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

ABSTRACT: 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.

SUBMITTER: Foudraine DE

PROVIDER: S-EPMC8218766 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Enzymatic modification of aminoglycosides by nucleotidyltransferases, acetyltransferases and/or phosphotransferases accounts for the majority of aminoglycoside-resistant Acinetobacter isolates. In this study, we investigated the relationship between aminoglycoside resistance and the presence of aminoglycoside-modifying enzymes in Acinetobacter baumannii clinical isolate groups with different resistance profiles. Thirty-two clinical A. baumannii isolates were included in this study. Acinetobacter isolates were divided into 4 groups according to results of susceptibility testing. The presence of genes encoding the following aminoglycoside-modifying enzymes; aph (3')-V1, aph (3')-Ia, aac (3)-Ia, aac (3) IIa, aac (6')-Ih, aac (6')-Ib and ant (2')-Ia responsible for resistance was investigated by PCR in all strains. The acetyltransferase (aac (6')-Ib, aac (3)-Ia) and phosphotransferase (aph (3')-Ia) gene regions were identified in the first group, which comprised nine imipenem, meropenem, and gentamicin-resistant isolates. The acetyltransferase (aac (6')-Ib, aac (3)-Ia), phosphotransferase (aph (3')-VI) and nucleotidyltransferase (ant2-Ia) gene regions were identified in the second group, which was composed of nine imipenem-resistant, meropenem-resistant and gentamicin-sensitive isolates. The acetyltransferase (aac (3)-Ia) and phosphotransferase (aph (3')-Ia) regions were identified in the fourth group, which comprised eight imipenem-sensitive, meropenem-sensitive and gentamicin-resistant isolates. Modifying enzyme gene regions were not detected in the third group, which was composed of six imipenem, meropenem and gentamicin-sensitive isolates. Our data are consistent with previous reports, with the exception of four isolates. Both acetyltransferases and phosphotransferases were widespread in A. baumannii clinical isolates in our study. However, the presence of the enzyme alone is insufficient to explain the resistance rates. Therefore, the association between the development of resistance and the presence of the enzyme and other components should be investigated further.

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

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