A Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant Enterobacteriaceae

Abstract

e Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla KPC carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ϳ11,109-Da MS peak corresponding to a gene product of the bla KPC pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla KPC -negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla KPC Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla KPC Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.

Carbapenem-Resistant Non-Glucose-Fermenting Gram-Negative Bacilli: the Missing Piece to the Puzzle

“…So far, this method has only been established for the bla KPC carbapenemase-bearing pKpQIL plasmid that was responsible for the CRE outbreak that occurred at the NIH Clinical Center in 2011. The principal advantage of this method is that it is inexpensive (if a MALDI-TOF instrument is already present), and it provides simultaneous organism and carbapenemase-bearing plasmid identification within 10 min from isolated colonies or 30 min from the time of automated detection of growth in spiked blood cultures (70,71). However, much more work is needed before it can be used generally, since it would need to detect many more carbapenemases.…”

Clinical Performance of Check-Direct CPE, a Multiplex PCR for Direct Detection of bla KPC , bla NDM and/or bla VIM , and bla OXA-48 from Perirectal Swabs

“…One swab from the paired culturettes was placed into 1 ml saline solution (Remel Inc., Lenexa, KS) and subjected to vortex mixing for 10 s. Two drops were inoculated onto HardyCHROM CRE agar (Hardy Diagnostics) and incubated for 18 to 24 h at 35°C without CO 2 . All morphologically different Gram-negative bacilli (GNB) underwent bla KPC /bla NDM PCR testing (http://www.cdc .gov/HAI/pdfs/labSettings/KPC-NDM-protocol-2011.pdf), identification via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics Inc., Billerica, MA [27]), and antimicrobial susceptibility testing using dried, custom-designed panels (Trek Diagnostics, Oakwood Village, OH). Saline suspensions were stored at 4°C for downstream culture of CPO/CRO culturenegative, Check-Direct CPE-positive samples.…”

Transplantation of human skin microbiota in models of atopic dermatitis

“…Colonies were then taken for species identification by mass spectrometry using MALDI-TOF analysis. Bacterial protein extraction for MALDI-TOF mass spectrometry using the BioTyper (v3.1, Bruker Daltonics Inc.) was performed by the NIH Clinical Center microbiology lab using previously described methods (46), instrument settings, and calibration (47,48). BioTyper identification was supplemented by additional mass spectra profiles provided by several NIH-developed databases (46,49,50).…”

“…Bacterial protein extraction for MALDI-TOF mass spectrometry using the BioTyper (v3.1, Bruker Daltonics Inc.) was performed by the NIH Clinical Center microbiology lab using previously described methods (46), instrument settings, and calibration (47,48). BioTyper identification was supplemented by additional mass spectra profiles provided by several NIH-developed databases (46,49,50). Ten of the healthy control R. mucosa strains reported in Figure 1 were identified based solely on the unique colony morphology, UV light reactivity, and Gram stain characteristics that had been observed in the other R. mucosa isolates identified by MALDI-TOF analysis.…”