Multilocus Sequence Typing of Streptococcus uberis Provides Sensitive and Epidemiologically Relevant Subtype Information and Reveals Positive Selection in the Virulence Gene pauA

Abstract

Control of the bovine mastitis pathogen Streptococcus uberis requires sensitive and epidemiologically meaningful subtyping methods that can provide insight into this pathogen's epidemiology and evolution. Development of a multilocus sequence typing (MLST) scheme based on six housekeeping and virulence genes allowed differentiation of 40 sequence types among 50 S. uberis isolates from the United States (n ‫؍‬ 30) and The Netherlands (n ‫؍‬ 20). MLST was more discriminatory than EcoRI or PvuII ribotyping and provided subtype data with better epidemiological relevance, e.g., by discriminating isolates with identical ribotypes obtained from different farms. Phylogenetic analyses of MLST data revealed indications of reticulate evolution between genes, preventing construction of a core phylogeny based on concatenated DNA sequences. However, all individual gene phylogenies clearly identified a distinct pauA-negative subtaxon of S. uberis for which housekeeping alleles closely resembled those of Streptococcus parauberis. While the average GC content for five genes characterized was between 0.38 and 0.40, pauA showed a considerably lower GC content (0.34), suggesting acquisition through horizontal transfer. pauA also showed a higher nonsynonymous/synonymous rate ratio (d N /d S ) (1.2) compared to the other genes sequenced (d N /d S < 0.12), indicating positive selection in this virulence gene. In conclusion, our data show that (i) MLST provides for highly discriminatory and epidemiologically relevant subtyping of S. uberis; (ii) S. uberis has a recombinatorial population structure; (iii) phylogenetic analysis of MLST data reveals an S. uberis subtaxon resembling S. parauberis; and (iv) horizontal gene transfer and positive selection contribute to evolution of certain S. uberis genes, such as the virulence gene pauA.

Virulence-associated genes and molecular typing of Streptococcus uberisassociated with bovine mastitis in northern Thailand

“…On the other hand, some isolates that were apparently clustered by PFGE may have different patterns of virulence genes, as illustrated in clusters VI, VII, VIII, XI, XII, XIII, and XVI ( Figure 3). Similar to this finding, Reinoso et al previously demonstrated a variation of virulence gene profiles among S. uberis isolates presenting identical PFGE patterns (30). This finding suggests that interpretation of genetic relatedness using only DNA banding pattern-based typing methods, such as PFGE, should be cautious, and genetically related isolates may not necessarily have identical functional genotypes or phenotypes.…”

Streptococcus mutans Clonal Variation Revealed by Multilocus Sequence Typing

“…In developing the MLST in this study, target genes were selected based on the complete genome sequence of S. mutans UA159 (1) and the ongoing complete genome sequencing of S. mutans NN2025 in our laboratories, on the genomic information from other bacteria, and on the published MLST schemes for other streptococci, such as S. pyogenes (4), S. uberis (44), S. suis (13), and S. pneumoniae (3), together with their locations in the genome. Finally, eight housekeeping genes were selected for this study.…”

Genetic diversity of Streptococcus uberis isolates from dairy cows with subclinical mastitis in Southern Xinjiang Province, China

“…The DNA was resuspended with 50 µl of TE buffer and then stored at 20 C. Six housekeeping and virulence genes were chosen for MLST. Primers for amplification of cpn60, pauA, oppF, sodA, and tuf were taken from the literature (Zadoks et al, 2005). For gapC, DNA sequence data from GenBank were used to design primers.…”

“…The method relies on the sequencing of internal fragments of certain genes. To our knowledge, 2 MLST schemes have been used to investigate genetic diversity of S. uberis, one based on the sequencing of 6 housekeeping genes and virulence genes (Zadoks et al, 2005) and the other based on the sequencing of 7 housekeeping genes (Coffey et al, 2006) (http://pubmlst. org/suberis/).…”