Among southern California facilities, there was significant inter-facility variation in MVB non-susceptibility (Pearson’s chi-squared test, p=0.005) but not in CZA, IPR, or PLZ non-susceptibility. Table 1. Cross-resistance to antimicrobials among carbapenem-resistant Klebsiella pneumoniae isolates from 21 Kindred long-term acute care facilities, August 1, 2014 – July 25, 2015 (N=459). Number of isolates and column percentages reported. 2021 Clinical &amp; Laboratory Standards Institute interpretive criteria are used for all agents except for PLZ, for which US Food and Drug Administration breakpoints are used. Abbreviations: CST = colistin. CZA = ceftazidime-avibactam. I = intermediate. IPR = imipenem-relebactam. MIC = minimum inhibitory concentration. MVB = meropenem-vaborbactam. PLZ = plazomicin. R = resistant. S = susceptible. Conclusion In this sample of CRKP isolates from LTACHs in 2014-2015, nearly 10% of isolates were non-susceptible to MVB or IPR, respectively, despite neither agent being commercially available at the time. In contrast, there was a low prevalence of non-susceptibility to CZA, which received initial US Food and Drug Association approval in 2/2015, and PLZ, which was not commercially available during the study period. Cross-resistance between CZA and carbapenem/beta-lactamase combination antibiotics was uncommon. Future studies should evaluate CRKP susceptibilities to new agents in post-marketing cohorts and identify risk factors for resistance. Disclosures Jennifer Han, MD, MSCE, GlaxoSmithKline (Employee, Shareholder) Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB))

Evaluation of cefiderocol activity for Gram-negative bacteria and performance of cefiderocol disk diffusion testing using multiple brands of Mueller Hinton Agar

Abstract Cefidericol is a cephalosporin-siderophore antibiotic for the treatment of multidrug resistant Gram-negative bacteria. Similar to other cephalosporin antibiotics, the lethal mechanism of action is due to inhibition of penicillin binding proteins leading to lysis of the bacteria. However, unlike previously developed antibiotics, the siderophore portion of cefidericol is able to bind iron and then be actively transported into the periplasmic space. To ascertain the feasibility of cefidericol antibiotic susceptibility testing in the Barnes-Jewish Clinical Microbiology Laboratory, we collected a cohort of multidrug Enterobacteriacae (5 Enterobacter cloace, 8 Escherichia coli, 12 Klebsiella pneumoniae), Pseudomonas aeruginosa (n=23), Stenotrophomonas maltophila (n=24), and Acinetobacter baumannii (n=25). We evaluated activity of cefidericol on these strains, and the performance of disk diffusion using three different brands of Mueller-Hinton Agar (BD, Hardy, and Remel). The reference method for comparison was an FDA-cleared broth microdilution panel containing cefidericol (ThermoFisher Scientific). Using CLSI breakpoints, we found that disk diffusion with BD agar had 96% categorical agreement for Enterobacterales, 100% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. We found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Finally, we found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Minor errors on any media never exceed 4% and there were no very major errors. Resistance to cefidericol within our cohort of selected antibiotic resistant bacteria was rare, one E. coli isolate and two P. aeruginosa isolates had minimal inhibitory concentrations (MICs) &amp;gt; 32 μg/mL. The highest MICs for one isolate of A. baumannii and one isolate S. maltophila was 8 μg/mL and 4 μg/mL, respectively, both of which were intermediate. There was no difference in the distribution of zone disk diffusion diameter for A. baumannii or Enterobacterales. However, there was a significant difference in the distribution of zone disk diffusion diameters for P. aeruginosa and S. maltophila on BD vs Hardy agar. The median for P. aeruginosa on BD is 25 mm while it is 29 mm on Hardy. The trend for S. maltophila is the opposite as the median for BD was 31.5 mm and 28.5 mm for Hardy. Use of FDA vs CLSI vs EUCAST breakpoints significantly changes outcome of susceptibility testing for broth microdilution and disk diffusion. As one example for broth microdilution of A. baumannii, we had one isolate intermediate using CLSI breakpoints, 4 resistant using EUCAST breakpoints, and 4 resistant and 3 intermediate isolates using FDA breakpoints. Our work demonstrates that cefedericol testing can be performed in a routine format, with certain organismal differences on Mueller-Hinton agar, and that different interpretative criteria significantly change outcomes.

ESBL-positive Escherichia coli and Klebsiella pneumoniae isolates from across Canada: CANWARD surveillance study, 2007-18.

ESBL-producing Escherichia coli and Klebsiella pneumoniae are pathogens of increasing importance in Canada and elsewhere in the world. The purpose of this study was to phenotypically and molecularly characterize ESBL-producing E. coli and K. pneumoniae clinical isolates obtained from patients attending Canadian hospitals over a 12 year period. Isolates were collected between January 2007 and December 2018 as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the CLSI (M100) phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent WGS. In total, 671 E. coli and 141 K. pneumoniae were confirmed to be ESBL producers. The annual proportion of ESBL-producing isolates increased for both E. coli (from 3.3% in 2007 to 11.2% in 2018; P < 0.0001) and K. pneumoniae (from 1.3% in 2007 to 9.3% in 2018; P < 0.0001). The most frequent STs were ST131 for E. coli [62.4% (419/671) of isolates] and ST11 [7.8% (11/141)] and ST147 [7.8% (11/141)] for K. pneumoniae. Overall, 97.2% of ESBL-producing E. coli and K. pneumoniae isolates were MDR. blaCTX-M-15 predominated in both ESBL-producing E. coli (62.3% of isolates) and ESBL-producing K. pneumoniae (48.9% of isolates). The proportion of ESBL-producing E. coli, especially ST131, and K. pneumoniae, especially ST11 and ST147, in Canada increased significantly from 2007 to 2018. Continued prospective surveillance of these evolving MDR and at times XDR pathogens is imperative.

In vitro activity of ceftazidime-avibactam and comparators against Enterobacterales and Pseudomonas aeruginosa isolates from Central Europe and Israel, 2014–2017 and 2018

Between 2014 and 2017, 6,662 Enterobacterales and 1,953 P. aeruginosa isolates were collected by 19 centers in four central European countries and Israel. A further 2,585 Enterobacterales and 707 P. aeruginosa isolates were collected in 2018 by 28 centers in seven European countries and Israel as part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) study. A central laboratory performed antimicrobial susceptibility testing using broth microdilution panels according to Clinical Laboratory Standards Institute (CLSI) guidelines. Susceptibility rates among Enterobacterales were highest to ceftazidime-avibactam (≥98.5%), colistin (≥97.3%), and meropenem (≥95.8%). Ceftazidime-resistant and multidrug-resistant (MDR) Enterobacterales subsets were highly susceptible to ceftazidime-avibactam (≥94.9%) and colistin (≥94.7%). Susceptibility rates to colistin among all P. aeruginosa were ≥97.4% and were ≥96.3% among ceftazidime-resistant and MDR subsets. Susceptibility rates to ceftazidime-avibactam were 91.9% (2014-2017), 86.3% (2018) and, in common with comparator agents, were lower among ceftazidime-resistant (≥51.7%) and MDR isolates (≥57.1%).