Development of Simplex-PCR assays for Accurate Identification of Nine Staphylococcal Species at Genus and Species Levels

Year 2018, Volume: 08 Issue: 03, 120 - 127, 15.09.2018

Bibek Ranjan Shome Krithiga Natesan Susweta Das Mitra Nimita Venugopal Bhuvana Mani Feroze Ganaie Rajeswari Shome Habibur Rahman

https://doi.org/10.5799/jmid.458462

Cited By: 2

Abstract

Objective: Species identification of Staphylococcus is the prerequisite for precise assessment of
microbial dynamics including their transmission and pathogenic significance in
the dairy herd environment. The present study aimed to develop simplex PCR
assays for rapid and specific identification of nine different Staphylococcus species.

Methods: Specific
primers targeting sodA gene for S. aureus, S. chromogenes, S. hominis,
S. haemolyticus, S. hyicus; gap gene for S. sciuri, S. auricularis, S. simulans;
and rdr gene for S. epidermidis were designed. The PCR assays were evaluated against
28 ATCC reference strains and 209 composite milk samples. Partial 16S rRNA sequencing was performed to
reconfirm the results.

Results: The
PCR assays allowed species level identification of 348 staphylococcal field
isolates recovered from 209 milk samples. The identification pattern was S. aureus (n=101), S. chromogenes (n=89), S.
epidermidis (n=57), S. sciuri
(n=43), S. haemolyticus (n=34), S. hyicus (n=13), S. hominis (n=5), S.
auricularis (n=3) and S. simulans (n=3). The PCR based species
identification was in 100% concordance with the partial 16S rRNA gene sequencing approach.

Conclusion: The simplex PCR assays can be used as a precise tool
for routine identification of Staphylococcus
species from bovine milk as Staphylococcus
species including coagulase-negative staphylococci is recognized a major cause
of bovine mastitis in different parts of the world including India. J Microbiol Infect Dis 2018; 8(3):120-127

Keywords

16S rRNA gene sequencing, Staphylococcus, Species-specific PCR

References

• 1. Bansal BK, Gupta DK. Economic analysis of bovine mastitis in India and Punjab – A Review. Ind J Dai Sci 2009; 62:337-344. 2. Sumathi BR, Veeregowda BM, Gomes AR. Prevalence and antibiogram profile of bacterial isolates from clinical bovine mastitis. Vet World 2008; 1:237-238. 3. Shome BR, Mitra SD, Bhuvana M, et al. Multiplex PCR assay for species identification of bovine mastitis pathogens. J Appl Microbiol 2011; 111:1349-1356. 4. Pyorala S, Taponen S. Coagulase-negative staphylococci- emerging mastitis pathogens. Vet Microbiol 2009; 134:3-8. 5. Braem G, De Vliegher S, Supre K, Haesebrouck F, Leroy F, De Vuyst L. (GTG)5-PCR fingerprinting for the classification and identification of coagulase-negative Staphylococcus species from bovine milk and teat apices: A comparison of type strains and field isolates. Vet Microbiol 2011; 147:67-74. 6. Zadoks RN, Watts JL. Species identification of coagulase-negative staphylococci: genotyping is superior to phenotyping. Vet Microbiol 2009; 134:20-28. 7. Park JY, Fox LK, Seo KS, et al. Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections. Vet Microbiol 2011; 147:142-148. 8. Rozen S, Skaletsky HJ. Primer-3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (Eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, USA; 2000.p.365-386. 9. Kuang Y, Tani K, Synnott AJ, et al. Characterization of bacterial population of raw milk from bovine mastitis by culture- independent PCR-DGGE method. J Biochem Engg 2009; 45:76–81. 10. Cremonesi P, Pisoni G, Severgnini M, et al. Pathogen detection in milk samples by ligation detection reaction-mediated universal array method. J Dairy Sci 2009; 92:3027-3039. 11. Supre K, Haesebrouck F, Zadoks RN, Vaneechoutte M, Piepers S, De Vliegher S. Some coagulase-negative Staphylococcus species affect udder health more than others. J Dairy Sci 2011; 94:2329-2340. 12. Patteet L, Goossens H, Ieven M. Validation of the MicroScan-96 for the species identification and methicillin susceptibility testing of clinical significant coagulase-negative staphylococci. Eur J Clin Microbiol Infect Dis 2012;31:747–751. 13. Shah MM, Iihara H, Noda M, et al. DnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus. Int J Syst Evol Microbiol 2007;57:25–30. 14. Supre K, De-Vliegher S, Sampimon OC, et al. Use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices. J Dairy Sci 2009; 92:3204–3210. 15. Chakravorty S, Helb D, Burday M, Connell C, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J Microbiol Methods 2007; 69:330-339. 16. Ghebremedhin B, Layer F, Konig W, Konig B. Genetic classification and Distinguishing of Staphylococcus Species Based on Different Partial gap, 16S rRNA, hsp60, rpoB, sodA and tuf gene sequences. J Clin Microbiol 2008; 46:1019–1025. 17. Heikens E, Fleer A, Paauw A, Florijn AC, Fluit AC. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol 2005; 43:2286-2290. 18. Capurro A, Artursson K, Waller KP, Bengtsson B, Ericsson-Unnerstad H, Aspan A. Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. Vet Microbiol 2009; 134:327–333 19. Gurtler V, Stanisich VA. New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiol 1996; 142:3-16. 20. Poyart C, Quesne G, Boumaila C, Trieu-cuot P. Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target. J Clin Microbiol 2001; 39:4296-4301. 21. Becker K, Harmsen D, Mellmann A, et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 2004; 42:4988–4995. 22. Schukken YH, Gonzalez RN, Tikofsky LL, et al. CNS mastitis: Nothing to worry about? Vet Microbiol 2009:134:9–14.