A Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Direct-from-Urine-Specimen Diagnostic for Gram-Negative Pathogens

Abstract

Urinary tract infections (UTIs) pose a major public health burden. The vast majority of UTIs are caused by Gram-negative bacteria. Current culture-based pathogen identification methods may require up to 24 to 48 h of incubation. In this study, we developed and evaluated a method for Gram-negative pathogen identification direct from urine, without culture, via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in approximately 1 h. Urine samples were collected (n = 137) from the University of Maryland Medical Center clinical microbiology laboratory. To identify bacteria direct from urine, two methods were evaluated. First, 1 μL of urine was directly spotted onto the MALDI target plate, and second, 1 mL of urine was centrifuged at 8,000 rpm for 5 min before processing using the fast lipid analysis technique (FLAT). Mass spectra were acquired on the Bruker MALDI Biotyper sirius system in the negative-ion mode. Results were compared to those of standard culture methods. When 1 μL of urine was directly spotted, positive agreement was 81.5% (101/124) and, after centrifugation, 94.4% (117/124) relative to that of standard culture methods. Negative agreement for both methods was 100% (13/13). The time to results for both of the specimen preparation methods using the FLAT extraction protocol was approximately 1 h, with minimal hands-on time required (<5 min). The ability to rapidly identify pathogens directly from urine, without the need for culture, allows for faster turnaround times and, potentially, improved patient outcomes. Overall, the FLAT extraction protocol, in combination with lipid A identification, provides a reproducible and accurate method to rapidly identify urinary pathogens. IMPORTANCE This study describes and evaluates a direct-from-urine extraction method that allows identification of Gram-negative bacteria via MALDI-TOF MS within 1 h. Currently, identification of urinary pathogens requires 24 h of culture prior to identification. While this method may not replace culture, we demonstrate its utility in screening for common urinary pathogens. By providing identifications in under 1 h, clinicians can potentially treat patients sooner with more-targeted antimicrobial therapy. In turn, earlier treatment can improve patient outcome and antimicrobial stewardship. Furthermore, MADLI-TOF MS is a readily available, easy-to-use diagnostic tool in clinical laboratories, making implementation of this method possible.

Keywords: Gram-negative bacteria; MALDI-TOF MS; MALDI-TOF mass spectrometry; UTI; fast lipid analysis technique; lipid A; urinary tract infection.

FIG 1

Flow diagram of the FLAT for both methods of specimen processing for direct-from-urine analysis. (A and B) Specimen preparation for FLAT without centrifugation (A) and with centrifugation (B). Both sample preparation methods have a hands-on time of less than 5 min.

FIG 2

Representative mass spectra from three pathogens, E. coli, Pseudomonas aeruginosa, and Klebsiella pneumonia, direct from an individual urine sample using the FLAT, followed by MS analysis, and a representative mass spectrum from individual urine samples that showed no growth (negative). An example of spectra from an E. coli harboring mcr-1 is shown at the bottom. All urine samples were previously characterized by the University of Maryland Medical Center clinical microbiology laboratory.