What's New in 2025: From the CLSI Subcommittee on Antimicrobial Susceptibility Testing

The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing (AST) held meetings during January and June 2025. Several breakpoint revisions and new breakpoints were introduced by ad hoc working groups (AHWGs) and subsequently approved by the full subcommittee.

Acinetobacter baumannii Breakpoint Revisions

Acinetobacter is a nonfermenting gram-negative organism known to cause health care–associated infections, including pneumonia, bacteremia, and urinary tract infections (UTIs).¹ An AHWG was formed to systematically review all Acinetobacter breakpoints after the US Food and Drug Administration (FDA) approval of sulbactam-durlobactam in 2023.² After prior review of ampicillin/sulbactam and minocycline breakpoints in 2024, the AHWG reviewed data for doxycycline, tetracycline, and aminoglycoside breakpoints in 2025.

Doxycycline

The original breakpoints for the tetracyclines and Acinetobacter spp were extrapolated from other organisms with no Acinetobacter-specific data. Unlike minocycline, no contemporary pharmacokinetic/ pharmacodynamic (PK/PD) data exist for doxycycline against Acinetobacter baumannii. The free area under the concentration-time curve (AUC)/minimal inhibitory concentration (MIC) target is unknown, and it cannot be assumed to be the same as minocycline because there are differences in the AUC/MIC targets for tigecycline and eravacycline. The current Infectious Diseases Society of America guidance³ for treatment of resistant gram-negative infections does not mention doxycycline as an option for the treatment of carbapenem-resistant A baumannii, and there are very limited clinical data^4,5 demonstrating its use or efficacy against A baumannii. In light of a lack of data supporting this breakpoint, a motion to remove the doxycycline breakpoints for Acinetobacter spp in the M100 document was made and approved by the full AST subcommittee (TABLE 1).

Tetracycline

The previous tetracycline breakpoints for A baumannii were approved only for urine isolates. Similar to doxycycline, sufficient contemporary data to support these breakpoints could not be located. Tetracycline urine concentrations after usual dosing were variable in the literature. Although some older case reports6 suggested successful treatment of Pseudomonas aeruginosa with tetracycline, almost no clinical data are published to support its use against A baumannii. With the exception of one paper⁷ that evaluated tetracycline efficacy using a mouse UTI model against a single strain (with no isolate MIC reported) of A baumannii, no PK/PD data were available. Although tetracycline demonstrated some efficacy in the mouse UTI model, it was significantly less active than minocycline and doxycycline.⁷ When reviewing the isolate MIC distribution data, no formal epidemiological cutoff value (ECV) could be set because data were available from only one laboratory (JMI Laboratories SENTRY Antimicrobial Surveillance Program; unpublished data). Using ECOFFinder⁸ and data from the aforementioned JMI data set, an unofficial ECV was set at approximately 8 μg/mL, which is one dilution above the previous urine susceptibility breakpoint of 4 μg/mL or less. A motion was made to remove the tetracycline urine breakpoints for Acinetobacter spp, and the vote passed the full subcommittee.

Minocycline Breakpoint Comment

With the removal of both doxycycline and tetracycline breakpoints, the remaining valid breakpoint for the tetracycline class is minocycline. Because minocycline is not found on many automated panels or is not present at a dilution low enough to apply the reduced susceptibility breakpoint (eg, ≤ 1 μg/mL) published in the 2025 edition of the M100 document, a comment has been added regarding prediction of minocycline susceptibility. This comment reads: “When available, it is recommended to test minocycline directly. If minocycline cannot be tested, isolates with doxycycline MICs ≤ 1 μg/mL or tetracycline MICs ≤ 4 μg/mL are considered susceptible to minocycline. Isolates with doxycycline MICs ≥ 2 μg/mL or tetracycline MICs ≥ 8 μg/mL should be tested against minocycline if that result is needed for treatment.”

Amikacin

Recent PK/PD data using the neutropenic mouse thigh infection model determined that the free-drug plasma PK/PD targets for a 1-log₁₀ reduction from baseline was a median AUC/MIC ratio of 12.2 for amikacin against A baumannii.⁹ Monte Carlo simulations using intravenous dosing of 20 mg/ kg every 24 hours and simulated data from patients with complicated UTI/acute pyelonephritis and hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) in clinical trials were conducted. The simulations used the PD target of 12.2 and determined that probability of PK/ PD target attainment (PTA) in the models was more than 90% for isolates with MICs up to 8 μg/mL. At MIC values greater than 16 μg/mL, PTA dropped below 75%. Sparse clinical data were reviewed, but amikacin was often part of combination therapy for A baumannii and no outcome data by MIC were located.¹⁰ Upon review of amikacin MIC distribution data using several data sets, the ECOFFinder suggested an ECV of 8 µg/mL for the purpose of breakpoint setting, which also agrees with where the European Committee on Antimicrobial Susceptibility Testing has set its ECOFF. A motion to accept the lowered amikacin MIC breakpoints of (susceptible [S] ≤ 8 μg/ mL; intermediate [I] = 16 μg/mL; resistant [R] ≥ 32 μg/mL) for Acinetobacter spp based on a dosage of 20 mg/kg daily was made and approved by the full committee. New disk diffusion breakpoints (S ≥ 20 mm; I = 17-19 mm; R ≤ 16 mm) for Acinetobacter spp were also approved.

Gentamicin and Tobramycin

On review of the current gentamicin and tobramycin breakpoints, no clinical data exist and it is unlikely that new PK/PD data will be generated. To assess these breakpoints, the AHWG evaluated whether the PK/PD target for amikacin could be extrapolated along with the gentamicin/ tobramycin PTA analyses for the other gram-negative organisms Enterobacterales and P aeruginosa, where PK/PD modeling targets do exist. In general, the target AUC/MIC exposures have been considered interchangeable/applicable to all aminoglycosides.¹¹ Experimental data from mouse studies have shown that the AUC/ MIC target for gentamicin and Escherichia coli is the same as that for tobramycin and E coli.

Using a 2-log10 kill target (AUC/MIC ratio of 19.2) for amikacin and Acinetobacter spp in the neutropenic thigh model, the stasis target (AUC/MIC ratio of 21.4) for gentamicin and Enterobacterales is similar.¹¹ Using these, it is likely that more than 90% PTA can be achieved for isolates with MIC of 2 μg/mL or less. Similarly, for tobramycin, if the PD targets behave similarly in A baumannii as in other gram-negative organisms, then more than 90% PTA can also be achieved for isolates with MIC of 2 μg/mL or less. These data, along with MIC distribution data demonstrating that the ECV for both tobramycin and gentamicin was 2 μg/mL, led the AHWG to suggest MIC breakpoints for both agents be reduced by one doubling dilution (S ≤ 2 μg/mL; I = 4 μg/mL; R ≥ 8 μg/mL) for Acinetobacter spp based on a dosage of 7 mg/kg daily. These breakpoints were formally approved by the full subcommittee.

New Aztreonam-Avibactam Breakpoint for Enterobacterales

Aztreonam-avibactam (ATM-AVI) was approved by the FDA in February 2025 in combination with metronidazole for use in patients 18 years or older who have limited or no alternative treatment options for the treatment of complicated intra-abdominal infections (cIAIs). The approved dosing is 1.5 g ATM with 0.5 g AVI every 6 hours infused over 3 hours. ATM is not hydrolyzed by metallo-β-lactamases, and AVI prevents hydrolysis of ATM by serine β-lactamases; therefore, ATM-AVI is effective against metallo-β-lactamase–producing isolates.

Upon review of MIC distribution data, it was determined that to avoid cutting into the wild-type distribution, based on ECV alone, the susceptibility breakpoint should be no lower than 0.5 μg/mL. PK/PD targets were derived from hollow fiber infection models and the mouse thigh and lung infection models testing both E coli and Klebsiella pneumoniae. Although data presented demonstrated the ATM-AVI was active against E coli with MICs of 8 to 16 μg/mL, there were less data showing activity against K pneumoniae isolates and isolates tested had lower ATM-AVI MICs of 0.5 μg/mL or less.¹² PTA analyses were performed for 5000 simulated patients with cIAI and HAP/VAP using steady-state plasma exposures. ATM-AVI achieved more than 90% PTA for isolates with MIC of 8 μg/mL or less, which incorporates the majority of the global distribution of MICs against Enterobacterales. Clinical outcomes data were available from 2 phase 3 clinical trials: the REVISIT trial (NCT03329092) and the ASSEMBLE (NCT03580044) study.^13,14 Clinical data from the REVISIT trial in participants with cIAI showed high rates of clinical cure and microbiological eradication against Enterobacterales in isolates with ATM-AVI MIC values of 2 μg/mL or less; however, there were no participants with infection from isolates with ATM-AVI MIC values of 4 μg/mL.¹³ After review of the data and extensive discussion among the subcommittee, a motion to accept the ATM-AVI MIC breakpoints (S ≤ 4/4 μg/mL; I = 8/4 μg/mL; R ≥ 16/4 μg/mL) for Enterobacterales was made and approved by the full committee. These breakpoints harmonize with the breakpoints approved by the FDA.

References

1.Kubin CJ, Garzia C, Uhlemann AC. Acinetobacter baumannii treatment strategies: a review of therapeutic challenges and considerations. Antimicrob Agents Chemother. 2025;69(8):e0106324. doi:10.1128/aac.01063-24

2.Parkinson J. FDA approves sulbactam-durlobactam for bacterial pneumonia. ContagionLive. May 23, 2023. https://www.contagionlive.com/view/fda-approves-sulbactam-durlobactam. Accessed September 8, 2025.

3.Tamma PD, Heil EL, Justo JA, Mathers AJ, Satlin MJ, Bonomo RA. Infectious Diseases Society of America 2024 guidance on the treatment of antimicrobial-resistant gram-negative infections. Clin Infect Dis. Published online August 7, 2024. doi:10.1093/cid/ciae403

4.Falagas ME, Vardakas KZ, Kapaskelis A, Triarides NA, Roussos NS. Tetracyclines for multidrug-resistant Acinetobacter baumanniiinfections. Int J Antimicrob Agents. 2015;45(5):455-460. doi:10.1016/j.ijantimicag.2014.12.031

5.Tuon FF, Yamada CH, de Andrade AP, Arend LNVS, Dos Santos Oliveira D, Telles JP. Oral doxycycline to carbapenem-resistant Acinetobacter baumannii infection as a polymyxin-sparing strategy: results from a retrospective cohort. Braz J Microbiol. 2023;54(3):1795-1802. doi:10.1007/s42770-023-01015-0

6.Musher DM, Minuth JN, Thorsteinsson SB, Holmes T. Effectiveness of achievable urinary concentrations of tetracyclines against “tetracycline-resistant” pathogenic bacteria. J Infect Dis.1975;131(suppl):S40-S44. doi:10.1093/infdis/131.supplement.s40

7.Obana Y, Nishino T, Tanino T. In-vitro and in-vivo activities of antimicrobial agents against Acinetobacter calcoaceticus. J Antimicrob Chemother. 1985;15(4):441-448. doi:10.1093/jac/15.4.44

8.ECOF finder. Clinical and Laboratory Standards Institute. https://clsi.org/resources/ecoffinder/. Accessed September 8, 2025.

9.Lepak AJ, Trang M, Hammel JP, et al; United States Committee on Antimicrobial Susceptibility Testing. Development of modernized Acinetobacter baumannii susceptibility test interpretive criteria for recommended antimicrobial agents using pharmacometric approaches. Antimicrob Agents Chemother. 2023;67(4):e0145222. doi:10.1128/aac.01452-22

10.Poulikakos P, Tansarli GS, Falagas ME. Combination antibiotic treatment versus monotherapy for multidrug-resistant, extensively drug-resistant, and pandrug-resistant Acinetobacter infections: a systematic review. Eur J Clin Microbiol Infect Dis. 2014;33(10):1675-1685. doi:10.1007/s10096-014-2124-9

11.Aminoglycoside in Vitro Susceptibility Test Interpretive Criteria Evaluations. United States Committee on Antimicrobial Susceptibility Testing. February 24, 2019. https://app.box.com/s/1hxc8inf8u3rranwmk3efx48upvwt0ww. Accessed September 8, 2025.

12.Crandon JL, Nicolau DP. Human simulated studies of aztreonam and aztreonam-avibactam to evaluate activity against challenging gram-negative organisms, including metallo-β-lactamase producers. Antimicrob Agents Chemother. 2013;57(7):3299-3306. doi:10.1128/AAC.01989-12

13.Carmeli Y, Cisneros JM, Paul M, et al; COMBACTE-CARE Consortium REVISIT Study Group. Aztreonam-avibactam versus meropenem for the treatment of serious infections caused by gram-negative bacteria (REVISIT): a descriptive, multinational, open-label, phase 3, randomised trial. Lancet Infect Dis. 2025;25(2):218-230. doi:10.1016/S1473-3099(24)00499-7

14.Daikos GL, Cisneros JM, Carmeli Y, et al. Aztreonam-avibactam for the treatment of serious infections caused by metallo-β-lactamase-producing gram-negative pathogens: a phase 3 randomized trial (ASSEMBLE). JAC Antimicrob Resist. 2025;7(4):dlaf131. doi:10.1093/jacamr/dlaf131