Cation-adjusted Mueller-Hinton Broth Research Articles

1680. Activity of the Novel Engineered Antimicrobial Peptide PLG0206 Against Enterobacterales isolates

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 in a phase 1 study. This study evaluated the activity of PLG0206 and comparator antimicrobials against Enterobacterales isolates from the IHMA repository collected from various world-wide locations in 2019. Methods Isolates tested included Citrobacter spp. (151), Enterobacter cloacae (152), Escherichia coli (300), Klebsiella pneumoniae (300), Morganella morganii (43), Proteus spp. (152), Providencia spp. (61) and Serratia marcescens (45). 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 Summary MIC and susceptibility data against Citrobacter spp., E. coli, E. cloacae, and K. pneumoniae are shown in the Table. PLG0206 retained activity against MDR isolates of Citrobacter spp. (26), E. coli (92), E. cloacae (45), and K. pneumoniae (132) with MIC50/90 values of 1/2, 1/2, 1/16 and 8/16 μg/mL, respectively. PLG0206 was essentially inactive against the Morganellaceae and Yersiniaceae isolates tested with MIC50 and MIC90 values of > 32 μg/mL (data not shown). Conclusion PLG0206 was active against isolates from the family Enterobacteriaceae, including MDR strains. The spectrum of activity of PLG0206 ranged from being most active against Citrobacter spp. and least active against K. pneumoniae. These data suggest that the novel antimicrobial peptide PLG0206 could be a potential treatment option against infections caused by Enterobacteriaceae isolates. Disclosures David Huang, MD, PhD, Peptilogics: Employee Jonathan Steckbeck, PhD, Peptilogics: I am CEO of Peptilogics.

1715. In Vitro Antibacterial Activity of Cefiderocol against Difficult-to-treat Resistant (DTR) Gram-negative Pathogens in United States from SENTRY Antimicrobial Surveillance Program in 2020/2021

Abstract Background Difficult-to-treat resistant (DTR) isolates are defined as resistance to all first-line high-efficacy, low-toxicity antibiotics (penicillins, cephalosporins, carbapenems and quinolones), leaving physicians with limited treatment options. Cefiderocol (CFDC) is a siderophore cephalosporin with activity against a wide variety of Gram-negative bacteria, including carbapenem-resistant Enterobacterales and non-fermenters. We evaluated the in vitro activity of cefiderocol against DTR isolates collected in United States as part of SENTRY surveillance program in 2020 and 2021. In vitro susceptibility of cefiderocol and comparator agents to DTR isolates Methods Susceptibility testing was performed by broth microdilution according to CLSI guidelines. All antibiotics were tested in cation-adjusted Mueller-Hinton broth (CAMHB) except for CFDC, for which iron-depleted CAMHB was used. Susceptibility rate (%) was determined according to CLSI breakpoints, and DTR pathogens were defined as being resistant to cefepime, ceftazidime ceftriaxone (only for Enterobacterales), imipenem, meropenem, ciprofloxacin and levofloxacin according to CLSI/FDA breakpoints. Results Among a total of 8,328 Enterobacterales, 2,241 Pseudomonas aeruginosa, and 586 Acinetobacter calcoaceticus-baumannii complex (ACB) clinical isolates from the United States, 50 Enterobacterales (0.6%), 36 P. aeruginosa (1.6%) and 114 ACB (19.5%) isolates showed a DTR phenotype, respectively. CFDC demonstrated its potent in vitro activity against these DTR isolates with MIC90 of ≤4 μg/mL with a susceptibility rate of ≥92% except for DTR ACB based on FDA breakpoint (75.4%). In contrast, MIC90s of β-lactam/β-lactamase inhibitor combination drugs showed lower activity (Table). Conclusion Cefiderocol demonstrated potent in vitro activity against DTR isolates of Enterobacterales, P. aeruginosa and ACB, indicating cefiderocol has high potential for treating infections caused by these difficult-to-treat strains. Disclosures Yoshinori Yamano, PhD, Shionogi: Employee Miki Takemura, n/a, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Christine M. Slover, PharmD, Shionogi: Employee Christopher M. Longshaw, PhD, Shionogi: Employee Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant.

660. In vitro activity of cefiderocol against difficult-to-treat resistance European Gram-negative bacterial pathogens from the multi-national sentinel surveillance study, SENTRY in 2020 and 2021

Abstract Background Difficult-to-treat resistance (DTR) organisms are defined as non-susceptible to all first-line high-efficacy, low-toxicity antibiotics (penicillins, cephalosporins, carbapenems and fluoroquinolones), leaving physicians with limited treatment options. Cefiderocol is a parenteral siderophore cephalosporin with potent activity against aerobic Gram-negative pathogens, including carbapenem-resistant strains. We evaluated the in vitro activity of cefiderocol and comparators against DTR pathogens collected in Europe by the SENTRY surveillance study in 2020 and 2021. Methods A total of 11434 clinical isolates of Gram-negative bacilli were systematically collected from 16 EU countries, Israel and Turkey in 2020 and 2021. Minimum inhibitory concentrations (MICs) were determined by broth microdilution for a panel of twenty-two antibiotics according to CLSI guidelines. All antibiotics were tested in cation-adjusted Mueller-Hinton broth (CAMHB) except for cefiderocol, for which iron-depleted CAMHB was used. Susceptibility was determined according to CLSI breakpoints, and DTR pathogens were defined as being resistant to cefepime, ceftazidime ceftriaxone, imipenem, meropenem, ciprofloxacin and levofloxacin according to CLSI breakpoints. Results Among 11434 Gram-negative isolates collected in 2020 and 2021, 792 (7.0%) were resistant to all 1st line therapy including cephalosporins, carbapenems and fluoroquinolone and could be defined as DTR. DTR was most frequently observed in Acinetobacter spp. (530/931, 56.9%), Enterobacterales (201/7739, 2.6%) and Pseudomonas aeruginosa (61/2440, 2.5. Based on CLSI breakpoints, cefiderocol was the most active antibiotic tested against DTR-Acinetobacter spp. (MIC90= 2mg/L, 97.4% susceptibility). Ampicillin/sulbactam was active in less than 1% of the DTR-Acinetobacter spp isolates. None of the drugs recommended by the IDSA for the treatment of resistant Gram-negative infections were as potent as cefiderocol (Table 1). Conclusion Cefiderocol was the only treatment option with demonstrated in vitro activity against more than 95% of all the tested DTR Gram-negative pathogens with limited treatment options. Disclosures Anne Laurence Santerre Henriksen, PhD, Shionogi: Contractor|UTILITY therapeutics Ltd: Advisor/Consultant Christopher M. Longshaw, PhD, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: 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 Miki Takemura, n/a, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi: Employee.

1721. In Vitro Activity of Cefiderocol against IMP-Producing Enterobacterales Isolated in Japan

Abstract Background Beta-lactam resistance is a major worldwide concern. Metallo-β-lactamases (MBLs) are a group of enzymes that can inactivate most commonly used β-lactam antibiotics including carbapenems and are not affected by new β-lactamase inhibitors. Therefore, treating infections caused by MBL producing strains is difficult. Imipenemase (IMP) type MBL is a rare family of acquired carbapenemases detected from Enterobacterales globally, however, it has been detected and become a major therapeutic concern in clinical settings mainly in East Asia including Japan [1]. Cefiderocol (CFDC) is a novel injectable siderophore cephalosporin antibiotic with potent activity against a wide variety of carbapenem resistant strains including both serine- and metallo-type carbapenemase-producers. We evaluated in vitro activity of CFDC and comparator agents against IMP-producing Enterobacterales isolated in Japan. Methods A total of 150 IMP-producing strains consisting of 48 Escherichia coli, 34 Klebsiella pneumoniae, 29 Klebsiella oxytoca, 21 Enterobacter cloacae, 7 Citrobacter freundii, 7 Serratia marcescens, 2 Providencia rettgeri, 1 Klebsiella aerogenes, and 1 Morganella morganii were tested. The minimum inhibitory concentrations (MICs) were determined for CFDC, ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam (I-R), meropenem (MEM), and amikacin (AMK) by broth microdilution according to CLSI guidelines. CFDC was tested in iron-depleted cation-adjusted Mueller-Hinton broth. Results MIC50 and MIC90 of CFDC were 0.25 and 1 µg/mL, respectively. MICs of CFDC for all tested strains ranged from ≤ 0.03 to 4 µg/mL, which showed all strains were susceptible to CFDC based on the CLSI breakpoint (≤ 4 µg/mL). On the other hand, MIC90s of CZA, C/T, I-R, MEM, and AMK were > 32/4, > 32/4, 8/4, > 32, and 4 µg/mL, respectively. The susceptibilities were 98.0% for AMK, 66.0% for I-R, and < 10% for CZA, C/T and MEM. Conclusion CFDC had potent in vitro activity against IMP-producing Enterobacterales collected from Japan, indicating CFDC has high potential for treating infections caused by these difficult-to-treat strains. Reference: 1. Logan, LK. et al., J Infect Dis. 2017 Feb 15;215(suppl 1):S28-S36. Disclosures Hidenori Yamashiro, Shionogi & Co., Ltd.: Employee Ryuichiro Nakai, n/a, Shionogi TechnoAdvance Research CO., LTD.: Employee Miki Takemura, n/a, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi: Employee.

1734. Susceptibility of Cefiderocol between US Census Regions against Gram-Negative Organisms collected from the SENTRY Surveillance Program: 2020-2021

Abstract Background Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with broad activity against Gram-negative (GN) bacteria, including multidrug-resistant organisms. GN bacteria such as Enterobacterales (ENT), Pseudomonas aeruginosa (PsA), Acinetobacter baumannii complex (ABC), and Stenotrophomonas maltophilia (StM) can be challenging to treat and are often carbapenem-resistant (CR). Regional susceptibility of CFDC and comparators were investigated against US GN isolates collected in 2020-2021 as part of the SENTRY Antimicrobial Surveillance Program. Methods GN pathogens were consecutively collected from 32 US hospitals between 2020 to 2021. Susceptibility testing was performed using the broth microdilution method. CFDC was tested in iron-depleted cation-adjusted Mueller-Hinton broth. FDA or CLSI breakpoints were used where available. Other agents tested included the beta-lactam/beta-lactamase inhibitor (BL/BLI) combinations ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, piperacillin-tazobactam, and ampicillin-sulbactam. CR-PsA, or CRAB was defined as meropenem resistant while CRE was defined as imipenem or meropenem resistant by CLSI breakpoints. Results A total of 8328 ENT, 2241 PsA, 586 ABC, and 404 StM were collected. For ENT and PsA, CFDC susceptibility between the 9 US Census regions remained above 98% susceptible by CLSI or FDA breakpoints in all regions (Table 1). Amongst the BL/BLI combinations, the majority had >90% susceptibility across regions except for piperacillin-tazobactam with 79.7% for ENT and 65.9% for PsA in the MidAtlantic by FDA breakpoints. For ABC and StM, CFDC susceptibility was > 88% across all regions. For CR pathogens, 96 CRE, 327 CR-PsA, and 199 CRAB were collected. CFDC had >87.5% susceptibility in all regions for CRE and CR-PSA. For CRAB, 4 regions were > 87.5% by FDA breakpoints and the lowest was 63.6% in 11 isolates in the Mountain region (Table 2). For CRAB, every BL/BLI had susceptibility ≤27.3% in every region. Ampicillin-sulbactam had 27.3% susceptibility in the Mountain region. Table 1 Susceptibility of Cefiderocol against Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii complex and Stenotrophomonas maltiphilia isolates in the cefiderocol program collected from medical centers in the USA Table 2: Susceptibility of cefiderocol against CRE, CR Pseudomonas aeruginosa, and CR Acinetobacter baumannii complex isolates in the cefiderocol program collected from medical centers in the USA Conclusion US GN isolates, including CR pathogens, had high susceptibilities to CFDC across the US census regions. CFDC remains an important treatment option for GN infections in all US census regions. Disclosures Frank H. Kung, PhD, Shionogi Inc: Employee Sean Nguyen, n/a, Shionogi: Employee Christine M. Slover, PharmD, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: 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 Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Miki Takemura, n/a, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi: Employee.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 μg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 μg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 μg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

In vitro Activity of Cefiderocol and Comparators against Carbapenem-Resistant Gram-Negative Pathogens from France and Belgium.

Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller–Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates.

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

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

Activity of Delafloxacin and Levofloxacin against Stenotrophomonas maltophilia at Simulated Plasma and Intrapulmonary pH Values.

ABSTRACTFluoroquinolones have become a popular treatment option for Stenotrophomonas maltophilia infections. Although levofloxacin is most commonly used, delafloxacin demonstrates comparable in vitro activity when evaluated under standard susceptibility testing conditions at neutral pH. At acidic pH, the activity of the anionic delafloxacin is improved, while the activity of the zwitterionic levofloxacin is reduced. Because the human respiratory tract has a pH of ~6.6 and is the most common site of S. maltophilia infection, it is vital to understand the activity of these agents in this environment. Therefore, levofloxacin and delafloxacin were tested against clinical S. maltophilia isolates via broth microdilution testing (n = 37) and time-kill analysis (n = 5) in neutral cation-adjusted Mueller-Hinton broth (CAMHB) (pH 7.3) and acidic CAMHB (aCAMHB) (pH 6.5). In CAMHB, MIC50 values were similar between levofloxacin and delafloxacin (8 mg/L versus 8 mg/L). In aCAMHB, levofloxacin MICs did not change, while delafloxacin MICs decreased by a median of 4 log2 dilutions (MIC50 values of 8 mg/L versus 0.25 mg/L). In time-kill analyses, levofloxacin and delafloxacin at the maximum drug concentration for the free drug (fCmax) were bactericidal against 3 and 2 isolates in CAMHB, respectively. In aCAMHB, levofloxacin was not bactericidal against any isolate, while delafloxacin was bactericidal against the same 2 isolates. Relative to CAMHB, levofloxacin activity was reduced by 2.5 log10 CFU/mL in aCAMHB, whereas delafloxacin activity was increased 2.7 log10 CFU/mL. Although the bactericidal activity of levofloxacin against S. maltophilia was attenuated in an acidic environment in this study, the increased potency of delafloxacin at pH 6.5 did not translate into improved bactericidal activity in time-kill analyses, compared to pH 7.3.IMPORTANCE Stenotrophomonas maltophilia most often infects the lungs, where the physiologic environment is naturally slightly acidic (pH ~6.6), compared to most parts of the body (such as the bloodstream), which have neutral pH values (~7.4). Pneumonia due to S. maltophilia is often treated with the antibiotic levofloxacin, despite the activity of levofloxacin being known to be impaired at acidic pH. Unfortunately, currently available methods for susceptibility testing of levofloxacin against S. maltophilia are performed at a neutral pH and therefore may not accurately represent the activity of levofloxacin at the site of infection in the lungs. A similar but newer antibiotic in the same class as levofloxacin, namely, delafloxacin, is not affected by being in an acidic environment and may actually work better at lower pH values. Therefore, the purpose of this study was to investigate whether one drug might be better than the other in this setting by testing each agent’s ability to kill S. maltophilia at pH 7.3 and pH 6.5. These findings could then be used to design confirmatory studies that may ultimately impact which drug is given to patients with lung infections due to S. maltophilia.

The Use of Resapolymyxin NP Test in Determining the Sensitivity of Colistin in Escherichia coli, and Klebsiella pneumoniae Isolates

Colistin is used as the last choice of drug in multidrug resistant gram-negative bacilli infections; therefore, accurate detection of colistin susceptibility has gained critical importance. Unfortunately, many of the widely used and practical methods in the clinical laboratory have various limitations for the determination of colistin susceptibility. This situation has led researchers to search for new methods to determine colistin susceptibility. In this study, the performance of the ResaPolymyxin NP test, which was developed for the rapid detection of colistin susceptibility of Pseudomonas aeruginosa and Acinetobacter baumannii isolates, was evaluated for various Gram-negative bacteria for the determination of colistin susceptibility. For this purpose, the colistin MIC values of 105 Escherichia coli, and 196 Klebsiella pneumoniae isolates were determined by broth microdilution (BMD) using cation-adjusted Mueller-Hinton broth and then ResaPolymyxin NP test was applied for each isolate. While 242 (%80.4) of the isolates included in the study were found to be susceptible to colistin with BMD, 214 (71.1%) of the isolates were found as sensitive to colistin with the ResaPolymyxin NP test. The categorical agreement rate for the ResaPolymyxin NP test was 85.7% for E.coli isolates, and 92.3% for K.pneumoniae isolates. The major error rate was 14.7% for E.coli, 10.8% for K.pneumoniae, whereas the very major error rate was 1.8% for K.pneumoniae. For ResaPolymyxin NP test, sensitivity, specificity, positive and negative predictive values were %98,3; %88.0; %66.7; and %99.5. In contrast to the available data about the ResaPolymyxin NP test, both the categorical agreement rate with BMD, and the very major and major error rates varied according to the isolate type, and it was concluded that the test performed relatively better in E.coli and K.pneumoniae isolates. Since the data obtained in this study are quite different from the previously published data, more comprehensive and multicenter studies are needed to evaluate the test effectiveness in order to recommend the use of the ResaPolymyxin NP test in clinical microbiology laboratories.

Development of a harmonized method for antimicrobial susceptibility testing of Bordetella avium using broth microdilution and detection of resistance genes.

In response to a request from the Clinical and Laboratory Standards Institute (CLSI), the objective of this study was to develop a harmonized method for broth microdilution susceptibility testing of Bordetella (B.) avium, the major causative agent of infectious coryza in poultry. To find a suitable test medium, growth curves with four epidemiologically unrelated B. avium isolates were created in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB+2.5% lysed horse blood and veterinary fastidious medium. All isolates showed good growth in CAMHB, therefore MIC values were determined using this medium and the homogeneity of the values was determined. An essential MIC agreement of 99.7% was calculated. Testing of a larger strain collection (n=49) for their susceptibility to 24 antimicrobials confirmed the suitability of the tested method and revealed some isolates with elevated MICs of florfenicol (n=1), streptomycin (n=2), tetracyclines (n=5), and trimethoprim/sulfamethoxazole (n=6). PCR assays detected the resistance genes aadA1, dfrB1, floR, sul1, sul2 and tet(A). The method used enables easy reading and a good reproducibility of MIC values for B. avium. Application of the tested method allows harmonized resistance testing of B. avium and identification of isolates with elevated MIC values.

Susceptibility of cefiderocol and other antibiotics against carbapenem-resistant, Gram-negative bacteria.

BackgroundCefiderocol is a promising antimicrobial agent against carbapenem-resistant, Gram-negative bacteria, but susceptibility data from the Chinese mainland are lacking. The aim of the present study was to test the susceptibility of cefiderocol against carbapenem-resistant, Gram-negative bacteria collected from Beijing, China.MethodsCarbapenem-resistant Klebsiella pneumoniae (CR-KP; n=105), carbapenem-resistant Acinetobacter baumannii (CR-AB; n=126), carbapenem-resistant Pseudomonas aeruginosa (CR-PA; n=74), and Stenotrophomonas maltophilia (SM; n=72) isolates were collected from inpatients at 4 tertiary hospitals in Beijing, China. Minimum inhibitory concentrations (MICs) for cefiderocol were determined using iron-depleted cation-adjusted Mueller Hinton broth (CAMHB), and for comparators using CAMHB, according to the recommended Clinical and Laboratory Standards Institute (CLSI) methodology. Carbapenemase and other β-lactamase gene profiles were determined using polymerase chain reaction (PCR).ResultsCefiderocol inhibited 100% of CR-KP and CR-PA, and 98.6% of the SM isolates at the susceptibility breakpoint concentration of 4 mg/L. However, the susceptibility rate for cefiderocol against CR-AB was only 62.7%, with MIC90 values as high as 128 mg/L. Nearly all the cefiderocol-susceptible CR-AB isolates were found to be positive for blaOXA-23 and blaTEM, whereas all the cefiderocol-resistant CR-AB isolates were found to be positive for the blaPER genes, in addition to blaOXA-23 and blaTEM.ConclusionsCefiderocol showed potent in vitro activity against CR-KP, CR-PA, and SM isolates collected from Beijing, China. However, the resistance rate for cefiderocol against CR-AB was higher than that reported by other research centers, and the presence of blaPER might contribute to resistance in non-susceptible CR-AB isolates.

Antimicrobial resistance profiles of human Brucella melitensis isolates in three different microdilution broths: the first multicentre study in Bosnia and Herzegovina

Brucellosis is a ubiquitous emergent bacterial zoonotic disease causing significant human morbidity in Bosnia and Herzegovina. So far, a high rate of resistant Brucella has been found worldwide. This study prospectively analysed the rates of resistance among human Brucella melitensis strains isolated in Bosnia and Herzegovina. This study included 108 B. melitensis isolates from 209 patients diagnosed at five medical centres in Bosnia and Herzegovina. The resistance profiles of the B. melitensis isolates for the 13 most commonly used antimicrobials were studied in standard Brucella broth (BB) and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 4% lysed horse blood or 5% defibrinated sheep blood. Of the 209 patients, B. melitensis blood cultures were positive for 111 (53.1%). Among the 108 isolates investigated, 91 (84.3%) were resistant to trimethoprim-sulfamethoxazole on BB, but not on either CAMHB. Nearly all isolates (>90%) were resistant to azithromycin on BB and both CAMHBs. We observed a high rate of B. melitensis resistance to azithromycin. The high rate of resistance to trimethoprim-sulfamethoxazole that we observed was related to BB, so an alternative broth should be used, such as the enriched CAMHBs in this study, for evaluating resistance to trimethoprim-sulfamethoxazole. Whole-genome sequencing studies are needed to understand the development of antimicrobial resistance in B. melitensis strains isolated from humans.

Assessment of In Vitro Cefiderocol Susceptibility and Comparators against an Epidemiologically Diverse Collection of Acinetobacter baumannii Clinical Isolates.

Cefiderocol is a catechol-substituted siderophore cephalosporin combining rapid penetration into the periplasmic space with increased stability against β-lactamases. This study provides additional data on the in vitro antimicrobial activity of cefiderocol and commercially available comparators against an epidemiologically diverse collection of Acinetobacter baumannii clinical isolates. Antimicrobial susceptibility was tested using pre-prepared frozen 96-well microtiter plates containing twofold serial dilutions of: cefepime, ceftazidime/avibactam, imipenem/relebactam, ampicillin/sulbactam, meropenem, meropenem/vaborbactam, ciprofloxacin, minocycline, tigecycline, trimethoprim/sulfamethoxazole and colistin using the standard broth microdilution procedure in cation-adjusted Mueller–Hinton broth (CAMHB). For cefiderocol, iron-depleted CAMHB was used. A collection of 113 clinical strains of A. baumannii isolated from Argentina, Azerbaijan, Croatia, Greece, Italy, Morocco, Mozambique, Peru and Spain were included. The most active antimicrobial agents against our collection were colistin and cefiderocol, with 12.38% and 21.23% of non-susceptibility, respectively. A high proportion of multidrug-resistant (76.77%) and carbapenem-resistant (75.28%) A. baumannii isolates remained susceptible to cefiderocol, which was clearly superior to novel β-lactam/β-lactamase inhibitor combinations. Cefiderocol-resistance was higher among carbapenem-resistant isolates and isolates belonging to ST2, but could not be associated with any particular resistance mechanism or clonal lineage. Our data suggest that cefiderocol is a good alternative to treat infections caused by MDR A. baumanni, including carbapenem-resistant strains.

In Vitro Time-Kill Studies of Trimethoprim/Sulfamethoxazole against Stenotrophomonas maltophilia versus Escherichia coli Using Cation-Adjusted Mueller-Hinton Broth and ISO-Sensitest Broth.

Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia, but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 μg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli isolates having the same MICs (0.25/4.75 to 4/76 μg/mL) in cation-adjusted Mueller-Hinton broth (CAMHB) and ISO-Sensitest broth (ISO broth). With the exception of the resistant isolates (4/76 μg/mL), which resulted in regrowth approaching the growth of the control, TMP/SMZ displayed significantly greater killing for E. coli than for S. maltophilia at each MIC. Against E. coli, the mean changes at 24 h were -4.49, -1.73, -1.59, and +1.83 log10 CFU for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 μg/mL, respectively. The area under the concentration-time curve for the free, unbound fraction of the drug (fAUC)/MIC ratio required for stasis and 1-log10 and 2-log10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia isolate regardless of the MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.

1072. In Vitro Antibacterial Susceptibility Testing of Sulopenem Against Category A and B Bio-threat Bacterial Pathogens

Abstract Background Sulopenem is a thiopenem β-lactam antibiotic being developed for the treatment of infections caused by multi-drug resistant bacteria. Sulopenem possesses potent activity against species of the Enterobacterales that encode ESBLs or AmpC-type β-lactamases that confer resistance to third generation cephalosporins. It has also demonstrated good in vitro microbiological activity against a range of bacterial pathogens including penicillin resistant S. pneumoniae, β-lactamase-producing H. influenzae and M. catarrhalis. Sulopenem is available as intravenous and oral pro-drug formulations, and its activity aligns with the most urgent drug-resistant antimicrobial threats defined by the CDC. Methods Bacterial inoculums were prepared by suspending colonies into cation adjusted Mueller Hinton broth (CAMHB) from 18-24 h (B. anthracis, B. pseudomallei and B. mallei plates incubated at 35ºC); or 36-48 h (F. tularensis and Y. pestis plates incubated at 35ºC and 28ºC, respectively). Sheep blood agar plates were used for B. anthracis and Y. pestis. Chocolate agar plates were used for F. tularensis, B. pseudomallei and B. mallei. Suspended cultures were diluted with CAMHB to achieve a turbidity equivalent to a 0.5 McFarland standard. MICs were determined by the microdilution method in 96-well microplates according to CLSI guidelines (Clinical and Laboratory Standards Institute, 2020). Antibiotic ranges used for sulopenem were 0.03 - 64 μg/mL and 0.004 - 8 μg/mL for the diversity strains of B. anthracis, F. tularensis, Y. pesis, B. mallei, and B. pseudomallei, based on a final well volume of 100 μl after inoculation. Results A summary of sulopenem MIC90 results versus bio-threat bacterial pathogens in presented in the table. Criteria for down selection into mice was met for all pathogens except F. tularensis. Sulopenem MIC90 Summary for Down Selection Criteria Conclusion Sulopenem is active in vitro against a number of bio-threat pathogens at concentrations likely to be achieved after oral dosing in humans and meets criteria to be tested in the murine model of B. anthracis, Y. pestis, B. mallei, and B. pseudomallei. Disclosures Michael Dunne, MD, Iterum Therapeutics (Board Member, Consultant, Shareholder) Steven I. Aronin, MD, Iterum Therapeutics (Employee, Shareholder)

1044. In vitro Activity of Tebipenem Against Relevant Clinical Isolates in the Presence of Pulmonary Surfactant

BackgroundTebipenem (TBP) is an orally administered broad-spectrum carbapenem antibiotic under development for the treatment of acute pyelonephritis and complicated urinary tract infections. This study evaluated the effect of bovine pulmonary surfactant (BPS) on the in vitro activity of TBP and ertapenem (ETP) against a recent collection of clinical isolates.MethodsA total of 10 isolates recovered from patients with infections in 2018 were tested for antimicrobial susceptibility to TBP and ETP in the absence or presence of 1%, 5%, or 10% BPS (Infasurf; ONY Biotech). These isolates included the following species: C. freundii, E. cloacae, E. coli, H. influenzae, H. parainfluenzae, K. pneumoniae, methicillin-susceptible S. aureus, M. catarrhalis, S. pneumoniae, and S. pyogenes. Isolates were tested with the appropriate broth microdilution method for each organism as specified by CLSI. For most organisms, MICs were determined in cation-adjusted Mueller-Hinton broth (CAMHB). CAMHB was supplemented with 2.5-5% lysed horse blood for streptococci and Haemophilus Test Medium broth for Haemophilus spp. Daptomycin (DAP) was tested against S. aureus ATCC 29213 as a positive control.ResultsAll isolates displayed TBP MIC values ranging from ≤0.004 to 0.06 mg/L in media without BPS. There were no observed MIC increases >2-fold in the presence of BPS. 4 of the 10 isolates displayed slightly higher (≥4-fold) ETP than TBP MIC values. The ETP MIC values ranged from 0.015-0.25 mg/L in media without BPS. Similarly, there were no observed instances of a >2-fold shift toward lower potency in the presence of BPS. For both TBP and ETP, MIC endpoint values were easily determined, except for in the case of the 2 Haemophilus strains growing in the presence of 5% or 10% BPS. For these conditions, resazurin was added to establish an MIC value. The MIC values found with this method did not differ from the MIC values found in either HTM media or HTM media with 1% BPS. As expected, the addition of BPS shifted DAP S. aureus MIC values to >8 mg/L for all 3 BPS concentrations.ConclusionTBP displayed potent activity against all isolates tested, as all observed MIC values were ≤0.06 mg/L. The addition of BPS to the testing medium did not affect the in vitro MIC values of TBP or ETP against these species.Disclosures S J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Abby L. Klauer, n/a, Cidara Therapeutics, Inc. (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Nicole Cotroneo, Spero Therapeutics (Employee, Shareholder) Ian A. Critchley, Ph.D., Spero Therapeutics (Employee, Shareholder) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)

1059. In vitro Activity of Exebacase (CF-301) against Staphylococcus aureus Causing Bacteremia in the United States, Including Multidrug-resistant Subsets

1059. <i>In vitro</i> Activity of Exebacase (CF-301) against <i>Staphylococcus aureus</i> Causing Bacteremia in the United States, Including Multidrug-resistant Subsets

1241. In Vivo Efficacy of Meropenem Against Metallo-ß-Lactamase (MBL)-Harboring Pseudomonas aeruginosa and Correlation to In Vitro Susceptibility Upon Addition of EDTA

BackgroundPrior investigations evaluating the predictive value of zinc-depleted media for MBL-susceptibility testing have focused on Enterobacterales. Therein, bacterial killing observed with meropenem (MEM) in vivo was concordant with its pharmacodynamic profile using MIC values determined in zinc-depleted media compared with conventional cation-adjusted Mueller-Hinton broth (CAMHB). This study aims to evaluate the exposure-response relationship of MEM against VIM- and NDM-harboring P. aeruginosa (PSA) using the murine thigh infection model and zinc-depleted MICs.MethodsMBL-harboring PSA isolates (VIM n=11; NDM n=10) were tested both in vivo (neutropenic murine thigh infection model) and in vitro (broth microdilution). The 24h murine thigh study was conducted with treatment groups receiving a humanized MEM 2g q8h (3h infusion) dose. Six different zinc-limited media were prepared by the addition of EDTA at concentrations ranging from 3 to 300 mg/L to CAMHB. MEM MICs were determined in triplicate in conventional CAMHB and zinc-limited media. Time > MIC values (generated in each zinc-depleted media) were then plotted against the change in 24h bacterial density count in an Emax model.ResultsAverage 0 h bacterial densities were 5.21 ± 0.40 and 5.13 ± 0.81 log10 CFU/thigh for NDM and VIM isolates, respectively. MEM resulted in -0.09 CFU reduction to +3.69 CFU growth against NDM isolates. MEM resulted in -2.59 CFU reduction to +4.81 CFU growth against VIM isolates. All MEM MICs in conventional CAMHB were >64 µg/mL for NDM and ranged from 8 to >64 µg/mL for VIM isolates. Increasing EDTA concentrations resulted in several-fold MIC reductions and on average, a larger magnitude of reduction was observed among VIM- (6-fold) compared with NDM-harboring PSA (4-fold) in CAMHB-EDTA 300 mg/L relative to CAMHB. For both NDM- and VIM-harboring PSA, an Emax model with MICs generated in CAMHB+EDTA 30 mg/L (r2 = 0.88) provided the highest correlation with MEM in vivo activity compared with CAMHB (r2 = 0.55).ConclusionResults indicate that MIC values generated in conventional CAMHB do not appropriately characterize the in vivo efficacy of meropenem against MBL-harboring PSA, and addition of EDTA (30 mg/L) to CAMHB appears to be a viable option for in vitro testing of these organisms.Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.)

1287. Double Disk Diffusion Study to Evaluate the Synergistic Effect Between Cefiderocol and Ceftazidime-Avibactam Against Cefiderocol-Non-susceptible Acinetobacter baumannii

Abstract Background Cefiderocol (CFDC), a siderophore cephalosporin, has broad coverage of Gram-negative bacteria and has been approved for clinical use in USA and Europe. The SIDERO-WT surveillance studies showed that CFDC shows &amp;gt;95% susceptibility against Acinetobacter baumannii. Against many of CFDC non-susceptible isolates, most of which had blaPER gene, the combination use of avibactam significantly decreased the MIC by broth microdilution (BMD). In this study, we evaluated the appropriate methodology to evaluate the synergistic effect by disk diffusion studies. Methods The susceptibility testing was conducted as recommended by the CLSI using CFDC non-susceptible isolates (MIC of &amp;gt;4 µg/mL based on CLSI breakpoint). The MIC by BMD was determined using iron-depleted cation-adjusted Mueller-Hinton broth, in the presence or absence of 4 µg/mL of avibactam. The disk diffusion was evaluated using Mueller-Hinton agar, and the synergy was evaluated by using disk stacking methods. For disk stacking methods, CFDC disk was placed on agar on which bacterial suspension of 0.5 McFarland units was spread, then the ceftazidime-avibactam (CZA) disks was stacked on the top, followed by adding a drop (30 µL) of saline on the stacked disks. As an alternative method, CZA was immersed in saline for 1 second instead of adding a drop of saline, followed by the stacking on the top. The disk zone size was determined after 24-hour incubation at 37°C. Results Against blaPER-positive A. baumannii which showed &amp;gt;64 µg/mL MIC of CFDC and CZA, CFDC MIC decreased to 0.25 µg/mL in the presence of avibactam. The disk diffusion methods also showed isolates resistant to CFDC and CZA and showed susceptiblilty disk zone to CFDC by stacking both disks. On the other hand, against blaNDM-positive A. baumannii which showed 64 µg/mL MIC of CFDC and CZA, the disk diffusion methods showed resistance even when stacking both disks. Against multiple isolates, the MIC of CFDC without or with avibactam was correlated well with the disk zone produced by CFDC disk alone or stacked with CZA disks, respectively (Figure). Conclusion The synergistic effect between CFDC and avibactam by BMD methods could be detected by disk stacking methodology using CFDC and CZA disks. Disclosures Yoshinori Yamano, PhD, Shionogi (Employee) Miki Takemura, MS, SHIONOGI &amp; CO., LTD. (Employee) Roger Echols, MD, Shionogi (Consultant) Christopher Longshaw, PhD, Shionogi (Employee)