Influence of PER-7 on cefiderocol susceptibility in clinical isolates of Acinetobacter baumannii producing OXA-23 or OXA-72 carbapenemases

Abstract

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Acinetobacter baumannii poses a significant challenge in clinical settings due to its ability to cause nosocomial infections and its extensive resistance to multiple antibiotics. Between March 2022 and September 2023, 759 out of 983 (77%) isolates of A. baumannii submitted to the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria produced an OXA-23 and/or OXA-72 carbapenemase.^1,2^ These infections are difficult to treat, as only a few effective antibiotics remain. One potential therapeutic option is cefiderocol, a catechol-substituted siderophore cephalosporin. However, in the mentioned time period, 181 (24%) isolates showed inhibition zone diameters <17 mm for cefiderocol, corresponding to the pharmacokinetic/pharmacodynamic (PK/PD) EUCAST breakpoint of >2 mg/L.^3^ We analysed 39 randomly selected OXA-23 (n = 31) or OXA-72 (n = 8) producing cefiderocol-resistant A. baumannii strains by PCR and/or whole-genome sequencing. Given that OXA-23 and OXA-72 are found in the vast majority of carbapenem-resistant A. baumannii in Germany, we specifically focused on cefiderocol-resistant isolates carrying these enzymes to ensure clinical and epidemiological relevance. Whole-genome sequencing revealed no mutations in previously identified resistance determinants such as PBP3, pirA, or piuA, and no NDM-type or other metallo-β-lactamases were detected, thereby excluding their involvement in the reduced cefiderocol susceptibility of these strains. All isolates harboured the extended-spectrum β-lactamase PER-7. PER-7 shows hydrolytic activity against broad-spectrum cephalosporins, aztreonam, and, to a limited extent, carbapenems.^4^ Previous studies by Poirel et al. reported cefiderocol-resistant A. baumannii isolates producing OXA-23 and PER-7, implicating a role for PER-7 in cefiderocol resistance.^5^ Heterologous expression in Escherichia coli TOP10 and Pseudomonas aeruginosa PAO1 raised cefiderocol MICs from ≤0.125 to 4 mg/L in E. coli, and from 0.5 to 8 mg/L in P. aeruginosa.^6^ However, blaPER-7 was not cloned into A. baumannii in the respective study, and the exact influence on cefiderocol resistance in this species remains not fully known.

Consequently, the aim of this study was to investigate the effect of PER-7 production on cefiderocol susceptibility in A. baumannii. Therefore, the blaPER-7 gene was amplified from A. baumannii NRZ-88408 and ligated into the expression plasmid pVRL-1 (BCCM, https://bccm.belspo.be) after XhoI and PstI digestion. The recombinant plasmid was transformed into E. coli DH5α, A. baumannii ATCC 17978, and four randomly selected, non-duplicate clinical A. baumannii isolates (NRZ-89738, NRZ-90235, NRZ-90408, NRZ-92586) producing OXA-23 or OXA-72, all with cefiderocol inhibition zone diameters ≥17 mm. Cefiderocol susceptibility was tested in triplicate by manual broth microdilution in iron-depleted, cation-adjusted Mueller-Hinton broth (concentration range: 0.03–32 mg/L).^7^

Heterologous expression of PER-7 led to increased MICs for cephalosporins and aztreonam in all strains, and a significant rise in carbapenem MICs in A. baumannii ATCC 17978, where MICs of imipenem and meropenem increased from 0.125 to 4 and 0.5 mg/L, respectively (Table 1). Similarly, the MIC for ertapenem increased from 1 to 4 mg/L. Despite these increases, MICs for meropenem and imipenem remained within the susceptible or susceptible, increased exposure range, indicating that blaPER-7 alone does not confer full resistance. In contrast, E. coli DH5α showed no change in carbapenem MICs. In the four clinical A. baumannii isolates, carbapenem resistance was already conferred by oxacillinases OXA-23 or OXA-72.^8^ PER-7 expression led to an increase in cefiderocol MICs in E. coli and all tested A. baumannii isolates (Table 1). MIC values rose to > 32 mg/L in A. baumannii, surpassing clinically relevant PK/PD thresholds (>2 mg/L) as defined by EUCAST.^3^ The increases in cefiderocol MICs observed in our in vitro models were consistent with resistance levels detected in the clinical PER-7 isolates. MICs for ceftazidime-avibactam and aztreonam-avibactam suggested that PER-7 is not fully inhibited by avibactam (Table 1). In E. coli DH5α, production of PER-7 led to a 1024-fold MIC increase for aztreonam (up to >64 mg/L), while MICs for aztreonam-avibactam increased only 62-fold, to 2 mg/L, indicating an incomplete inhibition. This is in line with the findings of Poirel et al., who showed that E. coli TOP10 with and without production of PER-7 showed aztreonam and aztreonam-avibactam MICs of >256 and 16 mg/L, respectively, which also indicates an incomplete inhibition. In A. baumannii, no inhibitory effect of avibactam was observed; however, this was expected, as both ceftazidime-avibactam and aztreonam-avibactam are no adequate drugs against A. baumannii due to intrinsic resistance mechanisms, such as the hydrolysis of ceftazidime and aztreonam by the chromosomally encoded ADC β-lactamase, and the inability of avibactam to cross the Acinetobacter outer membrane.^9^ This likely masked the effect of PER-7 inhibition. As cefiderocol was not tested in combination with avibactam in our study, any potential synergistic effects could not be evaluated.

In conclusion, our study confirms that the presence of PER-7 contributes to cefiderocol resistance in A. baumannii. The frequent localization of blaPER-7 on plasmids facilitates its rapid dissemination and transfer to other bacteria.^10^ These findings highlight the limited therapeutic options for infections caused by cefiderocol-resistant A. baumannii. To date, there is insufficient evidence to suggest that cefiderocol is an effective treatment option for such isolates.