A Simple Disk Stacking Plus Micro-Elution Method for Rapid Detection of the Synergistic Effect of Aztreonam and Ceftazidime/Avibactam Against Metallo-ß-Lactamase Producing Enterobacterales.

Purpose: 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-ß-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.

Bloodstream Infections in Patients with Rectal Colonization by Carbapenem-Resistant Enterobacteriaceae: A Prospective Cohort Study.

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 ß-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.

Co-occurrence of bla NDM-1 and mcr-9 in a Conjugative IncHI2/HI2A Plasmid From a Bloodstream Infection-Causing Carbapenem-Resistant Klebsiella pneumoniae.

Spread of the carbapenemase-encoding and mobilized colistin resistance (mcr) genes among Enterobacteriales poses a great threat to global public health, especially when the both genes are transferred by a single plasmid. Here, we identified a bla NDM-1- and mcr-9-co-encoding plasmid harbored by a clinical isolate of Klebsiella pneumoniae (KPN710429). KPN710429 was recovered from a blood sample from an inpatient in a tertiary hospital in China, and antimicrobial susceptibility testing showed that it was multidrug-resistant and only susceptible to aztreonam, colistin, and tigecycline. KPN710429 belongs to sequence type (ST) 1308 and capsular serotype KL144. The string test of KPN710429 was negative, and this strain didn't exhibit a hypervirulent phenotype according to serum-killing and Galleria mellonella lethality assessments. Whole-genome sequencing revealed the KPN710429 genome comprises a single chromosome and three plasmids. All virulence associated genes were harbored by chromosome. Most of its antimicrobial resistance genes, including bla NDM-1 and mcr-9 were carried by plasmid pK701429_2, belonging to the incompatibility (Inc) HI2/HI2A group and ST1. Comparative genomics assays indicates that pK710429_2 could be a hybrid plasmid, formed by a Tn6696-like bla NDM-1 region inserting into a mcr-9-positive-IncHI2/HI2A plasmid. pK710429_2 contained the conjugative transfer gene regions, Tra1 and Tra2, with some structural variations, and conjugation assays revealed that pK710429_2 was transferable. Although pK710429_2 lacked the qseB-qseC regulatory genes, mcr-9 expression was upregulated after pretreatment with colistin for 6 h, leading to colistin resistance in KPN710429. To our knowledge, this is the first report of a bla NDM-1- and mcr-9-co-encoding transferable plasmid harbored by a bloodstream-infection-causing K. pneumoniae strain in China. Effective surveillance should be implemented to assess the prevalence of the plasmid co-harboring carbapenemase-encoding gene and mcr-9.