Detection and Genotyping of SHV β-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed bla SHV genes was used for the identification and genotyping of SHV ␤-lactamases. For evaluation, bla SHV stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.

Putative Protein Biomarkers of Escherichia coli Antibiotic Multiresistance Identified by MALDI Mass Spectrometry

“…We were thus able to associate the mass peaks at 2009.51 m/z and 4434.70 m/z present in the proteomes of the isolates, which were grown in the presence of trimethoprim-sulfamethoxazole, as β-lactamases of the SHV type. This data corroborates the genotype obtained in isolates with resistance to this antibiotic and with the masses obtained in the study by Sturenburg et al [34]. In this sense, the detection of modifications in this family may become crucial in preventing newly emerged resistances from spreading.…”

“…Nevertheless, results with cross-validation of model generation were 88.43%, 79.41%, and 75.17% for Genetic Algorithm, Supervised Neural Network, and Quick Classifier, respectively, showing that the peaks were indeed accurately detected even if only by one algorithm. Peaks reported by Sturenburg et al for the strains exposed to tetracycline were not observed here [34]. These tetracycline-producing streptomyces present important signal and protein spectra in the mass range of 2 to 20 kDa.…”

“…The family of SHV-type β-lactamases is constantly evolving. Each type of SHV can be distinguished from all other types based on their peptide masses [33,34]. We were thus able to associate the mass peaks at 2009.51 m/z and 4434.70 m/z present in the proteomes of the isolates, which were grown in the presence of trimethoprim-sulfamethoxazole, as β-lactamases of the SHV type.…”

Non-Phenotypic Tests to Detect and Characterize Antibiotic Resistance Mechanisms in Enterobacteriaceae

“…In another investigation, Stürenburg et al implemented the MALDI-TOF MS to analyze fragment masses of base-specific cleavage of bla SHV PCR products after in vitrogenerated RNA transcription. Twenty-one Ent possessing different SHV-types were identified unambiguously and in perfect accordance to the PCR/sequencing results (Sturenburg, et al, 2006).…”

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

“…implemented the MALDI-TOF MS to analyze fragment masses of base-specific cleavage of bla SHV PCR products after in vitro -generated RNA transcription. Twenty-one Ent possessing different SHV-types were identified unambiguously and in perfect accordance to the PCR/sequencing results (Sturenburg, et al, 2006). …”