Cation-adjusted Mueller-Hinton Broth Research Articles

Effects of ZinT-Mediated Zinc Sequestration on Metallo-β-Lactamase Function in Escherichia coli

β-lactams are the most prescribed antibiotic class, but their clinical utility is threatened by the production of metallo-β-lactamase (MBL) enzymes by resistant bacteria. MBLs such as NDM-1 employ zinc ions as cofactors, allowing them to degrade β-lactams. The zinc chaperone ZinT transports zinc into the cytoplasm for intracellular functions, potentially decreasing the availability of zinc in the periplasm for MBLs. We hypothesize that when zinc is limited, ZinT could impair NDM-1 activity and make bacteria more susceptible to β-lactams. To determine whether ZinT and NDM-1 compete for periplasmic zinc, bacterial growth and NDM-1 activity were tested in zinc-poor M9 minimal media for NDM-1-producing Escherichia coli strains with different levels of zinT expression. In addition to wild-type and ΔzinT knockout strains, a recombinant zinT-containing plasmid was transformed into ΔzinT knockout cells to produce a complementation strain with increased zinT expression. Growth assays were conducted in M9 with various concentrations of the β-lactam antibiotic, meropenem. Under these conditions, the ΔzinT strain grew in significantly higher meropenem concentrations than the other strains tested. NDM-1 activity assays revealed that the ΔzinT strain hydrolyzed meropenem notably faster than wild-type cells, whereas the complementation strain showed almost no hydrolysis. This suggests that the increased ZinT production in the complementation strain effectively sequestered zinc from NDM-1. These differences in meropenem hydrolysis and bacterial growth were absent in zinc-sufficient Cation-Adjusted Mueller-Hinton Broth, providing evidence that ZinT and NDM-1 compete for periplasmic zinc under nutrient deficiency. Overall, our results elucidate the effects of competing zinc requirements under zinc-deficient conditions resembling the host environment during a bacterial infection. In these zinc-deficient environments, ZinT can deprive NDM-1 of zinc, causing impaired enzymatic activity and bacterial survival. By better understanding how MBLs function under physiological conditions, future investigations can more reliably explore countermeasures to the growing threat of antibiotic resistance.

Activity of antibiotics against Burkholderia cepacia complex in artificial sputum medium.

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant Gram-negative bacteria that cause life-threatening disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in Bcc infections, probably due in part to differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. To compare the activity of commonly used antibiotics under standard in vitro testing conditions with activity in conditions mimicking those found in vivo. Two Bcc strains were grown alone and with six different antibiotics (minocycline, ceftazidime, meropenem, tobramycin, levofloxacin, trimethoprim-sulfamethoxazole) in two different media: standard cation-adjusted Mueller-Hinton broth and an artificial sputum medium designed to simulate the environment in the lungs of people with CF through addition of components including mucin, free DNA and amino acids. Two different starting conditions were used for time-kill assays: a standard ∼5 × 106 cfu/mL inoculum, and a high-density inoculum in which bacteria were grown for 72 hours before addition of antibiotics. Growth detection was performed by colony enumeration and by detection of resazurin reduction. There were major discrepancies between standard susceptibility results and activity in our models. Some antibiotics, including ceftazidime, showed minimal activity in all time-kill assays despite low minimal inhibitory concentrations, while others, notably tobramycin, were more active in high-density growth conditions than in standard time-kill assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

Real-world performance of susceptibility testing for cefiderocol: insights from a prospective multicentre study on Gram-negative bacteria.

Cefiderocol is a novel siderophore-conjugated cephalosporin developed for the treatment of multidrug-resistant Gram-negative bacterial (GNB) infections. However, the current gold standard for cefiderocol susceptibility testing, broth microdilution (BMD) using iron-depleted cation-adjusted Mueller-Hinton broth, presents challenges for many microbiology laboratories. In this study, we evaluate the real-world performance of disc diffusion (DD) and a commercial BMD method (ComASP®) to test cefiderocol susceptibility in a series of isolates collected prospectively from severely ill patients in a multicentre study. The susceptibilities of 1472 isolates (632 Enterobacterales, 532 Pseudomonas aeruginosa, 84 Acinetobacter spp. and 224 Stenotrophomonas maltophilia) collected in 60 Spanish hospitals were analysed following the EUCAST 2023 and 2024 criteria. We assessed the performance of DD (cefiderocol 30 μg disc, Liofilchem) and a commercial BMD method (ComASP® Cefiderocol, Liofilchem). A total of 1408 and 1450 isolates were susceptible by DD and ComASP® BMD, respectively. Overall, the agreement between both methods was 96.9%. Forty-four isolates were resistant by DD but susceptible by ComASP® BMD, and two were susceptible by DD but resistant by ComASP® BMD (Acinetobacter baumannii isolates). Adoption of the updated 2024 EUCAST DD breakpoints and areas of technical uncertainty (ATUs) led to a decrease in susceptibility among Enterobacterales (95.3% versus 92.6%). DD is a straightforward, rapid and accessible method for routine determination of cefiderocol susceptibility in real-world practice. ComASP® BMD shows a high agreement with DD in susceptible isolates and may help to resolve DD interpretability concerns in isolates with susceptibility results within the ATU, but caution is warranted when testing resistant isolates.

Surveillance of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Clinical Isolates in a Tertiary Care Hospital.

Antibiotic resistance among Gram-negative bacterial isolates is increasingly observed. With the emergence of carbapenem-resistant and pan-resistant pathogens, treating these resistant infections is becoming more challenging due to the limited number of effective drugs. There is a desperate need for the discovery of new antibiotics with novel mechanisms of action. Cefiderocol is one such novel antibiotic with a unique siderophore-based mechanism of action, which has been recently approved for clinical use against drug-resistant pathogens. The present study aims to identify the in vitro activity of cefiderocol against major carbapenem-resistant clinical isolates, including those resistant to colistin. One hundred and one carbapenem-resistant clinical isolates were included in the study. Identification and antibiotic susceptibility testing were performed using the automated VITEK® 2 Compact (bioMérieux SA, Marcy-l'Étoile, France) identification and susceptibility testing system, except for colistin and cefiderocol. Colistin resistance in Enterobacterales and Pseudomonas aeruginosa was assessed using the agar dilution minimum inhibitory concentration method, while for Acinetobacter baumannii, broth microdilution method was employed. Cefiderocol susceptibility testing was conducted using the Kirby-Bauer disc diffusion method with 30 µg discs on standard Mueller-Hinton agar plates. For selected isolates, cefiderocol minimum inhibitory concentration detection was performed using broth microdilution with iron-depleted cation-adjusted Mueller-Hinton broth. Of the total 101 isolates, the majority (75, 74.25%) were Enterobacterales which includedKlebsiella pneumonia (42, 41.58%) and Escherichia coli (33, 32.67%), followed by Pseudomonas aeruginosa (13, 12.87%) and Acinetobacter baumannii (10, 9.9%). Only three (2.97%)of the isolates were Stenotrophomonas maltophilia. Most of the isolates were susceptible to cefiderocol, with only four (3.96%) isolates showing resistance. Colistin resistance was observed in six (6.12%) of the isolates. There was a good correlation between disc diffusion testing and broth microdilution testing for the detection of cefiderocol-resistant isolates. No cross-resistance with colistin was observed, as all colistin-resistant isolates were uniformly susceptible to cefiderocol Conclusion: Cefiderocol is highly effective with good in vitro activity against the majority of carbapenem-resistant pathogens. While some isolates do show resistance, it is relatively uncommon. Given its safety profile compared to colistin, cefiderocol can serve as an alternative to colistin to treat carbapenem-resistant infections and it may be considered even for the management of colistin-resistant cases. Disc diffusion testing is a reliable method for identifying cefiderocol-resistant isolates in routine clinical and diagnostic laboratories, especially in resource-limited settings.

Performance of modified colistin broth disc elution vis-a-vis broth microdilution method for susceptibility testing of Enterobacterales

Objectives: Recently, Clinical and Laboratory Standards Institute (CLSI) has approved colistin broth disc elution (CBDE) to be a supplemental test. This requires multiple discs and tubes to get the desired concentrations of colistin -1, 2, and 4 µg/mL and 10 mL volume of cation-adjusted Mueller–Hinton broth for a single isolate. The present study was aimed to evaluate the performance of CBDE in a microtiter plate format modified (mCBDE) with the reference method broth microdilution (BMD) for detection of colistin resistance in carbapenem-resistant Enterobacterales (CRE) isolates. Materials and Methods: One hundred and sixty non-duplicate clinical CRE isolates (May 2021–April 2022) were simultaneously subjected for BMD and mCBDE. For mCBDE, colistin 10 µg discs and Mueller–Hinton broth no-2 control cations were procured from HiMedia, Mumbai, and drug concentrations were prepared following CLSI-M100Ed31. Results of mCBDE were compared with reference BMD (Minimum inhibitory concentration [MIC] ≤2 µg/mL – intermediate and ≥4 µg/mL – resistant). Statistical Analysis: The performance of mCBDE was compared with BMD and expressed in terms of Categorical, essential agreement (EA), very major error (VME), and major error (ME). The sensitivity and specificity were calculated using Fisher’s contingency Table. Results: Of the 160 CRE isolates, 152 had exactly the same minimal inhibitory concentration (MIC) in both the tests with four isolates having higher and four having lower colistin MIC by mCBDE, giving a major error of 2.1% and VME of 5.5%. Categorical and essential agreement of mCBDE were 97.5% and 98.7%, respectively. Conclusions: mCBDE is an easy, economical, and reliable alternative test for determining colistin susceptibility for CRE isolates. Further, large-scale study is needed to strengthen our observation.

Impact of nutritional factors on in vitro PK/PD modelling of polymyxin B against various strains of Acinetobacter baumannii

The main objective of this study was to assess the effect of rich artificial cation adjusted Mueller-Hinton broth (CAMHB), on the growth of three Acinetobacter baumannii strains, ATCC 19606 and two clinical A. baumannii strains, either susceptible or resistant to polymyxin B (PMB), and on the PMB bactericidal activity. A pharmacokinetic (PK) / pharmacodynamic (PD) modelling approach was then used to characterize the effect of PMB in various conditions.Time-kill experiments were performed using undiluted CAMHB or diluted at 50%, 25% and 10%, with or without Ca2+ and Mg2+ compensation (known to affect PMB activity), and with PMB concentrations ranging from 0.25 mg/L to 256 mg/L based on the strain's MIC. For each strain, time-kill replicates were modelled using NONMEM®.Unexpectedly, CAMHB dilution by up to 10-fold did not affect the growth rate of any of the three strains in the absence of PMB. Yet, PMB bactericidal activity increased with medium dilution resulting in particular in a reduction of the apparent bacterial regrowth of the various strains observed typically after few hours of experiment. The data of each strain were well characterised by a PK/PD model with two bacterial sub-populations with different susceptibility to PMB (a more susceptible (S+) and a less susceptible (S−)). Relatively large, unexplained strain-to-strain variability was observed regarding the impact of medium dilution as well as cation compensation. Complementary experiments are needed to characterise the mechanism underlying this medium dilution effect.

NRX-101 (D-Cycloserine + Lurasidone) Is Active against Drug-Resistant Urinary Pathogens In Vitro.

D-Cycloserine (DCS) is a broad-spectrum antibiotic that is currently FDA-approved to treat tuberculosis (TB) disease and urinary tract infection (UTI). Despite numerous reports showing good clinical efficacy, DCS fell out of favor as a UTI treatment because of its propensity to cause side effects. NRX-101, a fixed-dose combination of DCS and lurasidone, has been awarded Qualified Infectious Disease Product and Fast Track Designation by the FDA. In this study, we tested NRX-101 against the urinary tract pathogens Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in cation-adjusted Mueller-Hinton broth (caMHB) and artificial urine media (AUM). Several strains were multidrug resistant. Test compounds were serially diluted in broth/media. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the test compound at which no bacterial growth was observed. DCS exhibited antibacterial efficacy against all strains tested while lurasidone did not appreciably affect the antibacterial action of DCS in vitro. In AUM, the MICs ranged from 128 to 512 mcg/mL for both DCS and NRX-101. In caMHB, MICs ranged from 8 to 1024 mcg/mL for NRX-101 and 32 to 512 mcg/mL for DCS alone. Our data confirm that DCS has antibacterial activity against reference and drug-resistant urinary pathogens. Furthermore, lurasidone does not interfere with DCS's antimicrobial action in vitro. These results support the clinical development of NRX-101 as a treatment for complicated urinary tract infections.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

2761. Activity of Cefiderocol and Comparator Agents Against Achromobacter and Burkholderia Isolates, Collected During 2020-2022 as Part of SENTRY Antimicrobial Surveillance Program

Abstract Background Achromobacter and Burkholderia species are opportunistic pathogens in individuals with an immunodeficiency, most notably cystic fibrosis. Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with broad activity against Gram-negative bacteria. In this study, the activity of CFDC and comparator agents was determined against isolates of Achromobacter and Burkholderia cepacia species complex, collected in 2020–2022 in Europe and the USA as part of the SENTRY antimicrobial surveillance program. Activity of cefiderocol and comparator agents against Achromobacter and Burkholderia Isolates Methods Minimum inhibitory concentrations (MICs) were determined according to CLSI guidelines against 167 Achromobacter and 160 Burkholderia cepacia species complex isolates, using broth microdilution with cation-adjusted Mueller-Hinton broth (CAMHB) for comparator agents and iron-depleted CAMHB for CFDC. Comparator agents included β-lactam/β-lactamase inhibitor combinations ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, piperacillin-tazobactam, as well as meropenem, ceftazidime, levofloxacin, amikacin, trimethoprim-sulfamethoxazole, and minocycline. Susceptibility was assessed for agents with CLSI breakpoints. For agents without established CLSI breakpoints, only MIC50, MIC90 and MIC ranges were reported. Results CFDC was the most potent agent tested against Achromobacter, showing MIC50 and MIC90 values of 0.03 and 0.25 µg/mL and all isolates were inhibited at ≤4 µg/mL (Table). Isolates from cystic fibrosis patients (n=15) showed higher MIC50 and MIC90 values for most agents, but CFDC MIC50 and MIC90 values remained low (0.06 and 0.25 µg/mL, respectively). Against Burkholderia cepacia species complex, CFDC was the most potent agent tested, with MIC50 and MIC90 values of 0.06 and 0.5 µg/mL, and 156 out of 160 isolates inhibited at ≤4 µg/mL. None of the comparator agents showed >90% susceptibility against these isolates (Table). Conclusion CFDC showed potent activity against a set of contemporary clinical Achromobacter and Burkholderia cepacia species complex isolates. These in vitro data suggest that CFDC could be an important treatment option for infections caused by these opportunistic pathogens. Disclosures Boudewijn L. DeJonge, PhD, Shionogi Inc.: Employee Sean T. Nguyen, PharmD, Shionogi: Employee|Shionogi, Inc: Employee Jason J. Bryowsky, PharmD, MS, Shionogi Inc.: Employee Christopher M. Longshaw, PhD, Shionogi BV: Employee Joshua Maher, PhD, AbbVie: Grant/Research Support|Affinity Biosensors: Grant/Research Support|AimMax Therapeutics, Inc: Grant/Research Support|Alterity Therapeutics: Grant/Research Support|Amicrobe, Inc: Grant/Research Support|Arietis Pharma: Grant/Research Support|Armata Pharmaceuticals, Inc: Grant/Research Support|Astrellas Pharma, Inc.: Grant/Research Support|Basilea Pharmaceutica AG: Grant/Research Support|Becton Dickinson And Company: Grant/Research Support|bioMerieux, Inc: Grant/Research Support|Boost Biomes: Grant/Research Support|Diamond V: Grant/Research Support|Fedora Pharmaceuticals, Inc: Grant/Research Support|Iterum Therapeutics plc: Grant/Research Support|Johnson & Johnson: Grant/Research Support|Kaleido Biosciences, Inc.: Grant/Research Support|Meiji Seika Pharma Co. Ltd.: Grant/Research Support|National Institutes of Health: Grant/Research Support|Pfizer Inc.: Grant/Research Support|Roche Holding AG: Grant/Research Support|Shionogi Inc.: Grant/Research Support|Summmit Therapeutics, Inc.: Grant/Research Support|Zoetis Inc: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Miki Takemura, n/a, Shionogi & Co., Ltd.: Stocks/Bonds Yoshinori Yamano, PhD, Shionogi HQ: Employee

2790. Activity of Cefiderocol and Comparator Agents Against Pediatric Isolates of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii-calcoaceticus species complex, and Stenotrophomonas maltophilia from the SENTRY Surveillance Program (2020-2022)

Abstract Background Cefiderocol (CFDC) is a siderophore cephalosporin with broad activity against Gram-negative bacteria including multi-drug resistant isolates. The in vitro activity of CFDC and comparator agents was evaluated against pediatric (0-17 years old) isolates collected in 2020–2022 as part of the SENTRY Antimicrobial Surveillance Program. In vitro Activity of Cefiderocol and Comparator Agents Against Pediatric Isolates Methods 2,249 Enterobacterales (ENT), 707 P. aeruginosa, 194 Acinetobacter baumannii-calcoaceticus complex (ABC) and 220 S. maltophilia from the USA and Europe were tested for susceptibility (%S) by broth microdilution with cation-adjusted Mueller-Hinton broth (CAMHB). Iron-depleted CAMHB was applied for CFDC. Comparators included newer β-lactam/β-lactamase inhibitor (BL/BLI) combinations ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam (I-R) and meropenem-vaborbactam (MVB) as well as ampicillin/sulbactam (SAM) meropenem (MEM) and colistin (CST). %S was interpreted according to 2023 CLSI & EUCAST breakpoints. Carbapenem non-susceptible (CarbNS) was defined as non-susceptibility to imipenem and MEM. Results All agents displayed >96 %S for ENT while CFDC (MIC50/90, 2/4 mg/L; 91.7 %S) was the most active agent against CarbNS ENT. P. aeruginosa susceptibilities to CFDC and BL/BLI combinations were >97.0%. CFDC was the most potent agent against CarbNS P. aeruginosa with MIC50/90 values of 0.12/0.25 mg/L and 100 %S & 98.5 %S per CLSI & EUCAST breakpoints, respectively. ABC susceptibility to CFDC was >97% per CLSI & EUCAST while the susceptibility for MEM was 87.1% (CLSI & EUCAST) and SAM was 80.9% (CLSI). CFDC (MIC50/90, 0.25/1 mg/L; 95.7 %S) displayed better in vitro potency in CarbNS ABC as compared to SAM (MIC50/90, 32/ >64 mg/L; 17.4 %S CLSI) and CST (MIC50/90, 0.5/ >8 mg/L; 78.3%S EUCAST). Among pediatric S. maltophilia, CFDC was the most active agent with MIC50/90, 0.06/0.25 and 100 %S per CLSI & EUCAST breakpoints. Conclusion CFDC was a highly active β-lactam against contemporary pediatric isolates of Enterobacterales, P. aeruginosa, ABC, and S. maltophilia, including CarbNS subsets for which treatment options are limited. These data suggest CFDC may be a valuable treatment for serious Gram-negative infections in pediatric patients. Disclosures Sean T. Nguyen, PharmD, Shionogi: Employee|Shionogi, Inc: Employee Boudewijn L. DeJonge, PhD, Shionogi Inc.: Employee Jason J. Bryowsky, PharmD, MS, Shionogi Inc.: Employee Anne Henriksen, PhD, Shionogi: Employee Christopher M. Longshaw, PhD, Shionogi BV: Employee Joshua Maher, PhD, AbbVie: Grant/Research Support|Affinity Biosensors: Grant/Research Support|AimMax Therapeutics, Inc: Grant/Research Support|Alterity Therapeutics: Grant/Research Support|Amicrobe, Inc: Grant/Research Support|Arietis Pharma: Grant/Research Support|Armata Pharmaceuticals, Inc: Grant/Research Support|Astrellas Pharma, Inc.: Grant/Research Support|Basilea Pharmaceutica AG: Grant/Research Support|Becton Dickinson And Company: Grant/Research Support|bioMerieux, Inc: Grant/Research Support|Boost Biomes: Grant/Research Support|Diamond V: Grant/Research Support|Fedora Pharmaceuticals, Inc: Grant/Research Support|Iterum Therapeutics plc: Grant/Research Support|Johnson & Johnson: Grant/Research Support|Kaleido Biosciences, Inc.: Grant/Research Support|Meiji Seika Pharma Co. Ltd.: Grant/Research Support|National Institutes of Health: Grant/Research Support|Pfizer Inc.: Grant/Research Support|Roche Holding AG: Grant/Research Support|Shionogi Inc.: Grant/Research Support|Summmit Therapeutics, Inc.: Grant/Research Support|Zoetis Inc: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Miki Takemura, n/a, Shionogi & Co., Ltd.: Stocks/Bonds Yoshinori Yamano, PhD, Shionogi HQ: Employee

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.

Assessment of cefiderocol disk diffusion versus broth microdilution results when tested against Acinetobacter baumannii complex clinical isolates.

The aim of this study was to evaluate the correlation between inhibitory zones and minimum inhibitory concentrations (MICs) when testing cefiderocol against Acinetobacter baumannii complex using disk diffusion and the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Four-hundred and sixty-eight non-duplicated A. baumannii complex clinical isolates were collected from 56 hospitals of the China Antimicrobial Surveillance Network from 2019 to 2021. BMD using iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) and standard disk diffusion methods were performed according to CLSI guidelines. Results were interpreted according to the CLSI and EUCAST breakpoints. Categorical agreement (CA), minor error (mE), major error (ME), and very major error (VME) were calculated for disk diffusion methods. The susceptibilities of all A. baumannii complex isolates by BMD were 98.7% (CLSI) and 97.6% (EUCAST). For all A. baumannii complex isolates, CA was 98.1% (CLSI) and 97.0% (EUCAST), with 0.9% (CLSI) and 1.9% (EUCAST) of VME, respectively. For the carbapenem-susceptible A. baumannii complex, the CA was 100%, with no mE or VME using both CLSI and EUCAST breakpoints. For carbapenem-resistant A. baumannii complex, CA was 97.5% (CLSI) and 96.2% (EUCAST), with 1.1% (CLSI) and 2.5% (EUCAST) of VME, respectively. Regarding the difficult-to-treat resistance A. baumannii complex isolates, CA was 97.6% (CLSI) and 95.7% (EUCAST), with 1.2% (CLSI) and 3.1% (EUCAST) of VME, respectively. Cefiderocol disk diffusion was difficult to assess in this study. Very few isolates were resistant to cefiderocol by BMD using CLSI breakpoint, and these were categorized as susceptible with the disk diffusion test. This study did, however, show that the main proportion of A. baumannii isolates were susceptible to cefiderocol by BMD, including carbapenem-resistant A. baumannii. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii is a major global health concern due to its high prevalence and limited treatment options. Cefiderocol is the only novel Food and Drug Administration (FDA)-approved β-lactam agent for the salvage treatment of carbapenem-resistant A. baumannii infection. Currently, a commercial automated susceptibility testing panel of cefiderocol is unavailable. Both the preparation of iron-depleted cation-adjusted Mueller-Hinton broth and the performance of broth microdilution are cumbersome in routine microbiology laboratories. A disk diffusion method is convenient for cefiderocol antimicrobial susceptibility testing, but limited data are available specifically for A. baumannii clinical isolates. Moreover, the Clinical and Laboratory Standards Institute published revisions to the A. baumannii cefiderocol disk diffusion breakpoints in 2022. Hence, we evaluated the performance of cefiderocol disk diffusion compared with the reference BMD against A. baumannii clinical isolates, especially those with cefiderocol zone diameters ≤ 14 mm.

A-233 Novel Microfluidic System for Antimicrobial Susceptibility Testing

Abstract Background Aiming to enhance antibiotic susceptibility testing (AST), BacterOMIC combines microdilution AST with cutting-edge microfluidics to quantitatively evaluate all clinically relevant antibiotics on a single disposable panel. In this study, we evaluated the performance of BacterOMIC in determining minimum inhibitory concentrations (MICs) of selected agents against Gram-positive and Gram-negative bacteria and in detecting the presence of extended spectrum beta-lactamase (ESBL) produced by Enterobacterales. Methods Using the BacterOMIC UNI Panel, we characterized bacterial strains collected from Polish hospitals in 2020–2022. We inoculated the BacterOMIC panels with bacterial suspensions in cation-adjusted Mueller-Hinton broth at a density of 5*10^5 CFU/mL. The panels then incubated in an analyzer for 16 h, and results were automatically calculated via built-in analytical software. For comparison, we obtained reference MIC data from laboratories contracted for determining strain MIC values via microdilution. We calculated the essential agreement (EA%) and bias according to ISO20776.We also qualitatively assessed several antibiotics via our panel. Categorical agreement (CA%) and discrepancies were determined for these antibiotics according to ISO20776.To assess ESBL detection, we compared BacterOMIC results with data obtained in our internal laboratory using the MIC Test Strip ESBL (Liofilchem). Results With the BacterOMIC system, CA% values exceeded 90% for amoxicillin, benzylpenicillin, ceftazidime/avibactam, ciprofloxacin, erythromycin, gentamicin, meropenem, moxifloxacin, tedizolid, and vancomycin. EA% values exceeded 90% for the following antimicrobials, with a bias of less than 30%. By comparing with our reference method, we found that BacterOMIC detected ESBL with a specificity of 92% (confidence interval [CI] 95%; 81%–98%) and sensitivity of 97% (CI 95%; 84%–99%). Conclusions Our data demonstrate that BacterOMIC provides reliable MIC and ESBL results for assessing the drug susceptibility of Gram-positive and Gram-negative bacteria against 25 agents in a single panel. Thus, the BacterOMIC system provides a valuable tool for automated AST systems.

Comparing minimum inhibitory concentrations of amikacin for pulmonary Mycobacterium avium complex disease: An analysis of culture media differences

Mycobacterium avium complex (MAC) is considered a paramount microbe, especially in East Asia, including Japan. The commonly used commercial Minimum Inhibitory Concentrations (MIC) assay using Middlebrook 7H9 (7H9) medium deviates from the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. Alternatively, measurement with cation-adjusted Mueller-Hinton broth (CAMHB) that conforms to CLSI standards is not yet widely available. Following the approval and commercialization of amikacin liposome inhalation suspension (ALIS) in 2021, a more precise evaluation of amikacin (AMK) susceptibility in MAC is necessary for treatment decisions. In the present study, 33 sputum samples were extracted from 27 patients, and MICs of AMK were compared between the frequently used 7H9 and the recommended CAMHB of the isolated MAC strains. The history of exposure to aminoglycosides for each sample was also added as clinical information. The findings indicated that there was only an 18% concordance rate in MIC between the two media, with 19 samples (58%) indicating lower MICs in 7H9 relative to CAMHB. The 17 samples had a history of exposure to aminoglycosides for periods ranging from 1.5 to 28 months. Specifically, 10 samples were exposed to amikacin by inhalation and intravenous injection, and the remaining seven samples had a history of ALIS inhalation. Samples with a prior utilization of aminoglycosides were significantly predisposed to developing resistance to ALIS compared to those without such a history (P = 0.046). Physicians are encouraged to scrutinize the findings of susceptibility testing utilizing CLSI-endorsed MIC assay using CAMHB medium to ascertain the optimal therapeutic approach.

Klebsiella pneumoniae co-harbouring bla NDM-1 , armA and mcr-10 isolated from blood samples in Myanmar.

Background. The spread of Enterobacteriaceae coproducing carbapenemases, 16S rRNA methylase and mobile colistin resistance proteins (MCRs) has become a serious public health problem worldwide. This study describes two clinical isolates of Klebsiella pneumoniae coharbouring bla IMP-1, armA and mcr-10.Methods. Two clinical isolates of K. pneumoniae resistant to carbapenems and aminoglycosides were obtained from two patients at a hospital in Myanmar. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. The whole-genome sequences were determined by MiSeq and MinION methods. Drug-resistant factors and their genomic environments were determined.Results. The two K. pneumoniae isolates showed MICs of ≥4 and ≥1024 µg ml-1 for carbapenems and aminoglycosides, respectively. Two K. pneumonaie harbouring mcr-10 were susceptible to colistin, with MICs of ≤0.015 µg ml-1 using cation-adjusted Mueller-Hinton broth, but those for colistin were significantly higher (0.5 and 4 µg ml-1) using brain heart infusion medium. Whole-genome analysis revealed that these isolates coharboured bla NDM-1, armA and mcr-10. These two isolates showed low MICs of 0.25 µg ml-1 for colistin. Genome analysis revealed that both bla NDM-1 and armA were located on IncFIIs plasmids of similar size (81 kb). The mcr-10 was located on IncM2 plasmids of sizes 220 or 313 kb in each isolate. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr genes.Conclusion. This is the first report of isolates of K. pneumoniae coharbouring bla NDM-1, armA and mcr-10 obtained in Myanmar.

In vitro activity of cefiderocol against Pseudomonas aeruginosa demonstrating evolved resistance to novel β-lactam/β-lactamase inhibitors.

Cefiderocol demonstrates excellent activity against MDR Pseudomonas aeruginosa; however, the activity against isolates from patients previously treated with β-lactam agents is unknown. We aimed to determine the activity of cefiderocol against P. aeruginosa collected before and after treatment with traditional β-lactams and new β-lactam/β-lactamase inhibitors. Cefiderocol MICs were determined in triplicate in iron-depleted cation-adjusted Mueller-Hinton broth and compared with β-lactam MICs tested by standard methods. All isolates underwent WGS analysis to identify mutations associated with resistance. One hundred and seventy-eight P. aeruginosa isolates were evaluated; 48% (86/178) were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam. The cefiderocol MIC50 and MIC90 were 0.12 and 1 mg/L, respectively. Median cefiderocol MICs did not vary against isolates classified as MDR, XDR, or those non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam when compared with non-MDR isolates. Against isolates collected from patients previously treated with ceftolozane/tazobactam, cefiderocol MICs were increased 4-fold compared with baseline. Cross-resistance to cefiderocol was identified in 21% (3/14) of patients who developed treatment-emergent resistance to ceftolozane/tazobactam. Overall, 6% (11/178) of isolates demonstrated cefiderocol MICs ≥2 mg/L, which were disproportionately collected from patients previously treated with ceftolozane/tazobactam (73%; 8/11). Isolates with reduced cefiderocol susceptibility harboured mutations in ampC, tonB-dependent receptors, the response regulator pirR and ftsI. Cefiderocol demonstrates excellent in vitro activity against P. aeruginosa isolates exposed to other novel β-lactam agents; however, some exceptions were identified. Cross-resistance between cefiderocol and ceftolozane/tazobactam was evident, but not with ceftazidime/avibactam or imipenem/relebactam. Reduced cefiderocol susceptibility was mediated by mutations in ampC and tonB-dependent receptors.

Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility.

Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes' expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.

Transcriptional profiling of extraintestinal Escherichia coli exposed to cranberry pomace extracts alone or in combination with ceftriaxone

Extraintestinal pathogenic Escherichia coli (ExPEC) includes several serotypes that have been associated with colibacillosis in poultry, as well as urinary tract infections and newborn meningitis in humans. This study investigated the antimicrobial activities of ceftriaxone (AXO) and cranberry pomace extracts (CRAN) alone or in combination (CC) against multidrug-resistant (MDR) ExPEC from broiler. The growth-inhibitory activity of CRAN and synergy tests by a checkerboard method were determined in cation-adjusted Mueller–Hinton broth (CAMHB). The transcriptomic profile of the MDR E. coli O7:H18 (ST38) grown in CAMHB supplemented with sub-inhibitory concertation of CRAN and AXO alone or in combination was obtained by RNA-seq. The MIC of CRAN for all isolates was 16 mg/mL. An additive activity was observed between 4 mg/mL of CRAN and 4 μg/mL of AXO. Compared to the control, the transcriptomic analysis revealed that 4 mg/ml of (1/4MIC) CRAN and its combination with 4 μg/mL of (1/8MIC) AXO (CC) exposures resulted in 727 and 712 differentially expressed genes, respectively (false discovery rate &amp;lt; 0.001 and log2-fold change &amp;gt; 2), in the studied E. coli. Major virulence genes including adhesins (fim, flg, csg, and yad), protectins (omp, tra, waa, and hly), secretion systems (hof, pho, and vir), and quorum sensing (lsr), which are energetically expensive for bacteria, were downregulated. Most importantly, 1/4MIC of CRAN or CC downregulated the β-lactamase blaCMY-2 and efflux pump including tolC, mdtEIJ, gadEW, and their regulator gene evgS, while upregulating the cysteine biosynthesis and oxidative stress-related regulatory genes including cys, dmlA, sbp, nrdGHI, soxSR, and rpoH. Downregulation of multiple enzymes involved in TCA cycles and upregulation of Fe–S cluster coordinated by Cys and Isc proteins reflect the regulation of energy metabolism of the studied E. coli upon CRAN or CC exposure. The downregulation of outer membrane protein genes that control permeability barriers, along with different antimicrobial resistance genes, demonstrates that CRAN may have the unique potential to enhance the antimicrobial activities of third-generation cephalosporins such as AXO against MDR E. coli.

1713. In Vitro Activities of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Against Mycobacterium avium Complex Isolates

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. We evaluated the effects of select culture conditions on MIC determinations of EBO against isolates of M. avium complex (MAC), as well as EBO MIC90 results with Middlebrook 7H9 broth compared to those with cation-adjusted Mueller Hinton Broth (CAMHB) for 51 MAC isolates. Methods Six strains of MAC were used to test the in vitro activity of EBO in different conditions in a broth microdilution (BMD) assay. Activity was compared in Middlebrook 7H9 and CAMHB with 5% OADC from different manufacturers. The effects of glycerol, cations, oxyrase, varying pH levels, and increasing inoculum sizes were tested. Finally, EBO in vitro activity was tested for 51 MAC isolates in a BMD assay in both Middlebrook 7H9 and CAMHB with 5% OADC. Results In general, manipulation of select culture conditions caused very little variation in EBO MIC values for the 6 MAC strains except for increasing the inoculum from ∼105 to 107 CFU/mL, which caused an approximately 64x increase in the MIC. Since 1 MAC isolate out of 6 was affected by the addition of casitone, we tested 51 MAC isolates in both the minimal media Middlebrook 7H9 and the complex media CAMHB. EBO had a narrow MIC range in both broths, 0.25-8 mg/L for all isolates. The EBO modal MIC, MIC50 and MIC90 for the entire MAC panel of 51 isolates was 2 mg/L, 2 mg/L, and 8 mg/L for CAMHB and 1 mg/L, 1 mg/L, and 4 mg/L for Middlebrook 7H9, respectively (Table 1). Three clarithromycin-resistant isolates had EBO MIC values of 0.5 mg/L, 1 mg/L, and 2 mg/L suggesting that clarithromycin resistance does not affect EBO in vitro activity. In addition, amikacin resistance as determined using the Clinical Laboratory Standards Institute (CLSI) IV amikacin breakpoint (MIC ≥ 64 mg/L) had no noticeable effect on EBO MIC values. Table 1EBO In Vitro Activity Against 51 Isolates of Mycobacterium avium complex Conclusion The MIC distribution for the 51 MAC isolates tested was similar in both media types, indicating that CAMHB can be used to test EBO MAC susceptibilities per CLSI guidelines. Clarithromycin- and amikacin-resistant isolates demonstrated no cross-resistance with EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

1681. Activity of the Novel Engineered Antimicrobial Peptide PLG0206 Against Staphylococci and Enterococci

Abstract Background PLG0206 is an investigational, engineered cationic antimicrobial peptide designed to overcome the shortcomings of other natural AMPs, such as toxicity and limited activity. PLG0206 has recently been shown to be well tolerated and safe when administered i.v. in a Phase 1 study. The initial proposed indication for this peptide is the treatment of periprosthetic joint infections via irrigation due to a broad spectrum of activity and anti-biofilm properties. This study evaluated the activity of PLG0206 and comparator antimicrobials against staphylococci and enterococci, causes of periprosthetic joint infections, from the IHMA repository of isolates collected from various world-wide locations in 2019. Methods Isolates tested included Enterococcus faecalis (77), E. faecium (75), methicillin-resistant Staphylococcus aureus (MRSA, 180), methicillin-susceptible S. aureus (MSSA, 121) and 152 coagulase-negative staphylococci (CoNS) comprised of S. epidermidis (113), S. haemolyticus (31), S. hominis (4), S. lugdunensis (1), S. saprophyticus (2) and S. simulans (1). Minimum inhibitory concentrations (MICs) were determined by CLSI broth microdilution in cation-adjusted Mueller Hinton broth (CA-MHB), except for PLG0206 which was tested in RPMI medium due to plate-reading difficulties with CA-MHB. The susceptibility of comparators was determined using the 2022 CLSI breakpoints. MDR was defined as resistance to ≥ 3 antibacterial classes. Results When tested in RPMI, 18 E. faecalis, 29 E. faecium, 9 S. aureus and 7 CoNS were unable to grow. Summary MIC and susceptibility data for PLG0206 and comparators just including those isolates able to grow in RPMI are shown in the Table. Identical MIC50 and MIC90 values were obtained for PLG0206 when tested against MDR E. faecium (38), MDR CoNS (63) and MDR MRSA (67). Insufficient numbers of MDR MSSA (7) and MDR E. faecalis (4) were tested to accurately evaluate. Conclusion PLG0206 was the most potent antimicrobial overall (based on MIC50 and MIC90) against enterococci and CoNS and compared well with the comparators against MRSA and MSSA. These data support the evaluation of this novel antimicrobial peptide as a treatment option for periprosthetic joint infections, including those caused by MDR strains. Disclosures David Huang, MD, PhD, Peptilogics: Employee Jonathan Steckbeck, PhD, Peptilogics: I am CEO of Peptilogics.