Molecular fingerprinting of bovine mastitis-associated Staphylococcus aureus isolates from India

Abstract

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC⁺, 44 (26.5%) isolates were mecA⁺, with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA⁺ but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates.

Figure 1

Map of India showing location of the six states from where bovine S. aureus isolates were obtained. For each state, the predominant spa type, sequence type and clonal complex are provided. GJ = Gujarat, KA = Karnataka, MH = Maharashtra, MG = Meghalaya, TG = Telangana, UP = Uttar Pradesh. The map was modified from one downloaded from the URL https://d-maps.com/carte.php?&num_car=24867&lang=en; from where up to 10 maps can be used per publication for free, as per their terms and conditions of use.

Figure 2

Minimum spanning trees showing spa types in each of the states. Each circle with different color represents spa type and size of the circle indicates the number of isolates for a particular spa type. Major spa type in each state is highlighted by a grey halo around the circle. The branch thickness (dotted lines, dashed lines and increasing thickness of solid lines in that order) indicates inverse of the distance between the types.

Figure 3

Minimum-spanning tree of MLST data from 166 S. aureus isolates in each state. Each circle with different color represents a different sequence type (ST) and the size of the circle is directly proportional to the number of isolates for a particular ST. Major clonal complex (CC) in each state is highlighted by a grey halo around the circle. The branch thickness (dotted lines, dashed lines and increasing thickness of solid lines in that order) indicates inverse of the distance between the STs.

Figure 4

PFGE dendrogram. The tree was generated from composite fingerprinting with 80% similarity between the isolates. The pulsotypes are designated by the letters A through F, and individual patterns within these groups are numbered.