Antimicrobial Susceptibility Testing Paradigms: Current Status and Future Directions

The development of new antimicrobial agents is essential to combat antimicrobial resistance. Reliable antimicrobial susceptibility testing (AST) methods should be established early and thoughtfully to ensure timely patient access to these agents. The standard reference method for AST is broth microdilution (BMD) in cation-adjusted Mueller-Hinton broth (CAMHB), as defined by CLSI M07 (CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 12th ed. CLSI Standard M07. Clinical and Laboratory Standards Institute, 2024) and ISO 20776-1 (ISO. Susceptibility Testing of Infectious Agents and Evaluation of Performance of Antimicrobial Susceptibility Test Devices-Part 1: Broth Micro-dilution Reference Method for Testing the In Vitro Activity of Antimicrobial Agents Against Rapidly Growing Aerobic Bacteria Involved in Infectious Diseases. ISO 20776-1. International Organization for Standardization, 2019). While some agents may require modifications to this method to better reflect clinical activity, such changes must be scientifically justified. Modifications aimed solely at producing lower minimal inhibitory concentration (MIC) values-or to make one antimicrobial agent appear superior to others-are not scientifically valid and are strongly discouraged. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) caution developers that unnecessary deviations from reference AST methods can lead to increased costs, regulatory hurdles, delays in test availability and reduced clinical adoption. Until joint guidance under development by CLSI and EUCAST is finalized, early and rigorous evaluation using the reference method is essential.

Introduction: Acinetobacter species in clinical isolates cause severe infections including meningitis, bloodstream infection, ventilator-associated pneumonia, and surgical site infections. Objectives: In the present study, we evaluated Acinetobacter drug resistance using both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility test methods. Materials and Methods: Clinical specimens of 128 patients who were admitted in three referral tertiary care teaching hospitals were enrolled in 2014. Blood and other sterile fluid samples, endotracheal secretion, ulcer, urine and other clinical specimen cultures were included, and microbial resistance of Acinetobacter isolates was determined and compared with disk diffusion and E-test antimicrobial susceptibility methods, using both the EUCAST and CLSI standards. Cohen’s kappa coefficient was also reported. Results: The highest percentage of resistance (96.9%) was found for meropenem and imipenem antimicrobials, and the lowest resistance (82.8%) was found for amikacin. The highest kappa agreement coefficient was for ciprofloxacin (kappa coefficient = 0.783), and the lowest kappa was for amikacin (kappa coefficient = 0.21). Conclusion: According to the results, it is better to consider amikacin as a choice in combination with another effective antimicrobial for treatment of drug resistant Acinetobacter.

Schizophyllum commune is a cosmopolitan, saprophytic basidiomycete known to cause respiratory tract infections following spore inhalation. Management of such infections remained poorly defined, and antifungal susceptibility testing may provide valuable guidance for therapeutic decisions. However, existing data are limited, and protocols are not optimized for non-sporulating molds such as S. commune, requiring methodological adaptations. This study aimed to (i) perform molecular characterization of isolates and (ii) determine antifungal susceptibility profiles of a collection of S. commune isolates using both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methods. A total of 113 fungal isolates were included, comprising environmental (n = 31), clinical French isolates (n = 74), and strains from fungal international collections (n = 8). Species identification was confirmed via the large subunit (LSU) region of the ribosomal DNA sequencing and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phylogenetic analysis was conducted on a subset of 20 isolates using partial sequences of three genes: LSU, EF-1α, and RPB2. Modifications to the CLSI and EUCAST broth microdilution methods included: inoculum preparation using standardized culture slices fragmented by bead-beating, incubation at 35°C for 96 h and endpoints reading at 100% of inhibition. Phylogenetic analysis confirmed that all tested isolates were S. commune and not S. radiatum. Amphotericin B (AMB) and voriconazole (VOR) demonstrated low geometrical mean minimal inhibitory concentrations (MICs) across both methods (EUCAST: AMB = 0.39 µg/mL, VOR = 0.24 µg/mL; CLSI: AMB = 0.1 µg/mL, VOR = 0.2 µg/mL). In contrast, terbinafine exhibited high MIC values (>8 µg/mL) in both protocols. Notable discrepancies were observed for posaconazole (POS), isavuconazole (ISA), and itraconazole (ITR), with MICs approximately two dilution steps higher in EUCAST compared to CLSI (POS: 4.22 µg/mL vs 0.9 µg/mL, ISA: 3.64 µg/mL vs 0.74 µg/mL, and ITR: 3.9 µg/mL vs 0.81 µg/mL, respectively). After standardization of both methods for this non-sporulating mold, VOR and AMB have the lowest MIC values.IMPORTANCESchizophyllum commune is a fungal pathogen increasingly associated with respiratory infections, yet therapeutic guidance remains unclear. This study provides the largest collection to date of clinical and environmental isolates (113 total) and applies standardized antifungal susceptibility testing using adapted EUCAST and CLSI protocols for this non-sporulating species. The results show that amphotericin B and voriconazole are the most active agents in vitro, while terbinafine is ineffective. These findings are critical for informing treatment decisions and interpreting susceptibility tests, especially in the absence of established guidelines. By introducing a reproducible methodology and delivering clinically relevant data, this work addresses a gap in medical mycology and supports improved management of rare fungal infections.

Contemporary tetracycline susceptibility testing: doxycycline MIC methods and interpretive criteria (CLSI and EUCAST) performance when testing Gram-positive pathogens

The main objective of susceptibility testing is to predict the outcome of treatment with the antimicrobial agents tested. The test results are generally reported to the treating physician, using the categories of susceptible, intermediate, and resistant. From the laboratory perspective, the key decisions involve the selection of a susceptibility testing method and the antimicrobial agents to be tested for each specimen and pathogen type. The principal methods used internationally are those of the Clinical and Laboratory Standards Institute (CLSI), based in the United States, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both organizations have a sophisticated approach to the setting of breakpoints (interpretive criteria) which permit the categorization of the susceptibility test results. This approach includes the collation and analysis of MIC distribution data, pharmacokinetic and pharmacodynamic data and associated target values, and clinical and bacteriological response rates from prospective clinical trials. This analysis is followed by the correlation of MICs and zone diameter distributions in order to establish breakpoints for commonly used methods. Modern susceptibility testing methods are often supplemented by specialized phenotypic and/or molecular testing to ensure that critical resistances and resistance mechanisms are detected.

We studied the antimicrobial activity of fosfomycin against 960 strains of commonly encountered bacteria associated with urinary tract infection using standard agar dilution and disk diffusion methods. Species studied included 3 common species of Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia; methicillin-susceptible and -resistant Staphylococcus aureus; and vancomycin-susceptible and resistant Enterococcus faecalis and E. faecium. MICs and inhibition zone diameters were interpreted in accordance with both the currently recommended Clinical and Laboratory Standards Institute (CLSI) criteria for urinary tract isolates of Escherichia coli and Enterococcus faecalis and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae. Tentative zone diameter interpretive criteria were developed for species not currently published by CLSI or EUCAST. Escherichia coli was uniformly susceptible to fosfomycin, as were most strains of Klebsiella pneumoniae and Enterobacter cloacae. A. baumannii was resistant to fosfomycin, while the prevalence of resistance in P. aeruginosa and S. maltophilia was greatly affected by the choice of MIC breakpoint. New tentative zone diameter criteria for K. pneumoniae, E. cloacae, S. aureus, and E. faecium were able to be set, providing some interim laboratory guidance for disk diffusion until further breakpoint evaluations are undertaken by CLSI and EUCAST.

To report reference method antimicrobial susceptibility testing results for recent clinical isolates of Gram-negative bacilli from Morocco. CLSI (Clinical and Laboratory Standards Institute) broth microdilution antimicrobial susceptibility testing was performed by a central laboratory for isolates of Enterobacterales (n=810), Pseudomonas aeruginosa (n=321), and Acinetobacter baumannii (n=191) collected in 2018-2020 by three hospital laboratories in Morocco. MICs were interpreted using both CLSI (2021) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) (2021) breakpoints. Molecular testing for β-lactamase genes was performed on isolates meeting defined screening criteria. Most isolates of Enterobacterales were susceptible (CLSI/EUCAST breakpoints) to amikacin (98.0%/96.2%), ceftazidime-avibactam (94.8%/94.8%), and meropenem (92.5%/94.2%). Of Enterobacterales isolates eligible for β-lactamase gene screening (n=210), 174 were ESBL-positive, 40 were metallo-β-lactamase-positive (all NDM), 39 were serine carbapenemase-positive (all OXA); and 7 isolates carried both OXA-48 and NDM-1. Amikacin (89.1%/89.1%) and ceftazidime-avibactam (88.2%/88.2%) were the most active agents tested against P. aeruginosa. Applying CLSI and EUCAST breakpoints, MDR rates were 21.9% and 29.3% for Enterobacterales and 18.4% and 21.8% for P. aeruginosa. Susceptible rates for amikacin, ceftazidime-avibactam, and meropenem were 93.2%/89.5%, 77.4%/82.3%, and 67.8%/80.2% for MDR Enterobacterales and 50.8%/57.1%, 40.7%/45.7%, and 27.1/32.9% for MDR P. aeruginosa. ≥70% of A. baumannii isolates were resistant to all agents tested (except colistin, EUCAST breakpoints only) including amikacin and meropenem. Newer β-lactam/β-lactamase inhibitor combinations such as ceftazidime-avibactam warrant testing and reporting for Enterobacterales and P. aeruginosa in Morocco given the presence of significant resistance to first-line β-lactams and fluoroquinolones, pervasive ESBLs and carbapenemases, and toxicity concerns associated with some second-line agents.

Background & objectives:The emergence of resistance to fluoroquinolones in enteric fever despite the pathogen being susceptible by in vitro laboratory results, led to repeated changes in Clinical and Laboratory Standard Institute (CLSI) guidelines for this class of antibiotics to have specific and sensitive interpretative criteria. In 2015, CLSI added pefloxacin disk diffusion criteria as a surrogate marker for fluoroquinolone susceptibility. This study was carried out to evaluate the use of pefloxacin as a surrogate marker for ciprofloxacin, ofloxacin and levofloxacin susceptibility in clinical isolates of Salmonella Typhi and S. Paratyphi A.Methods:A total of 412 strains of S. Typhi and S. Paratyphi A were studied for pefloxacin disk diffusion test as a surrogate marker for susceptibility to ciprofloxacin, ofloxacin and levofloxacin as per CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Molecular mechanisms of resistance to fluoroquinolones were also determined and correlated with pefloxacin susceptibility breakpoints.Results:Of the total 412 strains, 34 were susceptible to ciprofloxacin and 33 each to levofloxacin and ofloxacin using CLSI minimum inhibitory concentration (MIC) breakpoints. There was a positive correlation between MICs with correlation coefficients 0.917, 0.896 and 0.958 for the association between ciprofloxacin and ofloxacin, ciprofloxacin and levofloxacin and ofloxacin and levofloxacin, respectively (P <0.001). The sensitivity, specificity and positive predictive value of pefloxacin as a surrogate marker using ciprofloxacin MIC as a gold standard were 100, 99.5 and 94.4 per cent, while 100, 99.2 and 91.7 per cent taking ofloxacin and levofloxacin MIC as gold standard. Mutations in target genes correlated with the pefloxacin susceptibility results.Interpretation & conclusions:Our results showed that pefloxacin served as a good surrogate marker for the detection of susceptibility to ciprofloxacin, ofloxacin and levofloxacin in S. Typhi and S. Paratyphi A. Further studies are required to confirm these findings.

In 2007, 20 Enterococcus faecalis isolates from bloodstream infections diagnosed at seven hospitals in the North Denmark region were recorded by the regional clinical microbiology laboratory as being penicillin resistant (MICs ≥ 16 μg/ml) and ampicillin susceptible (MICs ≤ 4 μg/ml) using Etest (AB bioMerieux, Solna, Sweden). Seventeen and two isolates were additionally resistant to gentamicin (MICs ≥ 1,024 μg/ml) and imipenem (MIC = 32 μg/ml), respectively (Table ​(Table11). TABLE 1. Origin, MICs determined by Etest, and genotypes based on partial (gdh gene) or full multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) of 33 E. faecalis isolates from bloodstream infections in the North Denmark region in 2007 ... Due to the unusual susceptibility pattern obtained by Etest, the penicillin MICs were measured by broth microdilution (BMD) according to the Clinical and Laboratory Standards Institute (CLSI) (1). Twofold dilutions of penicillin G sodium salt (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) in the range between 0.5 and 32 μg/ml were prepared using Mueller-Hinton broth (MHB, Oxoid A/S, Greve, Denmark). Thirteen E. faecalis blood culture isolates from hospitalized patients obtained during the same year were randomly selected for comparative purposes based on their susceptibility to penicillin determined by Etest (MICs ≤ 12 μg/ml) (Table ​(Table1).1). All 20 isolates were reported as being penicillin resistant according to Etest and the 13 control isolates were found to be penicillin susceptible by BMD based on the CLSI interpretive MIC breakpoint (S ≤ 8 μg/ml) and fell into the wild-type E. faecalis MIC distribution (1 to 16 μg/ml), provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). However, using BMD according to CLSI standards, the penicillin MICs for the isolates that were reported to be penicillin resistant according to Etest were two- to eightfold higher (4 to 8 μg/ml) than those for the control isolates reported to be susceptible by Etest (1 to 2 μg/ml), with the exception of a single isolate displaying a MIC of 2 μg/ml. Gentamicin resistance (MIC ≥ 1,024 μg/ml) was confirmed in 17 of the 20 isolates displaying reduced susceptibility to penicillin, whereas the two isolates displaying imipenem resistance were susceptible to this agent (MICs ≤ 2 μg/ml). Multilocus sequence typing (MLST) (2) showed that most (17/20) E. faecalis isolates with reduced penicillin susceptibility belonged to sequence type 6 (ST6), whereas none of the control isolates belonged to ST6, as shown by partial (gdh gene) MLST typing. Pulsed-field gel electrophoresis (PFGE) (3) demonstrated closely related patterns (≤5-band difference) among the 17 ST6 isolates with reduced penicillin susceptibility (data not shown). Eight PFGE profiles (A1 to A8) were designated on the basis of minor band differences, and the most common profile (A1) was found in six isolates originating from four hospitals (Table ​(Table11). We report the occurrence in several Danish hospitals of a distinctive penicillin susceptibility phenotype in E. faecalis consisting of reduced penicillin susceptibility and full ampicillin susceptibility. Most isolates displaying this phenotype were additionally resistant to gentamicin, belonged to type ST6, and had indistinguishable or closely related PFGE patterns, indicating the occurrence of a clonal outbreak. Despite the increased MICs of penicillin and imipenem obtained by Etest, the ST6 isolates were susceptible according to CLSI standards. The results draw attention to the ambiguity of penicillin and imipenem MICs determined by concentration gradient diffusion assays such as Etest. In laboratories where these assays are routinely employed for susceptibility testing of E. faecalis, resistant isolates should be confirmed by broth dilution. As long as the mechanism leading to in vitro-reduced penicillin susceptibility in E. faecalis ST6 remains unknown, the use of penicillin should be considered with caution for treatment of infections caused by strains displaying this atypical phenotype.

Predicting doripenem susceptibility based on meropenem and imipenem interpretation for Pseudomonas aeruginosa

INTRODUCTION: Antibiotic resistance to Helicobacter varies by region. A 2001 report of H. pylori clinical trials noted 10.6% clarithromycin and 21.6% metronidazole resistance nationally. Regional clarithromycin resistance varied from 8.3% in the West to 13.0% in the Northeast and metronidazole resistance ranged from 22.1% in the Northeast to 27.5% in the Southeast. A 2015 Houston VAMC study noted resistance of 16.4% to clarithromycin and 20.3% to metronidazole. A recent clinical study found additional concerns for increasing resistance at both national and regional levels for H. pylori infection. METHODS: Isolates collected in a multi-center clinical trial (2017-2018) from de-identified patients were tested for minimal inhibitory concentrations (MICs) by agar dilution using Clinical and Laboratory Standards Institute (CLSI) methods. CLSI and EUCAST (The European Committee on Antimicrobial Susceptibility Testing) interpretive breakpoints were applied. MICs were obtained for amoxicillin, clarithromycin, metronidazole and rifabutin. Analysis provided resistance patterns for both national and regional levels. RESULTS: MICs were reported for 345 of 356 clinical isolates recovered from treatment-naïve patients at 55 centers (20 states) across the US. The regions were broken down as Northeast, Southeast, South, Central, and West. The MIC90 was 0.125 µg/mL for amoxicillin, 8 µg/mL for clarithromycin, 64 µg/mL for metronidazole, and ≤0.008 µg/mL for rifabutin. Across all US sites, resistance rates were: amoxicillin 6.7%; clarithromycin 17.4%; metronidazole 43.7% and rifabutin 0.0%. Regional assessment of amoxicillin, clarithromycin and metronidazole resistance was: Northeast 0.0% (n = 0), 31.6% (n = 6) and 36.8% (n = 7); Southeast: 6.1% (n = 8), 22.0% (n = 29) and 51.5% (n = 68); South 6.5% (n = 5), 11.7% (n = 9) and 45.5% (n = 35); Central 0.0% (n = 0), 44.4% (n = 4) and 22.2% (n = 2); and West 8.3% (n = 9), 11.1% (n = 12) and 35.5% (n = 38), respectively. CONCLUSION/DISCUSSION: Nationally, we found greater resistance rates for clarithromycin (17.4%) and metronidazole (43.7%) compared to previous reports from 2001 and 2015. Regionally, clarithromycin resistance was greater for the Northeast, Southeast and Central areas while metronidazole resistance was greater than previously reported across all regions. Amoxicillin resistance was low (6.7%: 0%-8.3%), and no resistance to rifabutin was reported. These emerging resistance patterns support the use of rifabutin based therapies for H. pylori eradication.

This is the first Portuguese multicenter observational and descriptive study that provides insights on the species distribution and susceptibility profiles of yeast isolates from fungemia episodes. Ten district hospitals across Portugal contributed by collecting yeast isolates from blood cultures and answering questionnaires concerning patients' data during a 12-month period. Molecular identification of cryptic species of Candida parapsilosis and C. glabrata complex was performed. The susceptibility profile of each isolate, considering eight of the most often used antifungals, was determined. Both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocols were applied. The incidence of 240 episodes of fungemia was 0.88/1,000 admissions. Fifteen different species were found, with C. albicans (40 %) being the most prevalent, followed by C. parapsilosis (23 %) and C. glabrata (13 %). Most isolates were recovered from patients admitted to surgical wards or intensive care units, with 57 % being males and 32 % aged between 41 and 60 years. For both the CLSI and EUCAST protocols, the overall susceptibility rates ranged from 74 to 97 % for echinocandins and from 84 to 98 % for azoles. Important resistance rate discrepancies between protocols were observed in C. albicans and C. glabrata for echinocandins and in C. parapsilosis and C. tropicalis for azoles. Death associated with fungemia occurred in 25 % of the cases, with more than half of C. glabrata infections being fatal. The great number of Candida non-albicans is noteworthy despite a relatively low antifungal resistance rate. Studies like this are essential in order to improve empirical treatment guidelines.

Background: Vancomycin is a critically important antimicrobial in human medicine, and vancomycin-non-susceptible enterococci represent a One Health concern when animal reservoirs contribute to the wider resistance ecology. We aimed to characterize vancomycin non-susceptibility among poultry-derived Enterococcus spp. from Hungary, using a combined phenotypic-genomic approach. Methods: Following a phenotypic pre-screen with antimicrobials authorized for poultry, 218 isolates with elevated minimum inhibitory concentrations (MICs) were selected for extended broth microdilution testing including vancomycin. Vancomycin susceptibility was interpreted using Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints and European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values (ECOFFs). Whole-genome sequencing was performed on a targeted multidrug resistant (MDR) subset (n = 42), enriched for elevated or borderline vancomycin MICs and stratified by region and host species (chicken, turkey), and resistance determinants were annotated against the Comprehensive Antibiotic Resistance Database (CARD) using stringent similarity/coverage thresholds. Results: Among the 218 pre-screened isolates (126 from chickens; 92 from turkeys), 196 (89.9%) met MDR criteria. For vancomycin, 15.6% of isolates were resistant and 9.2% intermediate by CLSI, while EUCAST ECOFF classification placed 34.9% in the non-wild-type group. The vancomycin MIC distribution was right shifted, with high-end MICs observed. In the sequenced subset, vancomycin-associated determinants consistent with the vanC pathway (including regulatory and auxiliary components) were detected in five isolates. Beyond vancomycin-related determinants, the WGS subset harbored common resistance genes consistent with the observed multidrug-resistant phenotypes. Conclusions: Vancomycin non-susceptibility was detected among pre-screened poultry-derived Enterococcus isolates in Hungary, and genomic analysis revealed vanC-associated and other peptide antibiotic resistance signatures. These findings support targeted One Health surveillance integrating MIC distributions with genomic resistance determinants in food animal reservoirs.

Clinical and Laboratory Standards Institute (CLSI) M100 ‘Performance Standards for Antimicrobial Susceptibility Testing (AST)’ and the European Committee on AST (EUCAST) ‘Breakpoint tables for interpretation of MICs and zone diameters’ guidelines for conducting and interpreting AST are revised yearly. The 2023 CLSI guideline introduces selective and cascade reporting methods for antibacterial agents as a part of strengthening antibiotic stewardship and changes in breakpoints for aminoglycoside (AG) in Enterobacterales and AG and piperacillin in Pseudomonas aeruginosa. Main changes in EUCAST include revised breakpoints for aminopenicillins in Enterobacterales, and detailed criteria reflecting the clinical situation and antibacterial agent administration method.

Oral amoxicillin/clavulanate is a community workhorse antibiotic, routinely prescribed for respiratory tract infections, skin infections as well as urinary tract infections (UTIs). Multiple adult and paediatric dose formulations of amoxicillin/clavulanate are available in different parts of the world. In adult formulations, clavulanic acid dose is restricted to 125 mg because of tolerability issues. Despite its popular use for 40 years, few pharmacokinetic/pharmacodynamic (PK/PD) studies were undertaken to justify the doses and breakpoints currently in use for various infections. Clavulanate has a minimal role in the combination’s use for respiratory infections. In the context of rising extended spectrum beta-lactamase (ESBL) prevalence globally, empirical and overuse of orally administered amoxicillin/clavulanate may select resistance in Gram-negative pathogens. The susceptibility test methods and interpretive criteria differ between the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Third-generation oral cephalosporins such as ceftibuten or cefpodoxime can be combined with amoxicillin/clavulanate to tackle UTIs involving ESBL producing Escherichia coli and Klebsiella spp. Clinicians who routinely prescribe amoxicillin/clavulanate in outpatient settings should be aware of potential benefits and limitations of this combination.