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Int.J.Curr.Microbiol.App.Sci (2021) 10(02): 122-128 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 10 Number 02 (2021) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2021.1002.015 Capsular Typing of Cap5k and Cap8k Genes in S. aureus Isolates from Wound Samples of Cattle Veenu Singhal*, Taruna Bhati, Pankaj Dhakarwal, Sudesh Sharma, Mahaender Milind and B. N. Shringi Department of Veterinary Microbiology and Biotechnology, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001 (Rajasthan) India *Corresponding author ABSTRACT Keywords Cattle, Capsular polysaccharide, cap5k and cap8k genes, Staphylococcus aureus Article Info Accepted: 12 January 2021 Available Online: 10 February 2021 The present study was implemented to evaluate S. aureus for their 2 major capsular polysaccharide genes namely cap5k and cap8k. Capsule is an important virulence factor of S. aureus which help the organism in its survival by evading host immune system specially phagocytosis. Thirty isolates of S. aureus were obtained from skin wounds of cattle in Bikaner, Rajasthan which were first identified by conventional cultural and biochemical properties and then confirmed by ribotyping based on 23S rRNA gene segment. All the 30 isolates were subjected to capsular typing by duplex PCR targeting cap5K and cap8K genes with specific primers. Capsular typing revealed 9 (30 per cent) isolates with cap5K and 15 (50 per cent) isolates with cap8K gene. numerous, structurally distinct CP, forming a basis for serotyping isolates (Visansirikul et al., 2020). Of the 11 capsular polysaccharides types, the majority of the human and animal staphylococci are of cap5 and cap8 genotypes (O'Riordan and Lee, 2004; Verdier et al., 2007). S. aureus strains which produce a high amount of capsular polysaccharides are relatively more resistant to nonspecific opsonophagocytic killing indicating that encapsulation might be an important immune evasion strategy for the organism (Nanra et Introduction Capsular polysaccharides (CP) produce by Staphylococcus aureus play an important role in its colonization, pathogenesis and confer resistance to phagocytosis by masking of surface proteins, the typical antigens that trigger an adaptive immune response and prolong its persistence in the bloodstream of the host (Keinhörster et al., 2019). Many bacterial species have been shown to produce CP, and a single species can produce 122 Int.J.Curr.Microbiol.App.Sci (2021) 10(02): 122-128 and Primer-R (5’-AGCTCAGCCTTAACGA GTAC-3’). al., 2013). The gene cluster for CP5 and CP8 type contains 16 open reading frames (cap5A through cap5P and cap8A through cap8P, respectively) the four of which are located in the central region (H-K) and are type specific. These 16 open reading frames encode for CP5 or CP8 biosynthesis, O-acetylation, transport and regulation (O’Riordan and Lee, 2004; Weidenmaier and Lee, 2017; Rausch et al., 2019). CP enhances virulence in abscess formation (Portoles et al., 2001), surgical wound infection (McLoughlin et al., 2006), and bovine mastitis (Zaatout et al., 2020). Cap5K and cap8k gene amplification The method of Verdier et al., (2007) was used for the amplification of cap5K and cap8K gene in duplex PCR with some modification. The sequences for 2 primers used for cap5k and cap8k gene according to Table 1: The amplification was carried out in thermocycler with initial denaturation at 94º C for 2 min and 30 cycles of denaturaion, annealing and extension at 94º C, 55º C and 72º C for 60 sec respectively. Final extension was done at 72º C for 5 min and reaction is terminated by holding at 4º C. The PCR products (for genus specific PCR) were subjected to electrophoresis at 4 V/cm for 45 minute in 1.5 per cent agarose gel prepared in 1 x TBE buffer containing 0.5 ng/ml of ethidium bromide. The PCR products (5µl) were run along with 100 b.p. DNA ladder (Invitrogen) and amplicons were visualized under UVP Gel Doc Bioimaging System. The objective of the present investigation was to carry out genotypic characterisation of S. aureus obtained from skin wounds in cattle from Bikaner, Rajasthan for capsular polysachharide (cap5k and cap8k) genes. Materials and Methods Sample collection A total of 63 swabs from wound samples with pus were collected from cattle, belonging to different veterinary clinics in and around Bikaner (Rajasthan). All samples were collected aseptically with sterile absorbent cotton swab soaked in nutrient broth from skin wounds in cattle and brought to the laboratory immediately over ice and processed for isolation and identification of S. aureus as per standard conventional methods (Cowan and Steel, 1975; Quinn et al, 1994). Results and Discussion Genotypic confirmation In the present investigation, all the 30 isolates from wound samples of cattle after their identification by conventional microbiological procedures were subjected to 23S rRNA based ribotyping for genotypic confirmation. The ribotyping produced an amplicon of 1250 bp in all the isolates (Fig.1) confirming them to be S. aureus. Ribotyping targeting 23S rRNA Extraction of S. aureus genomic DNA was carried out by method described by Nachimuttu et al., (2001) and quantification as per Sambrook et al., (1989). Further, the isolates were genotypically confirmed as per the method described by Straub et al., (1999) using species specific primers i.e. Primer-F (5’-ACGGAGTTACAAAGGACGA C-3’) Similar genotypic method based on 23S rRNA ribotyping for S. aureus identification has been used by other workers from the same laboratory during the previous year viz. Upadhyay et al., (2010); Rathore et al., (2012); Khichar and Kataria (2014); Yadav et 123 Int.J.Curr.Microbiol.App.Sci (2021) 10(02): 122-128 al., (2015a), Bhati et al., (2018), Singh et al., (2018) and Nirwan (2020) from the same study area successfully and Suleiman et al., (2012), Vazquez et al., (2013), Widianingrum et al., (2016), Hamid et al., (2017) and Sundareshan et al., (2017) from elsewhere. were non typable (Table-2, Fig.2). The amplicons obtained in the present study were similar to those obtained by Verdier et al., (2007), Upadhyay et al., (2010), Khichar and Kataria (2014) and Yadav et al., (2015b). In the present study, nine isolates were detected non typable for both of the genes which corroborated the earlier observations of Naidu et al., (1991) who recorded 28 per cent of the S. aureus of bovine mastitis origin as non-typeable for cap5 and cap8. Upadhyay et al., (2010) recorded 20 per cent of the S. aureus of bovine and caprine mastitis origin as non-CP5 or non-CP8. Yadav et al., (2015b) reported 9.37 per cent non-typable isolates for cap 5 or 8 genes and Ambroggio et al., (2018) found that none S. aureus isolates of Argentina carrying any of the cap genes. Cap typing In the present study, all the 30 isolates were subjected to detection of gene fragment in the K region for cap5K and cap8K genes by duplex PCR using specific primers. The result revealed that six (20 per cent) isolates produced amplicons of 361 bp indicating presence of cap5K gene, 12 (40 per cent) isolates produced amplicon of 173 bp suggesting presence of cap8K gene, three (10 per cent) isolates were detected with both types of amplicons suggesting presence of both the gene and nine (30 per cent) isolates Table.1 Primers used for PCR for S. aureus isolates from wound samples of cattle S. No. 1. Gene cap5K 2. cap8K Primer sequence (5´ to 3´) F-5’-GTCAAAGATTATGTGATGCTACT GAG-3’ R-5’-ACTTCGAATATAAACTTGAATCAA TGTTATACAG-3’ F-5’-GCCTTATGTTAGGTGATAAACC-3’ R-5’-GGAAAAACACTATCATAGCAGG-3’ Size (bp) 361 Annealing Temp. (°C) 55 173 55 Table.2 Capsular typing by PCR targeting cap5K and cap8K genes in S. aureus isolates from wound samples of cattle S. No. Type Isolate numbers 1. 2. Cap5K Cap8K 3 Both Total isolates ( per cent) VS6, VS15, VS19, VS24, VS25, VS27 6(20 per cent) VS1, VS5, VS8, VS9, VS10, VS11, 12(40 per cent) VS13, VS14, VS16, VS20, VS22, VS23 VS4, VS21, VS28 3(10 per cent) 4 Non typable VS2, VS3, VS7, VS12, VS17, VS18, 9(30 per cent) VS26, VS29, VS30 124 Amplicon size(bp) 361 bp 173 bp 361 bp, 173 bp - Int.J.Curr.Microbiol.App.Sci (2021) 10(02): 122-128 Fig.1 PCR amplicons of 23S rRNA ribotyping of S. aureus isolates M- Molecular marker (250bp) Fig.2 PCR amplicons of cap5K and cap8k genes of S. aureus isolates M- Molecular marker (100bp) However, a variable percentage of isolates were detected to possess one or both of the genes which corroborated the earlier observations of Ikawaty et al., (2010), Proietti et al., (2010), Khichar and Kataria (2014) and Salimena et al., (2016). isolates from bovine mastitis positive for cap8 gene and 33.6 per cent isolates positive for cap5 gene. On the contrary, a higher frequency of cap5 gene was detected by other scientists. Salimena et al., (2016) recorded cap5 gene in 80 per cent and cap8 gene in 20 per cent isolates. Khichar et al., (2016) demonstrated that among 18 (90 per cent) typable S. aureus isolates, 11 (55 per cent) were found to harbour cap5K gene and seven isolates (35 per cent) carried cap8K gene, while two isolates (10 per cent) were non-typable for either of the 2 genes. In our study, we recorded higher frequency of cap8K (40 per cent) as compared to cap5K (20 per cent) gene which is in complete agreement to observations of Ikawaty et al., (2010) who detected cap8 gene in 96.05 per cent isolates and cap5 gene in 3.94 per cent isolates from clinical mastitic case of bovine. Ote et al., (2011) recorded 68.6 per cent 125 Int.J.Curr.Microbiol.App.Sci (2021) 10(02): 122-128 In conclusion, there was considerable recovery of S. aureus from wound samples of cattle and all the isolates were typable using 23S rRNA ribotyping and could be identified with high specificity. An overall high prevalence of virulence genes cap5K and cap8K was observed. Hence pathogenic strains were recovered from wound samples of cattle in the present study. C., and Wolz, C. 2019. Function and regulation of Staphylococcus aureus wall teichoic acids and capsular polysaccharides. International Journal of Medical Microbiology, 309(6), 151333. Khichar, V., and Kataria, A.K. 2014. Capsular genotyping (cap5k and cap8k) of Staphylococcus aureus isolates from cattle with clinical mastitis. Human and Veterinary Medicine International Journal of the Bioflux society, 6(1): 3033. Khichar, V., Ruhil, S., Choudhary, V., Khyalia, M.K. and Kataria, A.K. 2016. 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Staphylococcus aureus persistence properties associated with bovine mastitis and alternative therapeutic modalities. Journal of Applied Microbiology, 129(5): 1102-1119. How to cite this article: Veenu Singhal, Taruna Bhati, Pankaj Dhakarwal, Sudesh Sharma, Mahaender Milind and Shringi, B. N. 2021. Capsular Typing of Cap5k and Cap8k Genes in S. aureus Isolates from Wound Samples of Cattle. Int.J.Curr.Microbiol.App.Sci. 10(02): 122-128. doi: https://doi.org/10.20546/ijcmas.2021.1002.015 128