Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Impact of bacterial species and baseline resistance on fosfomycin efficacy in urinary tract infections.

OBJECTIVES: To assess the antibacterial effects of a single 3 g oral fosfomycin dose on Escherichia coli and Klebsiella pneumoniae clinical isolates within a dynamic bladder infection model. METHODS: An in vitro model simulating dynamic urinary fosfomycin concentrations was used. Target fosfomycin exposure (Cmax = 1984 mg/L and Tmax = 7.5 h) was validated by LC-MS/MS. Pharmacodynamic responses of 24 E. coli and 20 K. pneumoniae clinical isolates were examined (fosfomycin MIC ≤0.25-128 mg/L). Mutant prevention concentration (MPC), fosfomycin heteroresistance, fosfomycin resistance genes and fosA expression were examined. Pathogen kill and emergence of high-level resistance (HLR; MIC >1024 mg/L) were quantified. RESULTS: Following fosfomycin exposure, 20 of 24 E. coli exhibited reductions in bacterial counts below the lower limit of quantification without regrowth, despite baseline fosfomycin MICs up to 128 mg/L. Four E. coli regrew (MIC = 4-32 mg/L) with HLR population replacement. At baseline, these isolates had detectable HLR subpopulations and MPC >1024 mg/L. All E. coli isolates were fosA negative. In contrast, 17 of 20 K. pneumoniae regrew post exposure, 6 with emergence of HLR (proportion = 0.01%-100%). The three isolates without regrowth did not have a detectable HLR subpopulation after dynamic drug-free incubation. All K. pneumoniae had MPC >1024 mg/L and were fosA positive. WGS analysis and fosA expression failed to predict fosfomycin efficacy. CONCLUSIONS: E. coli and K. pneumoniae isolates demonstrate discrepant responses to a single fosfomycin dose in a dynamic bladder infection in vitro model. Treatment failure against E. coli was related to an HLR subpopulation, not identified by standard MIC testing. Activity against K. pneumoniae appeared limited, regardless of MIC testing, due to universal baseline heteroresistance.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 ' ' )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.