A-094 A Post-COVID Epidemiological Shift: The Rise of NDM-KPC Co-Producing Enterobacterales in Brazil

Abstract Background The rapid emergence of carbapenem-resistant Enterobacterales (CRE) co-producing multiple carbapenemases represents a critical escalation in antimicrobial resistance worldwide, with increasing reports across South America. These organisms limit therapeutic options and challenge infection control strategies. This study aimed to characterize the epidemiology and distribution of carbapenem-resistant and co-carbapenemase-producing Enterobacterales isolates recovered from patients in 86 Brazilian healthcare centers comparing three epidemiological periods: pre-COVID-19 (2018-2019), COVID-19 (2020-2021-2022) and post-COVID-19 (2023-2024-2025). Methods An 8-year retrospective analysis used laboratory data from CRE isolates identified between January 2018 and October 2025 across 86 Brazilian healthcare centers. The data were obtained from the clinical microbiology section of a medical diagnostic laboratory. Double-carbapenemase production was screened phenotypically in isolates resistant to ertapenem and/or meropenem using a meropenem combined disk test with EDTA, phenylboronic acid (PBA), cloxacillin, and EDTA plus PBA. A double-carbapenemase phenotype was defined as an inhibition zone increase =5 mm with combined versus single inhibitors. Bacterial identification was performed by mass spectrometry, and antimicrobial MICs were determined using automated system, except for polymyxin B, which was tested by broth microdilution method, according to BrCAST guidelines. Carbapenemase genes were detected by PCR and the genetic relatedness of the strains was characterized by Pulsed-Field Gel Electrophoresis. Results A total of 40,146 CRE isolates were analyzed over 8-year study period. The frequency of isolates detected during the period was: 10.56% (4,242/40,146) in pre- COVID-19, 35% (14,071/40,146) in COVID-19 and 54.3% (21,833/40,146) in the post-COVID-19 period. In the same period, 733 CRE isolates harborig co-production of NDM and KPC were detected, including one (0.13%) isolate in pre-COVID-19, 23 (3.13%) isolates in COVID-19 and 709 (96.72%) isolates in the post-COVID-19 period. The prevalence of microorganisms detected was as follow: 95% Klebsiella spp., 2.32% Proteus spp., 1.22% Citrobacter spp., 0.81% Enterobacter spp., 0.41% Escherichia coli, 0.13% Providencia stuartii and Raoultella planticola, each. These microorganisms were isolated from urine (n=261), blood (n=122), surveillance swabs (n=230), catheter tips (n=19), skin and soft tissue samples (n=25), respiratory tract (n=50), bone (n=8) and others (n=18). In all of them, high-level resistance was observed to amoxicillin-clavulanic acid, ceftazidime, ceftriaxone, cefepime, ertapenem, meropenem, aztreonam and ceftazidime-avibactam. Among K. pneumoniae, 28%, 85% and 92% of isolates were resistant to polymyxin B, amikacin, and gentamicin, respectively. The PFGE analysis showed 21 clusters (pattern A to U) among the 70 KPN strains isolated from 15 hospitals. The clusters A and C were found in seventeen and fifteen KPN strains, respectively from the same hospital. Conclusion The marked increase in Enterobacterales co-producing carbapenemases observed during the study period, particularly in the post-COVID-19 era, suggests an important shift in the epidemiology of carbapenem resistance in Brazilian healthcare settings. The predominance of Klebsiella spp. and the identification of clonal clusters across multiple institutions indicate ongoing dissemination and potential establishment of these organisms within hospital networks. These findings highlight the urgent need for enhanced surveillance strategies and improved diagnostic approaches to accurately identify and contain the spread of these highly resistant organisms.