Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing

Abstract

Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database (http://pubmlst.org/neisseria) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis, was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.

Fig. 1.

Procedural steps involved in microbial typing by MALDI-TOF MS. Step 1, import of reference sequences into the system database; step 2, PCR and post-PCR biochemistry; step 3, MALDI-TOF MS spectrum and peak pattern comparison; step 4, tabulated typing results.

Fig. 2.

MALDI-TOF MS MLST typing statistics of 96 typeable N. meningitidis samples. For 97.6% of the sample alleles the software automatically assigned the correct top matching reference sequence, for 1.8% the correct matching reference was listed among a group of top matching references with equal score, and for 0.6% a wrong reference sequence was presented.

Fig. 3.

MALDI-TOF MS-based discovery of a mutation C→T in allele aroE9 at position 443. (A) Mutation-specific signal changes at 7,343.5 and 8,957.9 Da in the T-specific cleavage reaction of the forward RNA transcript. (B) Mutation-specific signal changes at 3,120.0 and 3,136.0 Da in the T-specific cleavage reaction of the reverse RNA transcript. (C) Mutation-specific signal change at 2,010.0 Da in the C-specific cleavage reaction of the forward RNA transcript.

Fig. 4.

Cluster analysis for the fumC allele. Shown is an unweighted pair group method analysis tree of base-specific cleavage and MALDI-TOF MS patterns (A) in comparison to an unweighted pair group method analysis tree derived from the primary sequences of the same sample set (B). Samples are labeled by allele and sample number (x_y). Alleles are color-coded, and partitions are numbered. Euclidean distance 2.8 is the cutoff for the degree of spectra similarity between identical samples. Clades defined by one tree but not by the other are highlighted by asterisks.