Application of Streptococcus uberis Multilocus Sequence Typing: Analysis of the Population Structure Detected among Environmental and Bovine Isolates from New Zealand and the United Kingdom

Abstract

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

Identification of Streptococcus uberis Multilocus Sequence Types Highly Associated with Mastitis

“…In this study, 95% of the isolates in GCC ST5 and GCC ST143 possessed hasA, whereas only 25% of the isolates in GCC ST86 possessed hasA gene with chisquare analysis demonstrating a significant link between the possession of hasA and the GCC type (P ϭ 0.005) ( Table 6). These results are in agreement with previous studies (6,42), which suggested a link between both GCC ST5s and GCC ST143s with hasA. Although hasA is not thought to be directly involved in the pathogenicity of S. uberis (17), it nevertheless appears to be a virulence marker gene.…”

“…The ST of four Australian ST60 isolates was identical to the ST of isolates originally identified in the United Kingdom (41). One ST184 isolate had a MLST profile identical to that of an isolate from New Zealand (42). In addition, two ST194 isolates recovered from different farms located 100 km apart in Victoria, Australia, were shown by PFGE analysis not to be clones.…”

“…The PCR conditions and the primers used are listed in Table 1. To further confirm species identification, 16S rRNA gene sequencing was also performed as described previously (42). The 16S rRNA gene sequences were compared with the reference sequence of S. uberis, strain HN1 (AB023576.1) using BLASTN at the NCBI (http://www.ncbi.nlm.nih.gov /BLAST/), and the individual isolates were designated as S. uberis if the 16S rRNA sequence was 100% identical to the reference sequence.…”

“…These methods include restriction fragment length polymorphism (23,27), pulsed-field gel electrophoresis (PFGE) (9,40,57), randomly amplified polymorphic DNA fingerprinting (61), and multilocus sequence typing (MLST) (6,42,62). These studies suggested that transmission of S. uberis occurs from environmental sources (40,61,63) and that feces could be a reservoir (61,63).…”

“…These studies suggested that transmission of S. uberis occurs from environmental sources (40,61,63) and that feces could be a reservoir (61,63). An earlier MLST study identified specific sequence types (STs) belonging to major clonal complexes (CCs) from milk samples and the cow's environment and suggested that these STs have the ability to survive in the environment and to establish intramammary infections (42).…”