Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a ß-lactam/ß-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane ß-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine ß-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of ß-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

Activity of omadacycline in vitro against Clostridioides difficile and preliminary efficacy assessment in a hamster model of C. difficile-associated diarrhoea.

OBJECTIVES: Antibiotics are associated with increased risk of Clostridioides difficile infection, which has limited treatment options. We assessed in vitro activity of omadacycline (an aminomethylcycline antibiotic) against the C. difficile infection strain and efficacy in a hamster model of C. difficile-associated diarrhoea. METHODS: Omadacycline, clindamycin, tigecycline, vancomycin, and metronidazole minimum inhibitory concentrations (MICs) for the infection-model strain (C. difficile ATCC 43596) were determined. Hamsters were pretreated with subcutaneous clindamycin (10 mg/kg) and infected 24 h later with C. difficile ATCC 43596; 24 h post infection, they received oral omadacycline (50 mg/kg/day), vancomycin (50 mg/kg/day), or vehicle for 5 days. Efficacy was reported as survival. RESULTS: Omadacycline was as active as tigecycline, vancomycin, and metronidazole (MIC 0.06 mg/L); clindamycin showed no activity. Median survival in hamsters was: 12 days, omadacycline; 2 days, vancomycin; 4 days, clindamycin pretreatment only. CONCLUSION: Omadacycline exhibited potent in vitro activity against C. difficile and showed efficacy in a model of C. difficile-associated diarrhoea.

Multicenter Clinical Evaluation of Vitek 2 Meropenem-Vaborbactam for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

The carbapenem/beta-lactamase inhibitor meropenem-vaborbactam (MEV) used to treat complicated urinary tract infections and pyelonephritis in adults was approved in 2017 by the U.S. Food and Drug Administration (FDA). Here, we evaluated Vitek 2 MEV (bioMérieux, Durham, NC) compared to the reference broth microdilution (BMD) method. Of 449 Enterobacterales isolates analyzed per FDA/CLSI breakpoints, the overall performance was 98.2% essential agreement (EA), 98.7% category agreement (CA), and 0% very major errors (VME) or major errors (ME). For 438 FDA intended-for-use Enterobacterales isolates, performance was 98.2% EA, 98.6% CA, and 0% VME or ME. Evaluable EA was 81.0%, but with only 42 on-scale evaluable results. Individual species demonstrated EA and CA rates of ≥90% without any VME or ME. When evaluated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, overall Vitek 2 MEV performance for Enterobacterales and Pseudomonas aeruginosa demonstrated 97.3% EA, 99.2% CA, 2.3% VME, and 0.6% ME (after error resolution: 97.3% EA, 99.4% CA, 2.2% VME, and 0.4% ME) compared to the reference BMD method. Performance for P. aeruginosa included 92.2% EA, 97.4% CA, 0% VME, and 3.0% ME (after error resolution: 92.2% EA, 98.7% CA, 0% VME, and 1.5% ME). Performance for Enterobacterales included 98.2% EA, 99.6% CA, 3.0% VME, and 0.2% ME. Evaluable EA was 80.6% but was based on only 67 evaluable results. These findings support Vitek 2 MEV as an accurate automated system for MEV susceptibility testing of Enterobacterales and P. aeruginosa and could be an alternate solution to the manual-labor-intensive reference BMD method.

Determination of MIC Quality Control Parameters for Exebacase, a Novel Lysin with Antistaphylococcal Activity.

Exebacase (CF-301), a novel, antistaphylococcal lysin (cell wall hydrolase) is the first agent of this class to enter late-stage clinical development (phase 3, NCT04160468) for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis. A multilaboratory Clinical and Laboratory Standards Institute (CLSI) M23-defined tier 2 quality control (QC) study was conducted to establish exebacase QC ranges for a new reference broth microdilution method. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were selected as reference QC strains. Broth microdilution MIC QC ranges for exebacase spanned 4 log2 dilutions and contained 99.2% of the MIC results generated for the two reference strains. The QC ranges for exebacase were defined as 0.25 to 2 µg/ml and 8 to 64 µg/ml against S. aureus ATCC 29213 and E. faecalis ATCC 29212, respectively, and were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. These QC ranges established for use with the reference broth microdilution method developed for exebacase susceptibility testing will ensure the test performance and accuracy of results generated during clinical development.

Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor Zoliflodacin.

This report describes the results of two different, multilaboratory quality control (QC) studies that were used to establish QC ranges for the novel gyrase inhibitor zoliflodacin against the ATCC strains recommended by the Clinical and Laboratory Standards Institute (CLSI). Following the completion of an eight-laboratory, CLSI document M23-defined tier 2 study, the agar dilution MIC QC range for zoliflodacin against the Neisseria gonorrhoeae QC strain ATCC 49226 was defined as 0.06 to 0.5 µg/ml and was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. This QC range will be used for in vitro susceptibility testing of zoliflodacin during phase 3 human clinical trials and surveillance studies, and eventually it will be implemented in clinical labs. In a separate study, broth microdilution MIC quality control ranges for zoliflodacin against additional QC strains were determined to be 0.12 to 0.5 µg/ml for Staphylococcus aureus ATCC 29213, 0.25 to 2 µg/ml for Enterococcus faecalis ATCC 29212, 1 to 4 µg/ml for Escherichia coli ATCC 25922, 0.12 to 0.5 µg/ml for Streptococcus pneumoniae ATCC 49619, and 0.12 to 1 µg/ml for Haemophilus influenzae ATCC 49247. These MIC QC ranges were also approved by CLSI for use in future in vitro susceptibility testing studies against organisms other than N. gonorrhoeae.

Multicenter Evaluation of the Xpert Carba-R Assay for Detection of Carbapenemase Genes in Gram-Negative Isolates.

This multicenter study evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative PCR test designed for the rapid detection of blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48 carbapenem resistance genes from bacterial isolates grown on blood agar or MacConkey agar. The results were compared to those obtained from bidirectional DNA sequence analysis of nucleic acid extracted from pure colonies. Isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii that tested as either intermediate or resistant to a carbapenem antibiotic were analyzed. A total of 467 isolates were evaluated, including prospectively collected clinical isolates, frozen isolates, and a group of contrived broth specimens sent by a central reference laboratory. The assay was run on the GeneXpert platform and took 48 min, with less than 1 min of hands-on time. Compared to the results of the reference methods, the overall sensitivity of the assay was 100% (95% confidence interval [CI], 99.0 to 100%) for isolates grown on both blood and MacConkey agars. Overall specificity was 98.1% (95% CI, 93.1 to 99.8%) and 97.1% (95% CI, 91.7 to 99.4%) for blood and MacConkey agars, respectively. This platform, previously demonstrated to be effective for the detection of carbapenemase genes in rectal swabs, is also adequate for the detection of these genes in bacterial colonies isolated from blood and MacConkey agars.

Rapid Identification of Five Classes of Carbapenem Resistance Genes Directly from Rectal Swabs by Use of the Xpert Carba-R Assay.

Carbapenemase-producing organisms (CPO) have been identified by global health leaders as an urgent threat. Detection of patients with gastrointestinal carriage of CPO is necessary to interrupt their spread within health care facilities. In this multisite study, we assessed the performance of the Xpert Carba-R test, a rapid real-time quantitative PCR (qPCR) assay that detects five families of carbapenemase genes (blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM) directly from rectal swab specimens. Using dual swabs, specimens from 755 patients were collected and tested prospectively. An additional 432 contrived specimens were prepared by seeding well-characterized carbapenem-susceptible and -nonsusceptible strains into a rectal swab matrix and inoculating them onto swabs prior to testing. Antimicrobial susceptibility testing, broth enriched culture, and DNA sequencing were performed by a central laboratory blind to the Xpert Carba-R results. The Xpert Carba-R assay demonstrated a positive percentage of agreement (PPA) between 60 and 100% for four targets (blaKPC, blaNDM, blaVIM, and blaOXA-48) and a negative percentage of agreement (NPA) ranging between 98.9 and 99.9% relative to the reference method (culture and sequencing of any carbapenem-nonsusceptible isolate). There were no prospective blaIMP-positive samples. Contrived specimens demonstrated a PPA between 95 and 100% and an NPA of 100% for all targets. Testing of rectal swabs directly using the Xpert Carba-R assay is effective for rapid detection and identification of CPO from hospitalized patients.

Impact of Variations in Test Method Parameters on In Vitro Activity of Surotomycin against Clostridium difficile and Surotomycin Quality Control Limits for Broth Microdilution and Agar Dilution Susceptibility Testing.

Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 µg/ml (standard for lipopeptide testing) is recommended. Quality control ranges for Clostridium difficile (0.12 to 1 µg/ml) and Eggerthella lenta (broth, 1 to 4 µg/ml; agar, 1 to 8 µg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.

Comparative in vitro antimicrobial activities of torezolid (TR-700), the active moiety of a new oxazolidinone, torezolid phosphate (TR-701), determination of tentative disk diffusion interpretive criteria, and quality control ranges.

This study assessed the spectrum of activity of torezolid (TR-700), the active moiety of torezolid phosphate (TR-701), and proposes tentative MIC and disk diffusion breakpoints as well as quality control ranges. The in vitro susceptibilities of 1,096 bacterial isolates, representing 23 different species or phenotypic groups, were determined for torezolid, linezolid, cefotaxime, and levofloxacin using Clinical and Laboratory Standards Institute (CLSI) broth microdilution MICs, minimum bactericidal concentrations (MBCs), agar dilution, and disk diffusion testing methods. Torezolid was very active against the majority of Gram-positive strains, including methicillin-susceptible and -resistant Staphylococcus aureus (MIC(50) = 0.25 microg/ml, MIC(90)

Inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against methicillin-resistant Staphylococcus aureus isolates collected from 1985 to 2007.

The inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against a collection of 479 methicillin-resistant Staphylococcus aureus isolates were assessed. The isolates were collected from U.S. and European hospitals from 1985 to 2007 and were primarily from blood and abscess cultures. The MICs and minimum bactericidal concentrations (MBCs) of the three agents were determined, and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. Tolerance was defined as an MBC/MIC ratio of > or = 32 or an MBC/MIC ratio of > or = 16 when the MBC was greater than or equal to the breakpoint for resistance. Tolerance to vancomycin and teicoplanin was observed in 6.1% and 18.8% of the strains, respectively. Tolerance to daptomycin was not observed.

In vitro antimicrobial activity of a new cephalosporin, ceftaroline, and determination of quality control ranges for MIC testing.

The spectrum of activity of ceftaroline was evaluated against 1,247 bacterial isolates representing 44 different species or phenotypic groups. For the majority of species, the activity of ceftaroline was comparable or superior to that of ceftriaxone. MIC and/or disk diffusion quality control ranges of ceftaroline were determined for five standard ATCC reference strains.

Proposed MIC and disk diffusion microbiological cutoffs and spectrum of activity of retapamulin, a novel topical antimicrobial agent.

Retapamulin, the first pleuromutilin antimicrobial agent approved for the topical treatment of skin infections in humans, was tested against 987 clinical isolates representing 30 species and/or resistance groups. MICs were determined along with disk diffusion zone diameters using a 2-microg disk. Population distribution and MIC versus disk zone diameter scattergrams were analyzed to determine microbiological MIC cutoff values and inhibition zone correlates. Minimum bactericidal concentrations were performed on a smaller subset of key species. The retapamulin MIC(90) against 234 Staphylococcus aureus isolates and 110 coagulase-negative staphylococci was 0.12 microg/ml. Retapamulin MIC(90)s ranged from 0.03 to 0.06 microg/ml against beta-hemolytic streptococci including 102 Streptococcus pyogenes, 103 Streptococcus agalactiae, 59 group C Streptococcus, and 71 group G Streptococcus isolates. The MIC(90) against 55 viridans group streptococci was 0.25 microg/ml. Retapamulin had very little activity against 151 gram-negative bacilli and most of the Enterococcus species tested. Based on the data from this study, for staphylococci, MICs of or=2 microg/ml with corresponding disk diffusion values of >or=20 mm, 17 to 19 mm, and or=15 mm can be proposed for susceptible-only microbiological cutoffs.

Caspofungin disk diffusion breakpoints and quality control.

Interpretive disk diffusion breakpoints for caspofungin are proposed by evaluating 762 isolates of Candida spp., representing 10 different species obtained as part of the caspofungin clinical trials. Standardized broth microdilution reference tests were compared to the zone diameters observed with 5-microg caspofungin disks produced by two different disk manufacturers. Disk diffusion breakpoints of >or=11 mm for susceptible are proposed. Compared to results from MIC testing, these zone diameters produced error rates that were

Prevalence of serotype 19A Streptococcus pneumoniae among isolates from U.S. children in 2005-2006 and activity of faropenem.

Of 393 isolates of Streptococcus pneumoniae from U.S. children collected in 2005-2006, nonvaccine serotypes accounted for 89.1%, with serotype 19A the most prevalent, representing 30.5% of all isolates. The MIC(90) of faropenem against serotype 19A isolates was 1 mug/ml, compared to > or =8 microg/ml against amoxicillin/clavulanate, cefdinir, cefuroxime axetil, and azithromycin.

National and regional assessment of antimicrobial resistance among community-acquired respiratory tract pathogens identified in a 2005-2006 U.S. Faropenem surveillance study.

Surveillance studies conducted in the United States over the last decade have revealed increasing resistance among community-acquired respiratory pathogens, especially Streptococcus pneumoniae, that may limit future options for empirical therapy. The objective of this study was to assess the scope and magnitude of the problem at the national and regional levels during the 2005-2006 respiratory season (the season when community-acquired respiratory pathogens are prevalent) in the United States. Also, since faropenem is an oral penem being developed for the treatment of community-acquired respiratory tract infections, another study objective was to provide baseline data to benchmark changes in the susceptibility of U.S. respiratory pathogens to the drug in the future. The in vitro activities of faropenem and other agents were determined against 1,543 S. pneumoniae isolates, 978 Haemophilus influenzae isolates, and 489 Moraxella catarrhalis isolates collected from 104 U.S. laboratories across six geographic regions during the 2005-2006 respiratory season. Among S. pneumoniae isolates, the rates of resistance to penicillin, amoxicillin-clavulanate, and cefdinir were 16, 6.4, and 19.2%, respectively. The least effective agents were trimethoprim-sulfamethoxazole (SXT) and azithromycin, with resistance rates of 23.5 and 34%, respectively. Penicillin resistance rates for S. pneumoniae varied by region (from 8.7 to 22.5%), as did multidrug resistance rates for S. pneumoniae (from 8.8 to 24.9%). Resistance to beta-lactams, azithromycin, and SXT was higher among S. pneumoniae isolates from children than those from adults. beta-Lactamase production rates among H. influenzae and M. catarrhalis isolates were 27.4 and 91.6%, respectively. Faropenem MICs at which 90% of isolates are inhibited were 0.5 mug/ml for S. pneumoniae, 1 mug/ml for H. influenzae, and 0.5 mug/ml for M. catarrhalis, suggesting that faropenem shows promise as a treatment option for respiratory infections caused by contemporary resistant phenotypes.

Comparative in vitro antimicrobial activity of tigecycline, a new glycylcycline compound, in freshly prepared medium and quality control.

The in vitro spectra of activity of tigecycline and tetracycline were determined for 2,490 bacterial isolates representing 50 different species or phenotypic groups. All isolates were tested simultaneously by broth microdilution using freshly prepared Mueller-Hinton broth and by disk diffusion. Portions of these data were submitted to the Food and Drug Administration (FDA) in support of the sponsor's application for new drug approval. In a separate study, MIC and disk diffusion quality control ranges were determined. The tigecycline MICs at which 50%/90% of bacteria were inhibited were (in microg/ml) as follows: for Streptococcus spp., 0.06/0.12; for Moraxella catarrhalis, 0.06/0.12; for Staphylococcus spp., 0.12/0.25; for Enterococcus spp., 0.12/0.25; for Listeria monocytogenes, 0.12/0.12; for Neisseria meningitidis, 0.12/0.25; for Haemophilus spp., 0.25/0.5; for Enterobacteriaceae, 0.05/2.0; for non-Enterobacteriaceae, 0.5/8.0. Tigecycline was consistently more potent than tetracycline against all species studied. The data from this study confirm the FDA-approved MIC and disk diffusion breakpoints for tigecycline for Streptococcus spp. other than Streptococcus pneumoniae, enterococci, and Enterobacteriaceae. Provisional breakpoints for Haemophilus spp. and S. pneumoniae are proposed based on the data from this study. The following MIC and/or disk diffusion quality control ranges are proposed: Staphylococcus aureus ATCC 29213, 0.03 to 0.25 microg/ml; S. aureus ATCC 25923, 20 to 25 mm; Escherichia coli ATCC 25922, 0.03 to 0.25 microg/ml and 20 to 27 mm; Pseudomonas aeruginosa ATCC 27853, 9 to 13 mm, Enterococcus faecalis ATCC 29212, 0.03 to 0.12 microg/ml; S. pneumoniae ATCC 49619, 0.015 to 0.12 microg/ml and 23 to 29 mm; Haemophilus influenzae ATCC 49247, 0.06 to 0.5 microg/ml and 23 to 31 mm; and Neisseria gonorrhoeae ATCC 49226, 30 to 40 mm.

Multicenter evaluation of the Etest and disk diffusion methods for differentiating daptomycin-susceptible from non-daptomycin-susceptible Staphylococcus aureus isolates.

Daptomycin is a novel cyclic lipopeptide that is approved by the U.S. Food and Drug Administration for the treatment of complicated skin and skin structure infections associated with Staphylococcus aureus and other gram-positive pathogens and also staphylococcal bacteremia, including right-sided endocarditis. The Clinical and Laboratory Standards Institute (CLSI) established "susceptible-only" interpretive criteria for broth microdilution (BMD) and disk diffusion (DD) testing of daptomycin in 2005. However, a series of S. aureus isolates have been recovered with daptomycin MICs in the nonsusceptible range (i.e., MICs of >1 microg/ml). The objective of this study was to determine the ability of the Etest and DD methods to differentiate daptomycin-susceptible from nonsusceptible isolates of S. aureus compared to the results of the CLSI BMD reference method. There was a good correlation between Etest MIC results and the results of BMD among laboratories (r = 0.86 to 0.88), with 95.3% of the Etest MICs within a +/-1 log(2) dilution of the BMD MIC result. A total of 92 of 102 (90.2%) non-daptomycin-susceptible isolates of S. aureus identified by BMD in two participating laboratories were also classified as nonsusceptible by Etest. However, the very major and major error rates reported by one of the participating laboratories were 13.5 and 4.0%, respectively, primarily due to the absence of an intermediate category. The DD method, however, did not reliably differentiate daptomycin-susceptible from non-daptomycin-susceptible isolates. In 2005, daptomycin disks were voluntarily removed from the market by Cubist Pharmaceuticals. The disk diffusion breakpoints were subsequently removed from the CLSI M100 standard in 2006.

Broth microdilution susceptibility testing of Brucella species: quality control limits for ten antimicrobial agents against three standard quality control strains.

Brucella broth without supplementation is the recommended medium for broth microdilution susceptibility tests of Brucella abortus, B. melitensis, and B. suis. Based on an eight-laboratory collaborative study using a pH-adjusted modification of this medium, we propose MIC quality control ranges for three control strains against 10 antimicrobials that are potentially efficacious for treating infections caused by these agents of bioterrorism.

Comparative in vitro antimicrobial activity of a new carbapenem, doripenem: tentative disc diffusion criteria and quality control.

OBJECTIVES: To determine the spectrum of activity of doripenem and to propose tentative MIC and disc diffusion breakpoints. METHODS: The in vitro susceptibilities of 2137 bacterial isolates, representing 28 different species, to doripenem, imipenem and meropenem were determined by the NCCLS broth microdilution and disc diffusion testing methods. RESULTS: The doripenem MIC(50)s/(90)s were (in mg/L) for Enterobacteriaceae, 0.06/0.25; Pseudomonas aeruginosa, 0.25/1; Haemophilus influenzae, 0.12/0.5; streptococci, 0.016/0.5 and for staphylococci, 0.06/4. Like other carbapenems tested, doripenem MIC(50)s/(90)s were >32/>32 and 0.5/32 mg/L for the enterococci and non-fermentative Gram-negative bacilli (excluding P. aeruginosa), respectively. Against members of the Enterobacteriaceae and H. influenzae, doripenem was generally more active than imipenem and the same as or slightly less active than meropenem. Values for the non-fermentative Gram-negative bacilli excluding P. aeruginosa were comparable for all three carbapenems. Doripenem MICs increased with increasing resistance to methicillin (staphylococci), penicillin (streptococci) and strains that were beta-lactamase-negative ampicillin-resistant (Haemophilus). Doripenem exhibits excellent activity against extended-spectrum beta-lactamase-producing strains of Escherichia coli and Klebsiella spp. The NCCLS disc diffusion test was performed simultaneously on all organisms. CONCLUSIONS: Assuming the MIC susceptible breakpoints for doripenem are < or =1 mg/L for the streptococci and < or =2 mg/L for all other genera, then disc diffusion zone diameter breakpoints can be proposed. In addition, MIC and/or disc diffusion quality control ranges of doripenem were determined for 10 ATCC reference strains.

Broth microdilution susceptibility testing of Francisella tularensis: quality control limits for nine antimicrobial agents and three standard quality control strains.

For broth microdilution susceptibility tests of Francisella tularensis, Mueller-Hinton broth with 2% Isovitalex is recommended. Using that medium, we studied three standard control strains tested with nine antimicrobial agents potentially efficacious for treating tularemia. An eight-laboratory collaborative study generated the data needed to propose appropriate MIC control limits.