Reference Broth Microdilution Methods Research Articles

Evaluation of VITEK® 2 AST cards (AST-N376 and AST-N397) for susceptibility testing of challenging Gram negatives

Due to the increasing diffusion of MDR/XDR Gram-negatives it is necessary to offer reliable antibiotic susceptibility testing (AST), which also include new drugs. Here we evaluated the performances of the VITEK®2 AST-N376 and the AST-N397 cards. A collection of 180 clinical Gram-negative bacteria, producing relevant resistance mechanisms, were tested using VITEK 2 and MERLIN, in parallel. Discrepancies between the 2 systems were solved by the reference broth microdilution method. The workflow timing of the VITEK®2 system was also assessed. Overall, the VITEK®2 cards proved to be reliable in determining AST for the molecules evaluated, even if compliance with ISO acceptance criteria for accuracy assessment was not reached for some combinations and showed a short hands-on time for panels preparation. In conclusion, VITEK®2 is a valid system that ensures accurate results for AST of the molecules evaluated in this study and speeds up the workflow in the laboratory of diagnostic microbiology.

Comparison of Disk Diffusion, E-Test, and Broth Microdilution Methods for Testing In Vitro Activity of Cefiderocol in Acinetobacter baumannii.

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant-A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer's guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii.

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.

A Selective Culture Medium for Screening Cefiderocol Resistance in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii.

Cefiderocol (FDC) is a siderophore cephalosporin with a broad spectrum of activity against many multidrug-resistant Gram-negative bacteria. Acquired resistance to FDC has been already reported among Gram-negative isolates, thus highlighting the need for rapid and accurate identification of such resistant pathogens, in order to control their spread. Therefore, the SuperFDC medium was developed to screen FDC-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. After testing several culture conditions, a selective medium was set up by supplementing an iron-depleted agar medium with 8 μg/mL of FDC and evaluated with a collection of 68 FDC-susceptible and 33 FDC-resistant Gram-negative isolates exhibiting a variety of β-lactam resistance mechanisms. The sensitivity and specificity of detection of this medium were evaluated at 97% and 100%, respectively. In comparison with the reference broth microdilution method, only 3% very major errors were found. In addition, excellent detection performances were obtained by testing spiked stools with a lower limit of detection ranging between 100 and 103 CFU/mL. The SuperFDC medium allows detection of FDC-resistant Gram-negative isolates regardless of their corresponding resistance mechanisms.

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.

Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam activities against multidrug-resistant Enterobacterales from United States Medical Centers (2018–2022)

A total of 35,360 Enterobacterales isolates were consecutively collected from 75 US medical centers in 2018–2022. Among these isolates, 2612 (7.4%) were categorized as multidrug-resistant (MDR). Isolates were susceptibility tested by reference broth microdilution methods. Carbapenem-resistant Enterobacterales (CRE) were screened for carbapenemase (CPE) genes by whole genome sequencing. The highest MDR rates was observed among Klebsiella pneumoniae (12.2%), followed by Raoultella spp. (10.9%) and Providencia stuartii (9.8%). Ceftazidime-avibactam and meropenem-vaborbactam were very active and showed identical susceptibility rates against MDR isolates (97.9%). Imipenem-relebactam (93.5% susceptible [S]) exhibited slightly lower susceptibility rates due to its limited activity against Morganellaceae family. The most active β-lactamase inhibitor combination (BLI) against CRE isolates (n = 310) was ceftazidime-avibactam (84.2%S), followed by meropenem-vaborbactam (81.9%S) and imipenem-relebactam (74.8%S). All 3 BLIs were very active against KPC producers and none were active against MBL producers. Ceftazidime-avibactam exhibited greater activity against OXA-48–type producers than meropenem-vaborbactam and imipenem-vaborbactam.

Comparison of Broth Microdilution, Disk Diffusion and Strip Test Methods for Cefiderocol Antimicrobial Susceptibility Testing on KPC-Producing Klebsiella pneumoniae

The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values (p value < 0.001) compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations.

Strategies for Updating Piperacillin-Tazobactam Breakpoints.

In this issue of the Journal of Clinical Microbiology, C. Manuel, R. Maynard, A. Abbott, K. Adams, et al. (J Clin Microbiol 61:e01617-22, 2023, https://doi.org/10.1128/JCM.01617-22) describe a multisite study evaluation of piperacillin-tazobactam (TZP) MIC testing on three U.S. Food and Drug Administration (FDA)-cleared antimicrobial susceptibility testing (AST) devices compared to the reference broth microdilution method for organisms belonging to Enterobacterales. Although overall performance of each of the three devices was comparable when applying either FDA or Clinical and Laboratory Standards Institute (CLSI) TZP breakpoints, failure to update to the current CLSI breakpoints may result in falsely categorizing as many as 20% of the TZP-resistant isolates as susceptible. The impact of not updating clinical breakpoints and strategies for implementation of updated breakpoints are discussed.

Performance of Ceftazidime-Avibactam 30/20-μg and 10/4-μg Disks for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

Ceftazidime-avibactam, a new β-lactam-β-lactamase inhibitor combination, is active against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa isolates and has became available for clinical use in China in the latter half of 2019. In this study, we evaluated the performance of the disk diffusion test with ceftazidime-avibactam 10/4-μg and 30/20-μg disks, compared with the reference broth microdilution method, with a collection of 467 Enterobacterales and 182 P. aeruginosa nonduplicate clinical isolates. The results of antimicrobial susceptibility testing indicated that the categorical agreement (CA) of ceftazidime-avibactam 10/4-μg disk testing for all tested Enterobacterales isolates was 99.8%, with 0.5% very major errors (VMEs) and no major error (ME). The CA of ceftazidime-avibactam 10/4-μg disk testing for all tested P. aeruginosa isolates was 87.9%, with 15.5% MEs and no VME. The CA of ceftazidime-avibactam 30/20-μg disk testing for all tested Enterobacterales isolates was 99.4%, with 1.5% VMEs and no ME. The CA of ceftazidime-avibactam 30/20-μg disk testing for all tested P. aeruginosa isolates was 91.8%, with 2.5% VMEs and 9.9% MEs. Overall, ceftazidime-avibactam 10/4-μg disk testing showed superior performance and was more suitable for assessment of the susceptibility of Enterobacterales and P. aeruginosa isolates. IMPORTANCE Multidrug-resistant Enterobacterales and P. aeruginosa strains have become a global public threat, with the emergence and prevalence of plasmid-mediated extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases disseminated worldwide. Ceftazidime-avibactam, which is commercially available, has shown excellent in vitro activity against multidrug-resistant and carbapenem-resistant Enterobacterales and P. aeruginosa isolates. Moreover, ceftazidime-avibactam has shown promise in treating infections caused by multidrug-resistant and carbapenem-resistant isolates. The disk diffusion test for ceftazidime-avibactam is the most common antimicrobial susceptibility testing method in most laboratories in China. The accurate detection of ceftazidime-avibactam susceptibility is of great significance for the rational clinical application of drugs. Here, we evaluated the performance of the ceftazidime-avibactam 10/4-μg and 30/20-μg disk diffusion tests, compared with the reference broth microdilution method, with clinical Enterobacterales and P. aeruginosa isolates.

Trends of β-Lactamase Occurrence Among Escherichia coli and Klebsiella pneumoniae in United States Hospitals During a 5-Year Period and Activity of Antimicrobial Agents Against Isolates Stratified by β-Lactamase Type.

The temporal and longitudinal trends of β-lactamases and their associated susceptibility patterns were analyzed for Escherichia coli and Klebsiella pneumoniae isolates consecutively collected in 56 United States hospitals during 2016-2020. Isolates (n = 19 453) were susceptibility tested by reference broth microdilution methods. Isolates that displayed minimum inhibitory concentration (MIC) values ≥2 mg/L for at least 2 of the following compounds-ceftazidime, ceftriaxone, aztreonam, or cefepime-or resistance to the carbapenems were submitted to whole genome sequencing for identification of β-lactamases. Longitudinal and temporal trends were determined by slope coefficient. New CTX-M and OXA-1 variants were characterized. Extended-spectrum β-lactamases (ESBLs) were detected among 88.0% of the isolates that displayed elevated cephalosporin/aztreonam MICs without carbapenem resistance. bla CTX-M-15 was detected among 55.5% of the ESBL producers. ESBL rates were stable over time, but significant increases were noted among bloodstream infection and K pneumoniae isolates, mainly driven by an increase in bla CTX-M. Carbapenem resistance and carbapenemase genes were noted among 166 and 145 isolates, respectively, including 137 bla KPC, 6 bla SME, 3 bla OXA-48-like, and 3 bla NDM. Ceftazidime-avibactam and carbapenems were very active (>99% susceptibility) against ESBL producers without carbapenem resistance. Ceftazidime-avibactam inhibited 97.0% of the carbapenem-resistant isolates. This agent and meropenem-vaborbactam inhibited 96.4% and 85.0% of the 2020 isolates, respectively. Overall, ESBL-producing isolates were stable, but an increase was noted for K pneumoniae isolates driven by CTX-M production. Carbapenem-resistant Enterobacterales rates decreased in the study period. The prevalence of metallo-β-lactamases and OXA-48-like remains low. Continuous surveillance of β-lactamase-producing isolates is prudent.

Evaluation of Broth Disk Elution Method to Determine Colistin Resistance in Klebsiella pneumoniae and Escherichia coli Strains.

The reference broth microdilution (rBMD) method for the determination of colistin resistance is very laborious and time consuming, and many manual errors can occur. There are also limitations in detection of colistin heteroresistance. Therefore, alternative methods with satisfactory performance are required for routine laboratory work. In our study, the colistin broth disk elution (CBDE) method recommended by the Clinical and Laboratory Standards Institute (CLSI) for the detection of colistin resistance in routine applications was compared with rBMD. The compatibility and error rates of the method were evaluated and its usability in routine laboratory studies was examined. Eighty-nine multidrug resistant Klebsiella pneumoniae and five Echerichia coli strains isolated from various clinical specimens were included in the study. Identification of strains and antibiotic susceptibility tests were performed with MALDI-TOF MS (bioMerieux, France) and Vitek-2 (bioMerieux) system. Minimum inhibitory concentration (MIC) was studied in 0.125 - 128 mg/L dilution range by using polystyrene microplate and colistin sulfate salt according to ISO-standard (20776-1) recommendations for the reference BMD test. The CBDE method was performed according to the CLSI recommendations. Isolates with MIC ≤ 2 mg/L were considered susceptible, while isolates with MIC > 2 mg/L were considered resistant according to EUCAST recommendations. The performance of the CBDE method was evaluated according to ISO criteria (Categorical agreement > 90%; major error and very major error rates < 3%). Categorical agreement for all 58 and 36 isolates found to be resistant and susceptible, respectively, by rBMD was found to be 100% with CBDE test. Since < 1 and > 4 µg/mL values could not be determined with the CBDE method, essential agreement (EA) could not be calculated. No major or very major errors were detected. Our results showed that the performance of the CBDE test is good when compared to the rBMD method. According to our data, we believe that the CBDE method can be used in routine laboratories for the detection of colistin resistance.

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.

124. Activity of Novel b-Lactam/b-Lactamase Inhibitor (BL/BLI) Combinations Against AmpC-Producing Species Collected in United States Hospitals

Abstract Background Therapeutic options for Enterobacterales species known to overexpress chromosomal AmpC (AmpC producers) are limited since these organisms can develop resistance to BLs during therapy. Novel BL/BLIs, such as meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), and imipenem-relebactam (IMR), display activity against isolates producing serine-carbapenemases, extended-spectrum β-lactamases, and AmpC enzymes. In this study, we evaluated the activity of novel BL/BLIs against a collection of AmpC producers collected in from US hospitals during 2021. Activity of newer BL/BLI combinations against AmpC producers Methods A total of 1,252 AmpC producers including were consecutively collected in 31 US hospitals. Isolates were susceptibility tested by reference broth microdilution methods. CLSI breakpoints were applied. Results The activity of new BL/BLIs is summarized displayed in the Figure. MEV (MIC50/90, 0.03/0.06 mg/L) and amikacin were the most active agents tested against these isolates, inhibiting 99.8%. CZA (MIC50/90, 0.12/0.5 mg/L) inhibited 99.5%. IMR (MIC50/90, 0.12/1 mg/L) was active against 95.9%. Cefepime and meropenem were active against 92.0% and 97.6% of these isolates, respectively. Piperacillin-tazobactam and ceftolozane-tazobactam (MIC50/90, 0.5/8 mg/L) displayed activity against 76.4% and 83.6% of the AmpC producers, respectively. Tigecycline was active against 96.8% of the isolates; only 53.8% of the isolates had a colistin MIC of ≤2 mg/L. A total of 39 (3.2%) AmpC producers were nonsusceptible to imipenem and/or meropenem. MEV (MIC50/90, 0.25/2 mg/L) was active against 92.3%. IRL (MIC50/90, 0.25/2 mg/L) and CZA (MIC50/90, 1/ &amp;gt; 32 mg/L) were active against 89.7% of the carbapenem nonsusceptible AmpC producers. Against cefepime-resistant AmpC producers (n=45; 3.6%), MEV, IMR, and CZA had 93.3%, 88.9%, and 86.7% activity. Conclusion Infections caused by AmpC producers often are challenging to treat. Understanding the activity of new BL/BLIs is important as the use of cefepime and meropenem can also lead to resistance to these agents. MEV, IRL, and CZA displayed good activity against AmpC producers. When analyzing carbapenem-nonsusceptible or cefepime-resistant isolates, MEV was slightly more active but more potent than other BL/BLI combinations. 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 Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support.

656. Comparative Activity of Newer β-Lactam/β-Lactamase Inhibitor Combinations against Pseudomonas aeruginosa isolates from United States Medical Centers (2020-2021)

Abstract Background P. aeruginosa (PSA) is one of the most common Gram-negative organisms causing infections in hospitalized patients. Prompt administration of an effective antimicrobial is crucial for patients with systemic PSA infection. We evaluated the in vitro activity of ceftazidime-avibactam (CAZ-AVI), ceftolozane-tazobactam (C-T), meropenem-vaborbactam (MEM-VAB), imipenem-relebactam (IMI-REL), and many comparator agents against contemporary PSA isolates from US hospitals. Methods 3,184 isolates were consecutively collected from 71 US medical centers in 2020-2021 and susceptibility tested by reference broth microdilution methods in a monitoring laboratory (JMI Laboratories). US FDA and CLSI breakpoints were applied. Results CAZ-AVI (97.0% susceptible [S]), C-T (98.0%S), IMI-REL (97.3%S), and tobramycin (TOB; 96.4%S) were the most active agents against the aggregate PSA isolate collection and retained good activity against piperacillin-tazobactam- (PIP-TAZ) nonsusceptible (NS), MEM-NS, and multidrug-resistant (MDR) isolates (Table 1). All other antimicrobials tested showed limited activity against PIP-TAZ-NS, MEM-NS, and MDR isolates. The most common infection types were pneumonia (45.9%), skin and skin structure infections (19.0%), urinary tract infections (UTI; 17.0%), and bloodstream infections (11.7%); CAZ-AVI, C-T, and IMI-REL showed consistent activity against isolates from these infection types. Susceptibility to PIP-TAZ and MEM were lower among isolates from pneumonia compared to other infection types (Table 2). CAZ-AVI remained active against 31.7% and 52.5% of C-T-NS and IMI-REL-NS isolates, respectively. C-T remained active against 54.7% and 68.9% of CAZ-AVI-NS and IMI-REL-NS isolates, respectively, and IMI-REL remained active against 64.2% and 64.8% of isolates NS to CAZ-AVI and C-T, respectively. Conclusion CAZ-AVI, C-T, and IMI-REL were highly active and exhibited similar coverage against a large contemporary collection of PSA isolates from US hospitals. Cross resistance among these new β-lactamase inhibitor combinations varied markedly, indicating that all 3 should be tested in the clinical laboratory. These 3 agents represent valuable therapeutic options for PSA infection treatment. Disclosures Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support SJ Ryan Arends, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Shionogi: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support 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.

1310. Antimicrobial Activity of Ceftazidime-Avibactam and Other New β-Lactamase Inhibitor Combinations Tested against Bacterial Isolates from Pediatric Patients from US Medical Centers (2020-2021)

Abstract Background The US FDA expanded the approval of ceftazidime-avibactam (CAZ-AVI) to include pediatric patients aged ≥ 3 months in 2019. We evaluated the in vitro activities of CAZ-AVI, ceftolozane-tazobactam (C-T), meropenem-vaborbactam (MEM-VAB), imipenem-relebactam (IMI-REL), and comparators against Enterobacterales (ENT; n=2,391) and P. aeruginosa (PSA; n=414) isolates causing infection in pediatric patients from US medical centers. Methods Among 21,332 organisms (1/patient) collected in 2020-2021 via the INFORM Surveillance Program, 2,805 (13.1%) were from pediatric patients (≤ 17 years-old [yo]). The isolates were consecutively collected from 69 US medical centers and susceptibility tested by reference broth microdilution methods. Susceptibility results were stratified by patient age (≤ 1 yo [1,040 isolates], 2-5 yo [580], 6-12 yo [651], and 13-17 yo [534], and by infection type. ENT isolates with an ESBL phenotype were screened for β-lactamase genes by whole genome sequencing. Results CAZ-AVI and MEM-VAB showed complete activity (100.0% susceptible [S]) against Enterobacterales, whereas C-T and IMI-REL showed limited activity against some organisms (Table). MEM, ceftriaxone (CRO), and gentamicin (GEN) were active against 99.8%, 87.5%, and 93.0% of Enterobacterales, respectively. Enterobacterales susceptibility to CRO varied from 84.7% (6-12 yo) to 89.8% (13-17 yo), susceptibility to GEN varied from 91.9% (6-12 yo) to 94.0% (≤ 1 yo), and susceptibility to the multidrug-resistant (MDR) phenotype varied from 3.7% (≤1 yo) to 4.8% (13-17 yo). CAZ-AVI (99.5%S), C-T (99.8%S), and IMI-REL (98.4%S) were the most active agents against the PSA collection, but CAZ-AVI and C-T showed greater activity than IMI-REL against PSA-resistant subsets. MEM-VAB and MEM exhibited similar activity against PSA (91.3% inhibited at ≤ 2 mg/L for both agents). PSA overall susceptibility for MEM was 91.3%, varying from 87.3% (13-17 yo) to 93.2% (2-5 yo).Piperacillin-tazobactam (PIP-TAZ) was 87.4%, varying from 85.1% (6-12 yo) to 90.1% (13-17 yo). Conclusion CAZ-AVI and MEM-VAB were the most active agents against Enterobacterales and CAZ-AVI. C-T was the most active agents against PSA. Susceptibility differences were observed among age groups. Disclosures Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Leonard R. Duncan, PhD, AbbVie: Grant/Research Support 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.

126. Frequency of Carbapenemases in United States Hospitals (2016-2020) and Activity of Meropenem-Vaborbactam and Comparator Agents Tested Against These Isolates

Abstract Background We used SENTRY Antimicrobial Surveillance Program data to evaluate the prevalence of carbapenemase enzymes in US hospitals and the activity of meropenem-vaborbactam (MEV) and comparators against these isolates. Methods A total of 22,725 Enterobacterales isolates were consecutively collected in 34 US hospitals and susceptibility tested by reference broth microdilution methods. CLSI breakpoints were applied. Carbapenem-resistant Enterobacterales (CRE) isolates, classified by resistance to imipenem or meropenem, were submitted to whole genome sequencing and data analysis for the detection of carbapenemases. Results Overall, 1.0% (222) of the isolates were CRE. A decrease in CRE prevalence was noted in 2020 (0.8%) compared to 2016 (1.2%). blaKPC variants were detected among 174 (78.4% of the CRE) isolates including 95 blaKPC-3, 75 blaKPC-2, and 1 each of blaKPC-4, blaKPC-6, blaKPC-58, and the Ω-loop variant blaKPC-59.blaSME was detected among 7 S. marcescens. Metallo-β-lactamases (MBLs; 4.0% of the CRE) and OXA-48-like (1.8%) were detected among 9 and 4 isolates, respectively: 7 harbored blaNDM-1/-5; 2 isolates carried blaVIM-1; and 4 isolates carried blaOXA-48/-232. MEV (MIC50/90, 0.03/2 mg/L) inhibited 93.2% of the CRE isolates and was the most active agent tested against all isolates. Comparator β-lactam agents had limited activity against these isolates (0 to 5%). Amikacin and tigecycline were the most active comparators and inhibited 78.8% and 95.0% of the CREs, respectively. Ceftazidime-avibactam (CAZ-AVI; MIC50/90, 1/4 mg/L) was tested against 169 (2017-2020) of these isolates, inhibiting 93.5%. Against 181 isolates producing class A serine-carbapenemases, MBV (MIC50/90, 0.03/0.5 mg/L) inhibited 98.9% of all isolates and CAZ-AVI inhibited 99.2% of the 129 isolates collected in 2017-2020. The activity of all agents was limited against MBL producers. Conclusion The occurrence of carbapenemases declined in the study period, but KPC-producing isolates are dominant among CRE from US hospitals. Small numbers of isolates carrying MBLs and/or OXA-48-like enzymes (&amp;lt; 0.1% of overall) were noted. MEV and CAZ-AVI are very active against isolates producing class A serine-carbapenemases. 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 Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Cory Hubler, BS, Melinta: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support.

2036. Dual VIM-producing Proteus mirabilis, Including a Novel VIM-75, Among Elderly Patients in a Medical Center from Hungary: Report from the 2020 SENTRY Antimicrobial Surveillance Program

Abstract Background Nonsusceptibility of Proteus mirabilis to imipenem allows this pathogen evade surveillance for carbapenemases. Metallo-beta-lactamases of the VIM family are widespread among Enterobacterales in Europe. We identified an outbreak of P. mirabilis carrying 2 VIM enzymes among elderly patients from a medical center in Hungary. Methods A total of 16 P. mirabilis isolates were received from Hungary during 2020 as part of the SENTRY Antimicrobial Surveillance Program. Isolates were susceptibility tested by the CLSI reference broth microdilution method. Carbapenem-nonsusceptible isolates were submitted to whole genome sequencing, screened for resistance mechanisms, and evaluated for core genome multi-locus sequence typing (cgMLST). Results Among the 16 P. mirabilis isolates from Hungary, 5 carbapenem-nonsusceptible, multidrug-resistant isolates (3 from urinary tract infections and 1 each from bloodstream infection and pneumonia) were identified from patients 66-92 years old. Four of these 5 isolates were listed as community acquired. All isolates were resistant to ceftriaxone (MIC, &amp;gt; 8 mg/L), cefepime (16- &amp;gt; 32), imipenem ( &amp;gt; 8), gentamicin ( &amp;gt; 16), levofloxacin (16- &amp;gt; 32), nitrofurantoin ( &amp;gt; 64), trimethoprim/sulfamethoxazole ( &amp;gt; 4), and plazomicin ( &amp;gt; 128), but susceptible to meropenem (0.25-0.5) and ertapenem (0.03-0.25). Isolates carried blaVIM-4 and blaVIM-75 on a class 1 integron within IS26 and separated by aac(6’)-IIc. IS26 was located on a compound plasmid carrying other resistance-encoding genes, including armA. The integron structure is identical to the blaVIM-4 and blaVIM-1-carrying integron described from a Vibrio cholerae isolated from a seagull in France in 2015 (KR262557). blaVIM-75 is a single amino acid variant (Q60R) of blaVIM-1. All isolates carried mutations in the QRDR (gyrA_S83I, parC_S84I). Based on cgMLST analysis, these 5 P. mirabilis isolates were highly similar, with only 7-19 SNPs detected. Conclusion The outbreak of multidrug-resistant P. mirabilis isolates carrying blaVIM-4 and blaVIM-75 in a Hungarian medical center is a cause of concern. Surveillance should be performed to understand the spread of and treatment options for infections caused by carbapenem-nonsusceptible P. mirabilis. Disclosures Lalitagauri M. Deshpande, PhD, Melinta: Grant/Research Support|Pfizer: Grant/Research Support Katalin Burián, MD, JMI Laboratories: Grant/Research Support Ilona Dóczi Csányi, MD, JMI Laboratories: Grant/Research Support 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.

1689. In Vitro Activity of Lefamulin Against Bacterial Pathogens Collected From Pneumonia Patients in United States and Latin America Medical Centers in 2020-2021

Abstract Background Lefamulin is a novel pleuromutilin protein synthesis inhibitor approved in the US, Canada, and Europe for the oral and intravenous treatment of community-acquired bacterial pneumonia (CABP) in adults due to typical and atypical pathogens. This study evaluated the in vitro activity of lefamulin and comparators against bacterial isolates from patients with community-acquired respiratory tract infections and hospitalized patients with pneumonia in the US and Latin America. Methods 1,907 unique isolates were collected within the SENTRY surveillance program from 29 medical centers in the US and 8 medical centers in Latin America (Argentina, Brazil, Chile, Colombia, Mexico, Panama). Isolates were susceptibility tested by CLSI reference broth microdilution methods. CLSI breakpoints (M100, 2022) were applied. Results Lefamulin inhibited 100% of S. pneumoniae isolates at or below its susceptible (S) breakpoint of ≤ 0.5 mg/L, regardless of resistance to other antibiotics used to treat CABP (MIC90 values of 0.12 or 0.25 mg/L, Table). The penicillin-resistant (R; 12.7%), azithromycin-R (43.7%), and tetracycline-R S. pneumoniae (20.4%) displayed reduced susceptibility to the other CABP drugs tested, except moxifloxacin ( &amp;gt; 98.2% S) and lefamulin (100% S). Lefamulin was highly potent against S. aureus, including MRSA, azithromycin-R, and moxifloxacin-R isolates, with 100% of isolates inhibited at or below the lefamulin S breakpoint of ≤ 0.25 mg/L. Susceptibility to azithromycin and moxifloxacin was particularly low for MRSA (13.9% and 25.0%, respectively). The fastidious Gram-negative H. influenzae and M. catarrhalis, of which 24.0% and 98.8%, respectively, were ß-lactamase positive, were S to lefamulin ( &amp;gt; 93%) and the other tested CABP drugs ( &amp;gt; 89%). Table. Susceptibility of pneumonia pathogens from US and Latin America to lefamulin and other CABP drugs Conclusion Lefamulin displayed potent in vitro activity against contemporary CABP pathogens from the US and Latin America. Its activity was unaffected by resistance to other antibiotic classes, including fluoroquinolones, macrolides, β-lactams, and tetracyclines. Lefamulin represents a valuable empiric treatment option for ambulatory and hospitalized patients with CABP, particularly when the causative pathogen is not identified or in settings with high prevalence of resistance. Disclosures Susanne Paukner, PhD, Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds SJ Ryan Arends, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Steven P. Gelone, PharmD, Nabriva Therapeutics: Board Member|Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds|Nabriva Therapeutics: Stocks/Bonds Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.