Carbapenems are considered a last-line class of antibiotics used for the treatment of infections caused by multidrug-resistant gram-negative bacteria. Owing to the lack of effective and safe alternative treatment options, carbapenem-resistant (CR) gram-negative bacteria, including CR-Enterobacterales (CRE) and CR-Pseudomonas aeruginosa (CRPA) cause a wide range of infections in hospitals of all sizes, leading to significant morbidity and mortality (Reyes et al., 2023; Ding et al., 2024). Some of the novel antimicrobial agents have not been available for clinical use in China, adding further complexity to the issue (Tamma et al., 2022; Zeng et al., 2023). The World Health Organization has categorized these pathogens as “Critical” priority, emphasizing the urgent need for novel antibiotics (Paul et al., 2022). Moreover, genetic and phenotypic differences in CR gram-negative bacteria, which vary based on region and population, have not yet been elucidated (Jiang et al., 2023). It is reported that the most prevalent clone of carbapenem-resistant K. pneumoniae (CRKP) circulating in China is ST11, while for CR-E. coli (CREC) and CRPA, the prevalent clones were ST410 and ST463 respectively (Zhang et al., 2023; Ba et al., 2024; Hu et al., 2024). Carbapenemase production, a key resistance mechanism among these pathogens, causes severe and often deadly infections with a higher 30-day mortality (Wei et al., 2022; Reyes et al., 2023). These carbapenemases can be divided into three Ambler classes: class A (e.g., Klebsiella pneumoniae carbapenemase, KPC), class B (metallo β–lactamases, MBLs), and class D (OXA-48-like carbapenemase). Rapid detection and differentiation of these carbapenemases is critical for the initiation of effective therapy; the identification and classification of carbapenemases have significant therapeutic, epidemiological, and infection-control implications. The drug combination ceftazidime/avibactam can be given to patients infected with class A and some class D carbapenemase-producing bacteria, but not to those infected with bacteria producing class B carbapenemases (Zeng et al., 2023). Furthermore, although bacteria producing OXA-48-like carbapenemases can be tested as susceptible to carbapenems, they are often associated with carbapenem treatment failure (Boyd et al., 2022).

In clinical laboratories, the detection of carbapenemases, particularly of CRPA, is challenging. Several of the existing phenotypic methods for screening carbapenemases, such as the Modified Hodge test, have low sensitivity for New Delhi metallo-beta lactamase (NDM) producers and do not distinguish between carbapenemase types (Zhou et al., 2018). Since 2017, the Clinical & Laboratory Standards Institute (CLSI) has recommended the modified carbapenem inactivation method (mCIM) for detecting carbapenemases in CRE; the CLSI expanded the scope of mCIM to CRPA in 2018 (CLSI, 2017; CLSI, 2018). In the new version of the CLSI guideline launched in 2023, carbapenemase phenotype testing was further emphasized (CLSI, 2023). Since 2018, EDTA-CIM (eCIM) has been recommended for identifying MBLs in CRE (CLSI, 2018), although it is not recommended for distinguishing carbapenemase in CR Pseudomonas isolates. Furthermore, the mCIM and eCIM methods require a broth incubation process that can be cumbersome (CLSI, 2023).

Commercial combined-disc tests (CDTs), which were among the first tests used in clinical laboratories to detect carbapenemases in CRE, utilize chemical compounds to specifically inhibit carbapenemases from different Ambler classes. Phenylboronic acid (PBA) inhibits class A carbapenemases, and EDTA inhibits class B carbapenemases (Haider et al., 2022). A similar method has been reported for discriminating between KPC and MBLs in Pseudomonas, and the cloxacillin test was introduced to help exclude over-expressing isolates; nonetheless, these methods utilize different inhibitors and interpretive criteria (Lopez-Hernandez et al., 2020).

Most phenotypic methods for carbapenemase screening use polymerase chain reaction (PCR) results as the reference (Gill et al., 2020). However, as PCR traditionally targets specific genes, it may generate false negatives if specific carbapenemase genes are not targeted, especially when novel variant genes emerge (Voulgari et al., 2020). Whole genome sequencing (WGS) method provides more comprehensive results when it is caused either a novel carbapenemase or by an AmpC enzymes combined with reduced permeability due to the alteration or down-regulation of porins (Di Pilato et al., 2022).

To investigate the genomic population structure of these pathogens in southern region of China and to improve carbapenemase screening, we determined the molecular characteristics and evaluated the performance of the mCIM and eCIM combination methods, as well as the commercial CDT method, to detect and distinguish carbapenemases among gram-negative bacteria collected in two tertiary hospitals in southern China, using WGS.

Monitoring the molecular epidemiology of carbapenem-non-susceptible isolates is key for controlling the spread these pathogens and detecting and distinguishing carbapenemases is crucial for clinicians when selecting appropriate antibiotic treatment (Tamma et al., 2022). To investigate the genomic population structure of carbapenem-non-susceptible isolates and to improve carbapenemase screening, we determined the molecular characteristics of these pathogens and evaluated the performance of the combined mCIM–eCIM method and commercial CDT method against the WGS.

We detected carbapenemases in 76.3% of the Enterobacterales isolates; this frequency is approximately the same as that of another epidemiology study of carbapenem non-susceptible Enterobacterales conducted from 2017 to 2019 in German (von Laer et al., 2022). However, the blaNDM carbapenemase gene, detected here among the K. pneumoniae isolates, is reported to occur much more frequently in strains from pediatric patients than in those from adult patients (Lee et al., 2022). Most of the CR-Pseudomonas isolates identified here were not carbapenemase-producing, unlike those identified in pediatric patients from other parts of China, South America, and Central America (Patil et al., 2023; Reyes et al., 2023). The carbapenem-resistance pattern observed here was similar to that observed in the USA (Reyes et al., 2023).

The genomic population structure of carbapenem-non-susceptible isolates varied based on region and population. In this study, the most prevalent carbapenem non-susceptible K. pneumoniae clones were st307 and st11. and the latter of which were all blaKPC-2 producing strains from adult patients at the First People’s Hospital of Zhaoqing. As depicted in literatures, the st11-blaKPC-2 clone is the predominant clones circulating among adult patients in China (Hu et al., 2020). Meanwhile, our findings reveal that the st307 clone was exclusively detected among pediatric patients, it may emerge as a major CRKP clones among pediatric patients in southern China, as reported in another children’s center in Shenzhen (Patil et al., 2021). However, although their STs was identical, they differed in their carbapenemase patterns, specifically, featuring metallo-β-lactamases in this study. Among P. aeruginosa in this study, the top three STs were st242, st244 and st385, which differed from most carbapenemase producing isolates among adult patients in other regions of China (Li et al., 2023). So, in this study, the molecular epidemiology of carbapenem non-susceptible isolates exhibited distinctive characteristics that are significant for understanding their prevalence and transmission.

The mCIM-eCIM combination test reportedly performs well against carbapenemase-producing Pseudomonas isolates, but not against imipenemase- and Sao Paulo metallo-β-lactamase-producing strains (Gill et al., 2020). Thus, in this study, we evaluated the performance of mCIM–eCIM combination test mainly using 29 non-carbapenemase-producing CR Pseudomonas clinical strains from southern China. The mCIM–eCIM combination exhibited excellent and reliable carbapenemase detection among the Enterobacterales and Pseudomonas isolates, with high sensitivity and specificity. Nonetheless, we obtained indeterminate results for some of the non-carbapenemase-producing strains, including CRE and CRPA isolates. The eCIM test’s classification ability may be hampered when class A and class B carbapenemases are coproduced because strains that achieve this coproduction effectively hydrolyze the substrates regardless of whether EDTA is added (Hao et al., 2022).

In China, the commercial CDT test has been widely used for Enterobacterales, while its use for Pseudomonas isolates has only recently been reported in a few studies (Zhang et al., 2022b). Here, the CDT accurately detected and distinguished carbapenemases in Enterobacterales. By using PBA and EDTA separately and simultaneously, the CDT test was also able to detect and classify carbapenemase coproduction in Enterobacterales. However, the CDT produced a false positive when used to test CRPA with Pseudomonas derived cephalosporinase, a type of chromosomal cephalosporinase ampC β-lactamase hyperproduction. The robustness of the CDT was substantially improved by including a cloxacillin test to exclude the influence of ampC β-lactamase hyperproduction when the CDT result was positive. The lower value of 95% CI of sensitivity of the CDT could have been higher had more carbapenemase-producing strains been included in this study, however the performance of the CDT with cloxacillin testing was no better than that of the mCIM–eCIM combination test among Pseudomonas isolates. We observed a strain of Enterobacter cloacae with blaCMH, a type of ampC β-lactamase produced false positive result with CDT test either. When we use CDT to detect the carbapenemase, if the strain often produces Class C β–lactamases, the cloxacillin test could be routinely carried out even among Enterobacterales isolates.

WGS can detect all kinds of carbapenemase genes, known or unknown, as with the co-occurrence of blaVIM-2, blaVIM-46 and blaIMP-8 in the Pseudomonas putida strain that we observed. In addition, another study found that some blaKPC-2 variants could not be detected using PCR (Ding et al., 2021). Co-occurring strains like this could contribute to the hidden dissemination of bacteria. Consequently, WGS, which more comprehensively identifies carbapenemase genes among CR gram-negative bacteria, may provide a better gold standard.

The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China displayed distinct characteristics. Both the mCIM–eCIM combination and CDT effectively detected and differentiated carbapenemases among the Enterobacterales isolates, whereas the former performed better than CDT among the Pseudomonas isolates. This novel attempt may thus improve the rapid and accurate detection and identification of carbapenemases, improving both therapy and infection control.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

BL: Funding acquisition, Writing – original draft. YC: Methodology, Writing – original draft. ZL: Methodology, Writing – original draft. XL: Methodology, Writing – original draft. HC: Methodology, Writing – original draft. HL: Methodology, Writing – original draft. HZ: Data curation, Writing – original draft. YX: Formal Analysis, Writing – original draft. LH: Funding acquisition, Writing – original draft. FG: Writing – original draft, Software. YL: Conceptualization, Writing – review & editing.