Pseudomonas aeruginosa (P. aeruginosa) is a nosocomial pathogen frequently encountered in healthcare-associated infections (HAIs) and difficult to treat due to its intrinsic or acquired resistance properties [1]. WHO (World Health Organization) has included carbapenem-resistant P. aeruginosa (CR-Pa) in the list of “high” priority pathogens that requires research and the development of new antibiotics [2]. According to the European Antimicrobial Resistance Surveillance Network (EARS-Net) 2024 report, the carbapenem resistance rate was reported as 18.6 % in invasive P. aeruginosa isolates [3]. In the Central Asia and Europe Antimicrobial Resistance Surveillance (CAESAR) program 2023 report, which includes 2021 data for Türkiye, it was reported that the CR-Pa rate in Türkiye was 39 % [4].

Although the most important and frequent cause of carbapenem resistance in P. aeruginosa is chromosomal mutations, the acquisition of mobile carbapenemase-encoding genes is extremely important for the spread of resistance [5]. Since the 1990s, reports of carbapenemase production among CR-Pa isolates have been increasingly published and carbapenemase production has become an important resistance mechanism [[6], [7], [8], [9]]. Carbapenemase genes cause stable and transferable resistance, which occurs clonally or through plasmid transfer of genes to susceptible bacteria. Therefore, it is very important to detect carbapenemase-producing isolates because these isolates spread more easily than non-carbapenemase producers and require more intensive infection control measures [10]. Although the majority of carbapenemases detected in P. aeruginosa isolates are metallo-β-lactamases (MBL), other groups of carbapenemases have been reported in recent years [11,12]. In studies conducted in our country, mostly VIM and GES type enzymes were reported [13,14]. There are also studies showing an increase in different carbapenemase genes such as blaNDM, blaKPC, blaIMP and blaOXA-48 [[15], [16], [17], [18]].

Molecular techniques that detect the presence of carbapenemase genes are the gold standard for demonstrating the presence of carbapenemases. They are particularly advantageous because they are rapid and can be performed directly from screening samples. However, these techniques have several disadvantages, such as requiring considerable expertise and the need for expensive instruments, in addition to the risk of missing unknown or emerging resistance genes. Various phenotypic methods are also available for the detection of carbapenemase producers, such as the carbapenem inactivation method (CIM) and its modifications, the CarbaNP test (CNPt) and its modifications. These tests are used in routine laboratories and are very useful in making therapeutic decisions in the clinic and preventing the spread of carbapenemase-producing organisms [[19], [20], [21], [22], [23]].

CNPt is a biochemical test for the rapid detection (<2 hours) of carbapenemase production in Gram-negative bacilli. The presence of carbapenemase is detected by the colour change of the indicator phenol red to the pH change caused by hydrolysis of imipenem in bacterial lysate prepared using Tris-HCl lysis buffer [24]. CNPt has been reported to be highly sensitive for the detection of Klebsiella pneumoniae carbapenemase (KPC) and MBL (NDM, IMP, VIM) producers. However, test sensitivity decreases for the detection of OXA-48-like carbapenemase producers. A major disadvantage of CNPt, especially for low-income countries, is the need for a commercial extraction buffer (B-PER II) used to obtain bacterial extracts. Pasteran et al. [25] proposed CarbaNP-direct test (CNPdt), which is simpler and more cost-effective to perform by using the bacterial colony directly, and in comparison studies, the test sensitivity was shown to be higher than that of CNPt. Although the performance of CNPdt, like CNPt, is lower in nonfermenters than in Enterobacterales, CNPdt sensitivity was reported to be 94.7 % and specificity 90.6 % in P. aeruginosa isolates [5].

New generation antibiotics, which are used as the last line of treatment, have varying efficacy depending on the carbapenemase type. Ceftazidime-avibactam (CZA) is highly effective against class A, class C and some class D carbapenemases, but ineffective against class B carbapenemases (MBL). The variable spectrum of activity against carbapenemase types has led to the necessity of carbapenemase detection as well as detection of carbapenemase type in order to ensure rapid initiation of the correct treatment and to prevent unnecessary drug use [[26], [27], [28], [29]]. Rapid and correct detection of MBL-producing P. aeruginosa isolates is critical for both infection control measures and determination of appropriate antibiotic therapy. Various phenotypic methods have been developed for MBL detection. Researchers have proposed an additional test to differentiate MBL isolates by adding ethylenediaminetetraacetic acid (EDTA) to tryptic soy brot after modified CIM, eCIM [30]. The test has a sensitivity of 76.8 % and specificity of 96.8 % and is widely used in laboratories for MBL detection. The major disadvantage is that it gives results in 18-24 hours and this test can only be interpreted in mCIM positivity. Another method is a test protocol in which a third tube containing EDTA is added to the CNPt with a sensitivity of 92.9 % and a specificity of 90.6 % [5]. In a study investigating the role of EDTA modification in CNPdt, it was shown that MBLs can be detected with 100 % specificity by modifying CNPdt with EDTA [31].

This study aimed to evaluate the detection of MBL-producing CR-Pa isolates by modifying CNPdt with EDTA and thereby predict CZA-resistant CR-Pa isolates on the same day of identification without waiting for AST results.