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

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.

Infections and dynamics of disease caused by the same pathogen differ from host to host and from one geographical location to another. Some clonal lineages of a particular pathogen tend to be specific to a host, and others appear to transmit easily between hosts. To gain knowledge about similarities among strains, sources of infection, modes of transmission and carriage of virulence and resistance genes, one can employ molecular typing methods which can reveal genetic diversity of the isolates of a particular pathogen.
Staphylococcus aureus is found associated with a variety of human infections, with some evidence for overlap in the distribution of certain clonal populations between humans and animals [1][2][3][4] . The organism is known to produce a number of virulence factors such as toxins and antibiotic resistance mediators which facilitate its survival in the host as well as in the environment 5,6 . In addition, methicillin-resistant S. aureus (MRSA), which encode multiple antimicrobial resistance genes, are a major problem in human medicine 7 . Bovine mastitis is a very common livestock production-related disease, and S. aureus is the major contributor, resulting in a range of manifestations, including a large proportion of subclinical and chronic cases 8,9 . Isolates from bovine mastitis have been shown to produce various types of hemolysins and enterotoxins, as well as Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin (TSST), although the proportions of the isolates expressing each type of these toxins vary widely [10][11][12] . Although early studies suggested much lower prevalence of MRSA among bovine mastitis-associated S. aureus compared to those isolated from human infections, recent studies have shown trends towards higher prevalence [13][14][15][16][17][18][19][20][21][22][23][24][25][26] . This has led to focus towards the longitudinal spread and sharing of particular MRSA clones and their related strains among and between animals and humans, in order to understand the consequence of animal-human interface in infections and diseases caused by MRSA.

Results
In this study, 166 bovine mastitis-associated S. aureus isolates from six different states of India were studied. The isolates included 98 from Karnataka, 19 each from Gujarat and Telangana, 12 from Uttar Pradesh and 5 from Maharashtra ( Fig. 1).
To examine virulence determinants with the potential for pathogenicity, we subjected the isolates to disk diffusion test, broth microdilution for MIC for determining susceptibility to oxacillin and to PCR for mecA and mecC genes. Out of the 166 isolates, 34 (20.4%) showed resistance to oxacillin, with MICs of 8 to 64 µg/mL, whereas mecA amplification was observed with 44 (26.5%) isolates (Supplementary Table S3); none of the isolates contained mecC (data not shown). Out of the 44 mecA-positive isolates, 20 showed oxacillin MIC of > 4 µg/ mL and 14 isolates showed oxacillin resistance in the absence of mecA or mecC (Table 1). By the definition of oxacillin susceptibility and presence of mecA, 24 of the 166 isolates (14.46%) could be categorised as OS-MRSA (Supplementary Table S4). Among 44 mecA + isolates, 32 were typeable for SCCmec I to V, and most of the isolates (26/32) belonged to SCCmec IVb, whereas 12 isolates did not amplify any bands for these types ( Table 1). The predominant MRSA clones were CC9-ST2454-SCCmecIVb-t7867 and CC97-ST2459-SCCmecIVb-t359 (Tables 1, 2).
PCR was also carried out to test the presence of the toxin genes hlg, tsst and pvl, which encode for the most common virulence determinants. The results showed that 56.25%, 28.75% and 47.5% of the isolates harboured hlg, tsst and pvl genes, respectively (Table 1). Notably, a high proportion of Karnataka isolates harboured hlg (87.7%) and pvl (82.2%) genes. Among these, isolates belonging to pulsotypes C2 and C3 had both hlg and pvl genes, and pulsotypes E5, E6 and E7 were positive for all the three genes ( Table 1). All of the B, G, H and I pulsotypes carried the pvl gene, although there were only one to six isolates in these pulsotypes, and among pulsotypes C and E, which each contained more than 50 isolates, pvl carriage ranged between 39.62% and 44.23% (Table 1).

Discussion
Mastitis is a major concern for the dairy industry, as it not only affects animal's health, but also results in direct and indirect losses 33 . Despite producing highest amount of milk, Indian dairy industry is crippled by mastitis 34 , with an estimated annual loss of INR 71.6551 billion (USD 1.6 billion) as of two decades ago 35 . Loss per incidence of INR 400 to 2086 (about USD 6 to 31) and annual loss per animal of INR 1500 to 1700 (about USD 22 to 25) have been reported [36][37][38][39] .
Although bovine mastitis is a multi-factorial, multi-etiological disease, S. aureus is one of the most common pathogens associated with it worldwide, including India [40][41][42] . Infection with S. aureus can lead to a wide range of manifestations including clinical as well as subclinical, chronic, persistent and recurrent mastitis. Transmission of S. aureus usually occurs from one animal to another through milking, but can also occur through contagion. Complete information on the population structure, virulence and resistance characteristics of mastitis-associated S. aureus is desired to monitor its dissemination among animal populations and for risk assessment, so as to improve interventions. This is the first study to report the genotypes of bovine mastitis-associated S. aureus from different locations in India, through the simultaneous application of spa typing, MLST and PFGE.
Among several genotypes of S. aureus, only a few appear to be associated with bovine mastitis 43 . As far as spa types are concerned, from bovine milk, t002, t008, t267, t359 and t6877 have been reported from Brazil, Canada and Japan [44][45][46][47] ; t127 and t2279 were reported to be common in China 48 . The spa types t267 and t359 were also observed to be predominant among our isolates from Karnataka, and have been previously reported from this state 29 ; other types such as t7867 and t3841 are also common in Karnataka 30 . On the other hand, t7867 was observed in all the states, suggesting that t267, t359 and t7867 may be commonly associated with bovine mastitis in India.
We observed a predominance of ST2454 and ST2459 among our isolates, although there were also 12 newly identified STs. Other types, ST72, ST245, ST239 and ST1687 have been previously reported from Andhra Pradesh, Telangana and Karnataka 29 , implying the existence of diverse genetic populations. Integrated data of our isolates showed that CC97-ST5414-t1965 and CC97-ST2459-t359 were predominant in the states of Gujarat and Karnataka, respectively, whereas CC9-ST2454-t7867 was frequent among isolates of the remaining four states studied. www.nature.com/scientificreports/ Early phenotypic studies from Europe and the US showed low prevalence of MRSA among livestock [13][14][15]18 . By specific culturing or PCR for mecA or mecC, other studies have reported a relatively lower prevalence of 0% to 9.7% from Europe, US, Japan and South Korea 17,19,20,25,26,45 , while higher prevalence ranging from 16.7 to 49.6% has been reported from Germany, Brazil, Japan, Sweden and China 16,[21][22][23][24] . We observed that 12.04% of our 166 isolates were methicillin-resistant both phenotypically and genotypically, whereas 14.46% were OS-MRSA. In total, 58 isolates (34.94%) were either oxacillin-resistant or mecA + . Earlier studies from India, which have performed either phenotypic or genotypic tests (not both), have reported a range from 5 to 27% [49][50][51][52] . It is possible that the high levels (34.94%) of MRSA within our isolates could just be a consequence of the application of both genopytic and phenotypic tests, but is similar to higher proportions observed in several parts of the world as stated above. It may, however, be noted that defining methicillin resistance has been problematic since a variety of conditions such as modifications in native penicillin-binding proteins, hyper-production of β-lactamase, or production of methicillinase can lead to resistance 53 . These studies together suggest that classifying an isolate as MRSA is complex, and in the context of a clinical setting, the phenotypic assay may be more important, although it is possible that genotypically positive, phenotypically negative isolates may still gain resistance in response to environmental stimuli such as encounter with salts or antibiotics [54][55][56] .
A large majority of our isolates, irrespective of methicillin resistance/susceptibility, clustered among CC97 and CC9. Historically, livestock-associated MRSA (LA-MRSA) were first reported from pig production systems, and found to belong to ST398 57 . Later, ST398 was also reported from bovine mastitis cases 58,59 , as well as other samples from various other livestock 57 . Majority of bovine mastitis-associated MRSA reported worldwide belong to CC398 60-63 , whilst less represented clones include those belonging to CC1, CC5, CC9, CC97, CC130 and a few others [64][65][66][67] . It is now apparent that strains belonging to CC9 are the predominant LA-MRSA in Asian countries whereas ST398 is predominant in Europe and North America 2,60-63 . In addition, CC97 MRSA have also been demonstrated to be predominant in dairy cattle and pigs in Italy 68 . On the other hand, a large proportion of MSSA from a number of countries have been reported to belong to CC398 and CC97 [68][69][70][71][72][73][74][75][76][77] . ST398, an ST which clusters under CC398, is also predominant among MSSA from Japan and China 78,79 . In addition, MSSA from China also belonged to ST97 and ST9-SCCmecXII-t899 whilst MRSA belonged to ST1-SCCmecI-t1939 62,80 . Whereas CC97 www.nature.com/scientificreports/ and CC9 were widely represented in our MRSA as well as MSSA isolates, we did not find any isolate belonging to ST398, suggesting commonality as well as variance with studies worldwide. The exact origin of the predominant strains in our study is not discernible since neither this is a longitudinal study, nor is there sufficient data from within India to compare and analyse. One of the major cluster of strains from our study was CC9-ST2454. This particular clone has been previously reported from pigs in China and from water buffalos in the Philippines 81,82 . It may be argued that our CC9-ST2454 strains originated from pigs. However, most of the places from where isolates were obtained for our study were from semi-urban or village areas from states which have very less pig population. In India, the majority of pig rearing occurs in the Northeast region and the state of Kerala. In fact, a little more than 70% of the pig population of India is in the Northeast region, and only the state of Meghalaya belonged to this geographical location within the isolates that we obtained. In addition, most of the other locations from where isolates were obtained were free from domesticated or feral pigs. Hence, we consider it highly unlikely that ST2454-SCCmecIVb MRSA in our study may have originated directly from pigs, although we cannot rule out transmission events which predate isolation of the organisms used in our study.
Majority (26/32) of our mecA-containing isolates were typed as SCCmecIV, which has also been reported in other studies elsewhere 83,84 . On the other hand, several recent studies have reported on oxacillin-susceptible mecA + S. aureus (OS-MRSA) 22,[85][86][87][88][89] . In our study, 14.46% of the isolates were OS-MRSA. In Karnataka, OS-MRSA belonged to CC97-ST2459-SCCmecIVb with diverse spa types. In other states, OS-MRSA belonged to CC9-ST2454-SCCmecIVb-t7867 or t4522. A report from China identified high incidence (35.9%) of OS-MRSA, and with SCCmecV-t267 whereas OS-MRSA isolates from Shanghai belonged to different spa and SCCmec types 22 . One study from India reported 48.7% OS-MRSA, with diverse STs and spa types 87 . Only two of our OS-MRSA isolates belonged t267 and the remaining belonged to diverse types indicating clonal diversity among OS-MRSA.  www.nature.com/scientificreports/ of the equipment and the requirement for technical expertise. In addition, the results of PFGE typing for strains from different laboratories is not easily comparable without an extensive cataloguing of the banding patterns. It may, however, be noted that the majority of ST2454 and ST2459 strains clustered to pulsotypes E and C (30/57 and 21/40), respectively, suggesting corroboration between the two typing methods. A large proportion of the isolates from Karnataka showed the presence of both hlg and pvl genes, suggesting pathogenic potential of these isolates. It has been hypothesized that different clones might display differential patterns of antimicrobial resistance and virulence factors 48 . We observed that isolates expressing the toxin genes hlg, tsst and pvl belonged to particular pulsotypes (C2, C3, E5, E6 and E7), lending credence to this hypothesis. It should be noted that C and E pulsotypes encompassed the largest groups with 52 and 53 strains, respectively, but only five sub-pulsotypes among these contained the majority of the isolates carrying the pvl gene, suggesting potential association between certain pulsotypes and virulence.
In conclusion, the majority of the isolates that we studied belonged to two major clonal complexes CC97 and CC9, similar to what has been observed globally. And yet, genetic diversity was evident as supported by the existence of more than 30 different genetic types (by any of the three typing methods). While heterogeneity at the micro-level would appear to support our hypothesis that mastitis-associated S. aureus strains from India show diversity, the diversity appeared to be within the two CCs. Among the oxacillin-resistant strains, the majority of the isolates belonged to CC97-ST2454-SCCmecIVb-t359 in Karnataka and CC9-ST2454-SCCmecIVb-t7867 in the other states except Maharashtra. These strains may therefore represent the predominant clones in these regions. Importantly, ST2454-and ST2459-SCCmecIVb appear to be the major MRSA clones in the country. However, a wider inference of the study is limited by the higher number of isolates from some states than others. Hence, more extensive, structured studies involving more number of states and isolates are required to better understand the genetic diversity of S. aureus isolates causing mastitis in India. This could help in elucidating strain variation, studying movement and transmission events, and in designing better control and prevention strategies for bovine mastitis.

Materials and methods
Isolates and genomic DNA isolation. For this study, 166 isolates of S. aureus curated at the Department of Microbiology, Veterinary College, Bangalore, Department of Microbiology, Veterinary College, Shivamogga, Karnataka State, and the National Institute of Animal Biotechnology, Hyderabad, were used. The study was therefore retrospective, and focussed on obtaining complete set of typing data for as many isolates as possible. The isolates had been obtained at various times from clinical and subclinical mastitis cases from six different states: Gujarat (Anand), Karnataka (North and South), Maharashtra (Mumbai), Meghalaya (unknown locations), Telangana (Hyderabad) and Uttar Pradesh (unknown locations) in India. Whereas details of the locations and dates are available for most of the isolates from Karnataka (Supplementary Table S1), no details are available for other locations as the isolates were obtained from collaborators. The isolates had been identified earlier as S. aureus by biochemical tests as well as by polymerase chain reaction (PCR), and were re-confirmed before beginning this study by PCR for the nuc gene 91 .
The isolates were cultured for 24 h at 35 °C on tryptic soy agar plates, and a single colony was further propagated in tryptic soy broth for 24 h. The cells were pelleted and genomic DNA was extracted using the HiYield Genomic DNA Mini Kit (Bacteria) (Real Biotech Corporation, Taiwan). Spa typing. The spa repeat region was amplified using primers and conditions as per RidomSpa server  Evaluation of methicillin susceptibility and the presence of mec genes. For these studies, methicillin-sensitive ATCC-29213 and methicillin-resistant ATCC-335913 strains of S. aureus were used as control strains. The antibiotic susceptibility of the isolates was evaluated by the disk diffusion test according to the Kirby-Bauer method 94 . Each isolate was subjected to testing three times along with a methicillin-sensitive and a -resistant reference strain (ATCC 29,213 and 335,913, respectively). Briefly, the isolates were inoculated into brain heart infusion broth (1 Ml; HiMedia) and incubated overnight at 37 °C. A suspension (0.5 mL) of the resultant culture was adjusted to 0.5 on McFarland scale using sterile phosphate-buffered saline (PBS, pH 7.2), and then spread plated onto Mueller-Hinton agar (HiMedia). Discs containing oxacillin or cefoxitin (HiMedia), as well as a sterile disc serving as a negative control, were gently placed on the dried inoculum, and the plates were incubated at 37 °C for 24 h. The zone of inhibition, inclusive of borders, was measured in mm and the breakpoints were applied as recommended for veterinary pathogens by the Clinical and Laboratory Science Institute 95 . All the isolates were subjected to amplification of mecA and mecC genes using primers and conditions described previously 96 . All the isolates which showed mecA amplification were subjected to SCCmec typing using multiplex PCR for SCCmec types I to V as described previously 97 .
Further, minimum inhibitory concentration (MIC) of oxacillin was determined for all the isolates which showed either mecA amplification or resistance in disc diffusion, following the broth micro-dilution method recommended by CLSI 95 .
Detection of toxin genes. Presence of toxin genes (hlg, tsst and pvl) was confirmed by individual PCRs using DNA template (100 ng) and gene-specific primer pairs (0.5 μM of each) added to a PCR Master Mix (Takara Bio, Japan), and amplification was performed with the following cycle: 15 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 1 min at 53 °C, and 1 min at 72 °C, with a final 10 min elongation step at 72 °C. PCR products were analysed by 2% agarose gel electrophoresis and UV transillumination. The primers were F: GCC AAT CCG TTA TTA GAA AATGC and R: CCA TAG ACG TAG CCA CGG AT for hlg, F: ACC CCT GTT CCC TTA TCA TC and R: TTT TCA GTA TTT GTA ACG CC for tsst, and F: GGC GCT GAG GTA GTC AAA AG and R: TCG GAA TCT GAT GTT GCA GT for pvl 98,99 .

Data availability
All data generated or analysed during this study are included in this article and its Supplementary Information files.