Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

Keywords: Campylobacter; chickens; matrix-assisted laser desorption/ionization mass spectrometry; wild birds.

Figure 1.

A. Dendrogram showing distances and grouping of Campylobacter isolates based on protein phenotypes created using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The 2 major subclades of C. jejuni subsp. jejuni are designated M1 and M2. Isolates with similar letters (A–E) had identical 16S ribosomal (r)DNA sequences. Color-coding refers to 3 clades seen in the distance matrix tree. B. Phylogenetic relationships of 13 C. jejuni subsp. jejuni isolates determined by whole genome sequence analysis. Three clades are identified (WGS1, WGS2, WGS3) in the distance matrix tree, and isolates with identical 16S rDNA sequences are identified as above (A–E).