Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method

Abstract

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1. This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Mycoplasma bovis in Nordic European Countries: Emergence and Dominance of a New Clone

“…Elsewhere in the world, ST29 has only been detected in Israel and Hungary. Interestingly, its closest subtype (ST30) was found in bison from Canada [28]. The observation of an ST linked to bison as the closest relative of the recent Danish isolates is very interesting and warrants further attention to help elucidate the origin of these isolates now present in Europe.…”

“…Only recent isolates were characterized by the loss of the adh-1 gene and they belong to three different MLST-subtypes (ST5, ST17, and ST94 in MLST-1a) suggesting this feature may be a consequence of evolutionary genome reduction associated with host adaption described in mycoplasmas, apparently irrespective of the genomic background. The issue of being unable to designate types for MLST-1a was recently addressed in an updated MLST scheme, which uses dnaA instead of adh-1 [28] (see https://pubmlst.org/mbovis/). It already comprises 464 entries and was shown to have a better discriminatory index than the "legacy" base or than the MLST scheme from Rosales et al (MLST-2 here) [27].…”

“…This study has contributed to the public database (both the genomic and MLST databases) by including a wider range of isolates from Europe. For instance, previously, only 33 isolates in the PubMLST set of 448 isolates studied by Register et al were of European origin [28]. We have been able to include older isolates, collected prior to 2000, that are currently less well represented in the MLST scheme and genomic data.…”

“…In order to assess their relative discriminatory ability on our set of strains and their maintained effectiveness to determine phylogenetic clusters in the genomic era, the MLST schemes from Register et al in its legacy or new version (MLST-1a, MLST-1b, respectively) [26,28], that of Rosales et al (MLST-2) [27], and the polC subtyping scheme proposed by Becker et al [24] were used to infer the M. bovis population structure (see all details in Table S1).…”

“…All isolates were assigned to subtypes (STs) according to the Multilocus Sequence Typing (MLST) database for M. bovis (https://pulmlst.org/bovis/; accessed on 7 August 2020) using the MLST package (https://github.com/tseemann/mlst) to retrieve individual allelic sequences from the genomes. The PubMLST website was developed by Keith and Jolley and is sited at the University of Oxford [50], while the scheme for M. bovis was developed and updated recently by Register et al (MLST-1a legacy and MLST-1b, new) [26,28,51]. ST assignment was performed for both the old MLST-1 (MLST-1a, based on genes adh-1, gltX, gpsA, gyrB, pta2, tdk, tkt) and the revised MLST-1 scheme (MLST-1b, same genes, but dnaA in place of adh-1).…”