Antimicrobial treatment of patients with bloodstream infections (BSI) is time-sensitive. In an era of increasing antimicrobial resistance, rapid detection and identification of bacteria with antimicrobial susceptibility are critical for targeted therapy early in the disease course. This study describes the performance of a rapid method for identifying and testing antimicrobial susceptibility of Gram-negative bacteria performed directly from blood culture bottles in a routine microbiology laboratory. A total of 284, 120, and 24 samples were analyzed by rapid identification (Rid), rapid susceptibility testing (RAST), and rapid broth microdilution for polymyxin B (rMIC), respectively, and compared with standard methods. Our protocol was able to identify 93% of isolates at the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We obtained 100% agreement for RAST compared to the standard method and 96% agreement for rMIC. Our protocol has proven to be an excellent tool for rapid identification of Gram-negative bacilli causing BSIs. It can also be used in microbiology laboratory routine along with RAST and faster polymyxin microdilution, especially for carbapenemase-producing bacteria, allowing for rapid, simple, accurate, and cost-effective diagnosis.

Carbapenem-Resistant Pseudomonas Aeruginosa (CRPA) is considered as one of the high priority pathogens by the World Health Organization. As CRPA carbapenemase producers have increased worldwide, the aim of this study was to evaluate the carbapenemase prevalence in CRPA at a tertiary care hospital in Brazil". All 395 CRPA identified in the period of September 2021 to May 2024 were evaluated by multiplex real-time polymerase chain reaction (qPCR-HRM) for the following carbapenemase genes: bla_KPC, bla_NDM, bla_OXA-48-like, bla_IMP, bla_VIM,bla_SPM and bla_GES. In the first period analyzed (September to December 2021), almost 70 % of the isolates were negative for the 7 tested genes, and the bla_NDM was found in 27.3 % of the CRPA. In the following semesters there was an increase of bla_NDM as follows: January to June of 2022 = 29.8 %; July to December of 2022 = 43.8 %; January to June of 2023 = 42.4 %; July to December 2023 = 58.9 % and January to May of 2024 = 59.5 % of bla_NDM. The prevalence of the other carbapenemases remained low. These results indicated an important increase of the bla_NDM gene, overcoming the CRPA non-carbapenemase producers in our institution.

Introduction: Infections caused by multidrug-resistant microorganisms have become increasingly common in hospital environments around the world. Gram-negative bacilli stands out among multidrug-resistant bacteria mostly due to the production of carbapenemase enzymes which lead to resistance to most β-lactam antibiotics including the carbapenems. As a consequence, polymyxins have been reintroduced in the clinic as a last resort to treat infections caused by Gram-negative bacilli resistant to carbapenems. However, the only reliable method to evaluate the susceptibility to polymyxins is the broth microdilution, a laborious and time-consuming technique. Among infections caused by multidrug-resistant bacteria, bloodstream infections are the most worrisome as they can lead to sepsis and septic shock with high mortality rates.Objective: Considering the severity of sepsis and the need for a treatment guided for the susceptibility test in vitro, this work aimed to evaluate a rapid method of polymyxins susceptibility either from colonies grown on agar or directly from positive blood culture bottles using the technology of MALDI-TOF.Methods: The method was based on the “direct on target microdroplets growth assay” (DOT-MGA) originally developed by Idelevich and collaborators with some modifications (Adapted DOT-MGA). Isolates of Enterobacterales and non-fermenting Gram-negative bacilli resistant to carbapenems were obtained from patients attending a tertiary care hospital in southern Brazil and tested as follows: 122 isolates from colonies grown on agar plates and 117 isolates directly from spiked positive blood cultures.Results: The adapted DOT-MGA presented 95 and 100% of categorical agreement considering the colonies grown on agar plates and directly from positive blood cultures, respectively.Discussion: The adapted DOT-MGA test proved to be a reliable technique to evaluate the susceptibility to polymyxins to be used in microbiology laboratories with the MALDI-TOF equipment.

Infections due to carbapenemase-producing Enterobacterales harboring more than one carbapenemase-encoding gene spreads mainly by plasmid and transposon mobilization. Analyze the mobile genetic elements carrying bla_KPC and bla_NDM of K. pneumoniae carbapenemase co-producers (KpKN). K. pneumoniae isolates with reduced susceptibility to carbapenems were obtained from 2016 to 2023. To evaluate the genetic environment of KpKN, 22 isolates were selected for antimicrobial susceptibility testing and whole-genome sequencing (WGS). The bla_KPC-2 gene was carried mainly by IncN/IncFIB, a novel co-integrated plasmid in the Tn4401b transposon. The bla_NDM-1 was disseminated in the only two KpKN isolates recovered before 2020 by IncHI1B/IncFIB plasmid type, in the Tn3000 transposon. Noteworthy, isolates obtained since 2020 showed the bla_NDM-1 gene carried by IncA/C in an IS26-flanked pseudo-composite transposon containing ISCR1, which also had genes that confer resistance to sulfonamides, aminoglycosides, macrolides, quinolones, amphenicols, tetracyclines, rifampicin, sulfonamide, and trimethoprim. The isolates belong mainly to ST11 and ST16. The plasmid and transposon changes during the different periods could be related to higher dissemination of the bla_NDM-1 and the large number of resistance genes present in the IS26-flanked transposon may have increased the co-selection of this plasmid through the wide use of antimicrobials during the pandemic.