To establish and evaluate a simple disk stacking plus micro-elution (DSE) method that can be routinely performed to rapidly detect the synergistic effect between aztreonam (ATM) and ceftazidime/avibactam (CZA) against metallo-β-lactamase (MBL)producing carbapenem-resistant Enterobacterales (CRE). Methods: The DSE method was established, and a total of 32 MBL-producing CRE isolates collected from multiple centers were tested for ATM-CZA synergy. The results obtained after 8 h of incubation were compared with those obtained by a reference checkerboard assay (CBA) after 18~24 h. Reproducibility experiments were completed on three separate days. Results: The reproducibility study showed that the results of the DSE method were precise. Compared with CBA, the DSE method exhibited excellent performance, with 92.8% sensitivity, 100.0% specificity 93.8% categorical agreement, 0.0% very major error, 0.0% major error, and 6.2% minor error over three days of testing. Conclusion:The DSE method is a simple, rapid and practical method for ATM-CZA combination testing. Further evaluation should be completed to improve its clinical application.

Purpose Carbapenem-resistant Enterobacteriaceae (CRE) infection has become a concerning threat, especially in hospital settings; however, its phenotypic characterization, association with rectal colonization and subsequent bloodstream infections (BSI) remain to be clarified. This study aimed to investigate the incidence and risk factors of CRE infection in rectal CRE carriers and to understand the clonality of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and their association with subsequent BSI in these patients. Patients and Methods This was a prospectively designed cohort study. Hospitalized patients treated at our institution from April 2019 to October 2020 with intestinal CRE carriage were screened at admission and weekly thereafter until death or discharge from the hospital. Stool and blood samples were obtained for strain growth and mass spectrometry. The colonization and clinical infection isolates were analyzed by antimicrobial susceptibility testing to identify CRE. The clonality of the CRE strains and their corresponding clinical infection strains was studied by whole-genome sequencing to explore the mechanism of drug resistance and evaluate possible transmission. CRE-associated risk factors were analyzed in combination with epidemiological data. Results Of the 1203 patients, 85 were colonized by CRE and 21 developed CRE infection, of whom 13 developed CRE bloodstream infection (BSI). Ninety-one CRE strains were isolated from the rectal specimens of the 85 patients. Tracheotomy and chemotherapy in the past three months were independent risk factors for CRE infection in intestinal CRE carriers. ST11-KL64 (92.3%, 24/26) was the most dominant capsule and multilocus sequence typing (MLST) type among clonal CRKP isolates. Single-nucleotide polymorphism clustering showed homology of representative colonization and infection CRKP strain pairs (n=13) in the same patient. One group of leading clones was endemic in surgical intensive care units (ICUs). Twenty-four CRKP strains carried β-lactamase K. pneumonia carbapenemase 2, and 73.1% (19 strains) of CRKP carried mucoid phenotype regulator genes A2 and iucABCD. Conclusion In summary, intestinal CRE colonization was detectable at an elevated rate among hospitalized patients and prevalent in ICU patients, with potential rapid horizontal transmission, providing evidence that CRE BSI infection in hospitalized patients might be due to their colonized strains and indicates the correlation between intestinal colonization and BSI.

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