Regional profiling for determination of genotype diversity of mastitis-specific Staphylococcus aureus lineage in Canada by use of clumping factor A, pulsed-field gel electrophoresis, and spa typing

Abstract

One of the major concerns in global public health and the dairy industry is the emergence of host-specific virulent Staphylococcus aureus strains. The high degree of stability of the species genome renders detection of genetic microvariations difficult. Thus, approaches for the rapid tracking of specialized lineages are urgently needed. We used clumping factor A (clfA) to profile 87 bovine mastitis isolates from four regions in Canada and compared the results to those obtained by pulsed-field gel electrophoresis (PFGE) and spa typing. Twenty-five pulsotypes were obtained by PFGE with an index of discrimination of 0.91. These were assigned to six PFGE lineage groups A to F and seven spa types, including two novel ones. Group A had 48.3% of the isolates and group D had 43.7% of the isolates, while only 8% of the isolates were variable. The results of antimicrobial susceptibility testing indicated that all isolates were sensitive to methicillin and the non-beta-lactam antibiotics, while three isolates were resistant to penicillin and one isolate was resistant to tetracycline. All isolates had the clfA gene and belonged to 20 clfA repeat types with an index of discrimination of 0.90. The dominant clfA types, types X, Q, C, and Z, formed 82% and 43% of PFGE groups A and D, respectively, and had copy numbers that varied only within a narrow range of between 46 and 52 copies, implying clonal selection. The rest were variable and region specific. Furthermore, the dominant groups contained subpopulations in different regions across Canada. Sequence information confirmed the relatedness obtained by the use of clfA repeat copy numbers and other methods and further revealed the occurrence of full-repeat deletions and conserved host-specific codon-triplet position biases at 18-bp units. Thus, concordant with the results of PFGE and spa typing, clfA typing proved useful for revealing the clonal nature of the mastitis isolate lineage and for the rapid profiling of subpopulations with comparable discriminatory powers.

FIG. 1.

Pulsed-field gel electrophoresis of SmaI-digested total DNA from S. aureus isolates. The dendrogram shows estimates of the genetic relatedness of the PFGE types. The 25 PFGE patterns were dividable into six major lineage groups, designated by the letters A through F, and these were further subdivided into sublineages, indicated by numerals associated with group letters. The colored boxes highlight the PFGE pattern types.

FIG. 2.

(A) Whole clfA R-domain global multiple nucleotide sequence alignment patterns of isolates of the 25 PFGE patterns by using the Geneious Bioinformatics package (version 4.6) with a cost matrix of 65% similarity (5.0/4.0) and free end gaps. A combined guide tree based on neighbor joining (by use of the Tamura-Nei distance model) was used. Common transversions are highlighted by base colors, as follows: green is T, blue is C, red is A, and yellow is G. Identity and sequence coverage are indicated by color, and the most common consensus sequence was selected. Standard strain abbreviations were used. (B) Local multiple-sequence alignment of selected human and animal isolates showing only one region of differential selection of amino acid codon triplets by isolates from the two hosts. In repeat units 1 to 5, the nucleotide codon triplets in animal/human strains, respectively are shown in the following patterns: unit 1 Ser, AGC/AGT; unit 2, GAC/GAT, GAC/GAT, and AGT/AGC; unit 3, GAC/GAT and AGT/AGC; unit 4, GAC/GAT, GAC/GAT, AGT/AGC, and GAC/GAT; and unit 5, GAC/GAT and AGT/AGC.

FIG. 3.

Phylogenetic relationships of the isolates shown as a phylogram of the clfA R-domain sequences with conserved 3′ and 5′ regions on the basis of the neighbor-joining program in the Geneious Bioinformatics package (version 4.6). Distances were estimated by use of the Tamura-Nei model by using bootstrapping as the resampling method, which had a support threshold of 50%.