Aztreonam

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.

Carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CR-PA) producing metallo-β-lactamases (MBLs) cause severe nosocomial infections with no defined treatment. The combination of aztreonam (ATM) with ceftazidime-avibactam (CZA) is a potential therapeutic option, but there is no approved, feasible testing method for use in clinical laboratories to assess the activity of two antimicrobials in combination. Here, we evaluate the performance of four ATM-CZA combination testing methods, as follows: broth disk elution (DE), disk stacking (DS), strip stacking (SS), and strip crossing (SX). We used 10 clinical, representative Enterobacterales and 6 P. aeruginosa isolates harboring MBL, Guiana extended-spectrum beta-lactamase (GES), or non-MBL enzymes. Four of these isolates were from clinical cases treated by ATM-CZA. All CRE producing NDM and CR-PA producing GES that were resistant to ATM and CZA alone were susceptible to the ATM-CZA combination. P. aeruginosa generating NDM or VIM remained resistant to ATM-CZA, likely due to non-β-lactamase mechanisms, and all other isolates were susceptible to ATM or CZA alone. The most accurate, precise, and reproducible methods of low complexity were disc elution and both strip methods (SX and SS) using MIC test strips (MTS) , all with 100% sensitivity and specificity, followed by Etest with SX (95.83% sensitivity, 100% specificity) and SS (87.5% sensitivity, 100% specificity). DS had the lowest performance. DE is particularly valuable in low-resource settings that routinely use disks. MTS yielded higher categorical agreements by SX (94%) and SS (84%), relative to Etest by SX (90%) and SS (82%). P. aeruginosa results yielded the majority of the errors. These methods may allow laboratories to inform clinical decision making like combination therapy for severe infections caused by extensively drug-resistant Enterobacterales.

BackgroundAztreonam/avibactam is one of the last therapeutic options for treating infections caused by NDM-like-producing Enterobacterales. However, PBP3-modified and NDM-producing Escherichia coli strains that co-produce CMY-42 have been shown to be resistant to this drug combination. The aim of our study was to assess the in vitro activity of cefepime/taniborbactam and cefepime/zidebactam against such aztreonam/avibactam-resistant E. coli strains.MethodsMIC values of aztreonam, aztreonam/avibactam, cefepime, cefepime/taniborbactam, cefepime/zidebactam and zidebactam alone were determined for 28 clinical aztreonam/avibactam-resistant E. coli isolates. Those isolates produced either NDM-5 (n = 24), NDM-4 (n = 2) or NDM-1 (n = 2), and they all co-produced CMY-42 (n = 28). They all harboured a four amino acid insertion in PBP-3 (Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys).ResultsAll strains were resistant to aztreonam/avibactam and cefepime, as expected. The resistance rate to cefepime/taniborbactam was 100%, with MIC50 and MIC90 being at 16 mg/L and 64 mg/L, respectively. Conversely, all strains were susceptible to cefepime/zidebactam, with both MIC50 and MIC90 at 0.25 mg/L. Notably, all strains showed low MICs for zidebactam alone, with MIC50 and MIC90 at 0.5 mg/L and 1 mg/L.ConclusionsOur data highlighted the excellent in vitro performance of the newly developed β-lactam/β-lactamase inhibitor combination cefepime/zidebactam against aztreonam/avibactam-resistant E. coli strains, suggesting that this combination could be considered as an efficient therapeutic option in this context. Our study also highlights the cross-resistance between acquired resistance to aztreonam/avibactam and the cefepime/taniborbactam combination.

Members of the Stenotrophomonas maltophilia complex are intrinsically multidrug-resistant pathogens that disproportionately affect critically ill patients. Aztreonam-avibactam (ATM-AVI), FDA approved in 2025, combines aztreonam (ATM; stable to L1 β-lactamase) with avibactam (AVI; an L2 β-lactamase inhibitor). Aztreonam plus ceftazidime-avibactam (ATM-CZA) has been used as a surrogate for ATM-AVI, but direct comparisons between the two regimens are lacking. Twenty-three clinical S. maltophilia complex isolates underwent broth microdilution (BMD) testing for ATM, ceftazidime, ATM-AVI, and ATM-CZA, with gradient diffusion performed in parallel for ATM-AVI and ATM-CZA. Static 24-h time-kill assays at humanized steady-state Cmax concentrations evaluated bactericidal activity (≥3-log₁₀ reduction). Semi-mechanistic pharmacodynamic modeling characterized growth kill dynamics by resistance determinants (blaL1, blaL2, smeABC). ATM-CZA and ATM-AVI MIC50/90 values for BMD were 1/2 and 2/4 mg/L, respectively. Both regimens were bactericidal against most isolates, with a mean paired difference of 0.09 log₁₀ colony-forming units (CFU)/mL (P = 0.83). Isolate-level variation was evident: ATM-AVI sustained killing, whereas ATM-CZA permitted regrowth for MD17639, -4.86 vs 0.13 log₁₀ CFU/mL, P = 0.019; MD17061, -2.61 vs 0.64 log₁₀ CFU/mL, P P = 0.026), MD17047 (-4.27 vs -2.64; P = 0.021), and UK4 (-3.47 vs -1.58; P = 0.017). Modeling predicted ATM-AVI benefit in blaL2 and ATM-CZA benefit against blaL1 or smeABC dominant isolates, and diminished activity of both when mechanisms coexisted. ATM-AVI and ATM-CZA show comparable in vitro activity against S. maltophilia complex. Isolate-level heterogeneity warrants further study of genotype-phenotype relationships.