MALDI-TOF MS-Based Approaches for Direct Identification of Gram-Negative Bacteria and BlaKPC-Carrying Plasmid Detection from Blood Cultures: A Three-Year Single-Centre Study and Proposal of a Diagnostic Algorithm

Abstract

The rapid identification of pathogens of bloodstream infections (BSIs) and the detection of antibiotic resistance markers are critically important for optimizing antibiotic therapy and infection control. The purpose of this study was to evaluate two approaches based on MALDI-TOF MS technology for direct identification of Gram-negative bacteria and automatic detection of Klebsiella pneumoniae carbapenemase (KPC) producers using the Bruker MBT Subtyping IVD Module in a large routine laboratory over a three-year period. MALDI-TOF MS analysis was performed directly from blood culture (BC) bottles following bacterial pellet recovery by Rapid MBT Sepsityper® Kit and on blood agar 4-h subcultures. Automated detection of blaKPC-carrying pKpQIL-plasmid by Bruker MBT Subtyping Module was evaluated in BCs tested positive to K. pneumoniae or E. coli. The results were compared with those obtained with conventional reference methods. Among the 2858 (93.4%) monomicrobial BCs, the overall species identification rates of the Rapid Sepsityper and the short-term subculture protocols were 84.5% (n = 2416) and 90.8% (n = 2595), respectively (p < 0.01). Excellent specificity for KPC-producers identification were observed for both MALDI-TOF MS protocols. The pKpQIL plasmid-related peak was detected in overall 91 of the 120 (75.8%) KPC-producing isolates. Notably, 14 out of the 17 (82.3%) K. pneumoniae isolates carrying blaKPC variants associated with ceftazidime/avibactam resistance and tested negative by the immunocromatography assay, were correctly identified as KPC-producers by MALDI-TOF MS. In conclusion, combination of both Rapid Sepsityper and short-term subculture protocols may represent an optimal solution to promptly identify more than 95% of Gram-negative bacteria causing BSIs. MALDI Biotyper® platform enabled a reliable and robust automated detection of KPC producers in parallel with species identification. However, integration of molecular or immunocromatographic assays are recommended according to local epidemiology.

Prevalence and Impact on Mortality of Colonization and Super-Infection by Carbapenem-Resistant Gram-Negative Organisms in COVID-19 Hospitalized Patients

“…First, in the first wave of the pandemic, due to the severity of the SARS-CoV-2 infection, a higher proportion of deaths within the study occurred in the early stages of hospitalization; therefore, these patients had less time to develop superinfections and to obtain microbiological investigation results. In addition, the successful implementation of rapid diagnostics coupled with infectious counselling intervention for the timely and appropriate use of new drugs such as ceftazidime/avibactam and cefiderocol may suffice to counteract the putative positive influence of such infections on mortality [ 34 , 42 , 43 ].…”

A Review of Carbapenem Resistance in Enterobacterales and Its Detection Techniques

“…Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in optical techniques has recently become popular for identifying pathogens and resistant bacteria due to its distinct fingerprint spectra [ 104 , 118 ]. To determine the antimicrobial resistant profile in pathogens, MALDI-TOF MS identifies (1) the antimicrobial-resistant clonal group (e.g., carbapenem-resistant E. coli ), the modified antimicrobial drug (e.g., carbapenemase activity), the modified antimicrobial target (e.g., lipid A modification), the direct detection of the AMR determinant (e.g., KPC-2 β-lactamases), and biomarkers co-expressed with the AMR determinants (e.g., bla KPC carrying plasmid) [ 124 ]. This technique identifies specific resistant profiles (e.g., KPC and MBLs) of bacteria at the species and genus level from single isolated colonies within 1–4 h, with 72.5–100% sensitivity and 98–100% specificity; however, it has issues regarding OXA-48 identification [ 40 , 123 , 124 ].…”

“…To determine the antimicrobial resistant profile in pathogens, MALDI-TOF MS identifies (1) the antimicrobial-resistant clonal group (e.g., carbapenem-resistant E. coli ), the modified antimicrobial drug (e.g., carbapenemase activity), the modified antimicrobial target (e.g., lipid A modification), the direct detection of the AMR determinant (e.g., KPC-2 β-lactamases), and biomarkers co-expressed with the AMR determinants (e.g., bla KPC carrying plasmid) [ 124 ]. This technique identifies specific resistant profiles (e.g., KPC and MBLs) of bacteria at the species and genus level from single isolated colonies within 1–4 h, with 72.5–100% sensitivity and 98–100% specificity; however, it has issues regarding OXA-48 identification [ 40 , 123 , 124 ]. Further, the combination of automation and the implementation of a user-friendly interface recently made MALDI-TOF MS popular in clinical laboratories [ 123 , 125 , 126 , 127 ].…”

Current viewpoint on the epidemiology of nonfermenting Gram-negative bacterial strains

“…Burkholderia cepacia complex has been associated with colonization and infection in patients with cystic fibrosis, and importantly, with the development of the Cepacia syndrome in this patient population, an acute necrotizing pneumonia that bears high mortality [ & ]. The apparent emergence is partly due to advanced microbiological identification systems, such as the Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) [44]. Finally, it should be noted that most of the larger studies originate from microbiology laboratory data or retrospective clinical data, and do not seem to consider clinical criteria to rule out colonization potential [16,45,46].…”

“…In addition, reports for several other uncommon NFGNB as cause of HCAIs have emerged in the recent years, mostly as case reports or case series [15 ▪▪ ,41,42 ▪ ,43 ▪ ]. The apparent emergence is partly due to advanced microbiological identification systems, such as the Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) [44]. Finally, it should be noted that most of the larger studies originate from microbiology laboratory data or retrospective clinical data, and do not seem to consider clinical criteria to rule out colonization potential [16,45,46].…”