Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid detection of β-lactam resistance in Enterobacteriaceae derived from blood cultures

Abstract

The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one β-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS β-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the β-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C β-lactamases. Discrimination was more difficult in species expressing class A β-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.

FIG 1

Results of the ampicillin hydrolysis assay. The logRQ values for ampicillin are displayed in a box plot diagram. For comparison with conventional susceptibility testing, boxes that represent E. coli strains with MIC values of >8 μg/ml are colored in red. Boxes representing E. coli strains with MIC values of ≤8 μg/ml (susceptible breakpoint according to CLSI and EUCAST) are colored in green. Blood culture flask 92 contained one susceptible and one resistant E. coli strain.

FIG 2

β-Lactamase assay with cefotaxime. Results for isolates that constitutively produce AmpC enzymes. All isolates with logRQ values of less than −0.5 were susceptible to 3rd-generation cephalosporins. Isolates with stable hyperproduction of AmpC β-lactamase had a moderately elevated hydrolysis rate. One isolate with an additional plasmid-encoded CTX-M-type β-lactamase exhibited logRQ values comparable to those of the other ESBL producers.

FIG 3

Eighty-five blood culture flasks contained Enterobacteriaceae that typically express class A β-lactamase genes but do not have chromosomally encoded AmpC activity. The logRQ values for cefotaxime are displayed in a box plot diagram. In SHV- and TEM-negative isolates, the logRQ values were comparable to those for isolates with low AmpC expression. Cefotaxime-susceptible isolates with class A β-lactamase activity showed a wider dispersion of the logRQ values. All of the isolates that were phenotypically resistant to cefotaxime due to either ESBL or AmpC enzyme activity showed logRQ values of >0. An arbitrary logRQ cutoff value of 0 was set.

FIG 4

Cefotaxime logRQ values from subcultures on Columbia blood agar. LogRQ values were elevated compared to those from blood culture fluid. No differences in the logRQ values between AmpC hyperproducers and ESBL positive strains were observed. The cutoff was set at a logRQ value of 0.