The emergence and rapid dissemination of colistin-resistant carrying the plasmid-mediated gene have created an urgent need to develop specific screening methods. In this study, we evaluated four assays based on the inhibition of MCR-1 activity by EDTA: (i) a combined-disk test (CDT) comparing the inhibition zones of colistin and colistin (10 μg) plus EDTA (100 mM); (ii) reduction of colistin MIC (CMR) in the presence of EDTA (80 μg/ml); (iii) a modified rapid polymyxin Nordmann/Poirel test (MPNP); and (iv) alteration of zeta potential (R = ZP/ZP). We obtained encouraging results for the detection of MCR-1 in isolates recovered from human, food, and animal samples, using the following assay parameters: ≥3 mm difference in the inhibition zones between colistin disks without and with EDTA; ≥4-fold colistin MIC decrease in the presence of EDTA; R of ≥2.5; and the absence of metabolic activity and proliferation, indicated by unchanged color of phenol red in the presence of colistin-EDTA, in the MPNP test. In this regard, the CDT, CMR, R, and MPNP assays exhibited sensitivities of 96.7, 96.7, 95.1, and 96.7% and specificities of 89.6, 83.3, 100, and 100%, respectively, for detecting MCR-1-positive Our results demonstrate that inhibition by EDTA and zeta potential assays may provide simple and inexpensive methods for the presumptive detection of MCR-1-producing isolates in human and veterinary diagnostic laboratories.

This study evaluates the accuracy of liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detecting carbapenem hydrolytic activity among SPM-1-, GIM-1-, and GES-5-producing Pseudomonas aeruginosa isolates and OXA-143-, IMP-10-, and OXA-58-producing Acinetobacter baumannii isolates. Class A and B carbapenemase activities were rapidly detected by MALDI-TOF in a 2-h assay. However, an extended incubation time was necessary for detection of carbapenem-hydrolyzing class D ␤-lactamase (CHDL) activity in Acinetobacter spp. Over the last two decades, the therapeutic options to treat infections caused by Gram-negative rods have been narrowed by bacterial acquisition of carbapenemase-encoding genes (1). The rapid detection of clinically significant carbapenemases is important for establishing adequate therapy and controlling their spread. Recently, the detection of class A or B carbapenemase activity has been accomplished by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (2, 3). Although MALDI-TOF MS seems to be a promising tool, there is a lack of evidence showing its usefulness in testing a broader range of carbapenemases. To date, the published studies have mainly focused on KPC-2-and NDM-1-producing Enterobacteriaceae, IMP-1-, VIM-1-, and VIM-2-producing Pseudomonas aeruginosa, and OXA-23-and OXA-24-producing Acinetobacter baumannii isolates (2-4). In the current study, we evaluated the performance of the MALDI-TOF MS assay in detecting carbapenemase activity of GES-5-, GIM-1-, or SPM-1-producing P. aerugi- 128-Ͼ256 64-Ͼ256 64 (7/11) 100 (11/11) 100 (11/11) 100 (11/11) 100 (11/11) 100 (11/11) IMP-1 1 Ͼ256 32 Ͼ256 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) VIM-1 2 256-Ͼ256 256 256 0 (0/2) 100 (2/2) 100 (2/2) 100 (2/2) 100 (2/2) 100 (2/2) GIM-1 4 Ͼ256 128-256 256-Ͼ256 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) Negative control 6 4-Ͼ256 0.5-16 Ͻ0.06-32 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) a ETP, ertapenem; IPM, imipenem; MEM, meropenem; MHT, modified Hodge test; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight mass spectrometry; LC-MS, liquid chromatography-mass spectrometry.

Background Carbapenemase-producing K. pneumoniae have become a global priority, not least in low-middle income countries. Here, we report the emergence and clinical impact of a novel KPC-K. pneumoniae ST16 clone in a Clonal Complex (CC)258 endemic setting. Methods In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infections (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates were MLST-typed. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole genome sequencing (WGS), and had their virulence tested in a Galleria larvae model. Results One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patient’s severity scores were high and baseline clinical variables were not statistically different across ST’s. In multivariate analysis, ST16 (OR 21.4; CI95% 2.3-202.8; p=0,008) and septic shock (OR 11.9; CI95% 4.2-34.1; p<0,001) were independent risk factors for fatal outcome. ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule and Yersiniabactin virulence determinants. ST16 clone was highly pathogenic in the larvae model. Conclusions Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring.

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The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.