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. 2025 Mar 20:16:1542029.
doi: 10.3389/fmicb.2025.1542029. eCollection 2025.

Study on the invitro synergistic susceptibility and biofilm inhibition mechanism of ceftazidime-avibactam combined with aztreonam against carbapenem-resistant Klebsiella pneumoniae

Guangfen Wang #  1 , Hui Zhang #  2 , Qiaoping Wu  3 , Jianqiang Xu  3 , Xuedan Qiu  3 , Jinyuan Chen  3 , Fujie Cui  3 , Jian Zhou  4 , Qingcao Li  3
Affiliations

Study on the invitro synergistic susceptibility and biofilm inhibition mechanism of ceftazidime-avibactam combined with aztreonam against carbapenem-resistant Klebsiella pneumoniae

Guangfen Wang et al. Front Microbiol. .

Abstract

Objective: This study aims to investigate the synergistic effects and biofilm inhibition mechanisms of ceftazidime-avibactam (CZA) combined with aztreonam (ATM) against carbapenem-resistant Klebsiella pneumonia (CRKP) commonly found in the local clinical setting, providing new insights for clinical anti-infective strategies.

Methods: We selected a total of 150 non-duplicate clinical isolates of CRKP from multiple hospitals in Ningbo. Common carbapenemase genes were detected using PCR. Broth microdilution and time-kill assays were used to evaluate the in vitro synergistic effects of CZA and ATM, both individually and in combination, on CRKP isolates with different enzyme types, and the fractional inhibitory concentration index (FICI) was calculated. The crystal violet staining method and bacterial cell permeability assay were employed to assess the impact of CZA, ATM, and their combination on the cell structure and biofilm formation capacity of CRKP. Real-time quantitative PCR (qRT-PCR) was used to measure the expression levels of biofilm-related genes (Luxs, mrkA, wbbM, pgaA, and wzm) in CRKP under treatment with CZA, ATM, or their combination.

Results: The comparison of synergistic indices for different enzyme-type CRKP strains with CZA and ATM combination therapy showed a statistically significant difference (p < 0.01). The time-kill assay indicated that the time-kill curves for strains carrying blaKPC-2 and blaNDM-1 resistance genes were similar between the monotherapy and combination therapy groups, while the CZA + ATM combination therapy group showed a significant decrease in bacterial concentration after 4-8 h of cultivation compared to the CZA and ATM monotherapy groups. The crystal violet staining and bacterial cell permeability assays demonstrated that the CZA + ATM combination significantly reduced biofilm formation and increased cellular structure disruption in CRKP. The qRT-PCR results showed that CZA combined with ATM notably decreased the expression levels of biofilm-related genes Luxs, mrkA, wbbM, pgaA, and wzm in CRKP.

Conclusion: The combination of ATM and CZA shows a strong synergistic antibacterial effect against CRKP strains with various enzyme types, with particularly notable synergy in strains carrying the blaKPC-2 resistance gene. Additionally, this combination significantly disrupts the cellular structure of CRKP and inhibits biofilm formation.

Keywords: CRKP; aztreonam; carbapenemase; ceftazidime-avibactam; combined drug sensitivity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cumulative rate curves of CZA and ATM monotherapy and combination therapy. After the combination of CZA and ATM, the sensitivity rate significantly increased.
Figure 2
Figure 2
(A) Time sterilization curves of CRKP carrying blaNDM resistance gene by different concentrations of ATM. (B) Time sterilization curves of CRKP carrying blaNDM resistance gene with different concentrations of CZA. (C) Time sterilization curves of CRKP carrying blaKPC resistance gene by different concentrations of ATM. (D) Time sterilization curves of CRKP carrying blaKPC resistance gene with different concentrations of CZA.
Figure 3
Figure 3
(A) Time sterilization curves of ATM, CZA monotherapy, and combination on CRKP carrying blaNDM resistance gene. (B) Time sterilization curves of ATM, CZA monotherapy, and combination on CRKP carrying blaKPC resistance gene.
Figure 4
Figure 4
The absorbance of different enzyme types of CRKP biofilms stained with crystal violet after ATM, CZA monotherapy, and combination treatment. The combined use of ATM and CZA inhibits the formation of biofilms carrying blaNDM and blaKPC resistance genes in CRKP. *p < 0.01, determined by the student’s t-test.
Figure 5
Figure 5
Combination of CZA and ATM contributes to the damage of bacterial cell membranes and the leakage of bacterial ALPs. The absorbance at 510 nm integrity by detecting ALP. The chart indicates that the combination of two drugs acts on CRKP carrying blaNDM and blaKPC resistance genes, an increase for alkaline phosphatase in the medium after exposure to the specified compound. *p < 0.01, determined by the student’s t-test.
Figure 6
Figure 6
(A) The expression levels of biofilm formation related genes in CRKP carrying blaKPC resistance gene by ATM, CZA monotherapy, and combination. (B) The expression levels of biofilm formation related genes in CRKP carrying blaNDM resistance gene by ATM, CZA monotherapy, and combination, *p < 0.01, determined by the student’s t-test.
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