Standardization of multiplex PCR for identification of ten staphylococci species and their prevalence in bovine mastitis

TAWHEED AHMAD SHAFI; BALJINDER KUMAR BANSAL; SHUKRITI SHARMA; NARINDER SINGH SHARMA

doi:10.56093/ijans.v92i1.120909

Authors

TAWHEED AHMAD SHAFI Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India
BALJINDER KUMAR BANSAL Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India
SHUKRITI SHARMA Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India
NARINDER SINGH SHARMA Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India

https://doi.org/10.56093/ijans.v92i1.120909

Keywords:

Bovine mastitis, Multiplex PCR, Non-aureus staphylococci

Abstract

To assess the clinical impact, epidemiology of intra-mammary infections, accurate diagnosis, and choosingappropriate antimicrobial therapy, the staphylococci group should be studied correctly up to the species level. Inthis study, a multiplex PCR (mPCR) was developed to identify 10 important Staphylococcus species that wasapplied to study the prevalence of staphylococci species in bovine mastitis from five agro-climatic regions ofPunjab, India. A two tube mPCR assay consisted of 5 species each, with one reaction including primer pairs ofS. epidermidis, S. hemolyticus, S. hominis, S. warneri and S. saprophyticus, and another reaction consisted primerpairs of S. aureus, S. intermedius, S. chromogenes, S. sciuri and S. capitis was standardized. Prevalence studyrevealed S. aureus (35.76%) as most prevalent species among all staphylococci followed by S. chromogenes (16.56%),S. haemolyticus (15.23%), S. epidermidis (5.30%), S. sciuri (1.99%), S. capitis (0.66%), S. hominis (0.66%),S. saprophyticus (0.66%) and rest were other staphylococci (23.18%). The mPCR developed in this study could beused for identification of staphylococci of various origins including those from human specimens. The high prevalenceof S. chromogenes and S. haemolyticus in the present study suggest emergence of these non-aureus staphylococciin bovine mastitis in Punjab (India).

Downloads

Download data is not yet available.

References

Becker K, Heilmann C and Peters G. 2014. Coagulase-negative staphylococci. Clinical Microbiology Reviews 27(4): 870–926. Bes M, Guerin-Faublee V, Meugnier H, Etienne J and Freney J. 2000. Improvement of the identification of staphylococci isolated from bovine mammary infections using molecular methods. Veterinary Microbiology71: 287–94. DOI: https://doi.org/10.1016/S0378-1135(99)00163-7

Braem G, De Vliegher S, Supré K, Haesebrouck F, Leroy F and De Vuyst L. 2011. (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. Veterinary Microbiology 147: 67–74. DOI: https://doi.org/10.1016/j.vetmic.2010.05.044

Brakstad O D D G, Aasbakk K and Maeland J A. 1992. Detection of Staphylococcus aureus_nuc pcr und primer. Journal of Clinical Microbiology 30(7): 1654–60. DOI: https://doi.org/10.1128/jcm.30.7.1654-1660.1992

Cheng J, Qu W, Barkema H W, Nobrega D B, Gao J, Liu G, De Buck J, Kastelic J P, Sun H and Han B. 2019. Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds. Journal of Dairy Science 102(3): 2416–26. DOI: https://doi.org/10.3168/jds.2018-15135

Condas L A Z, De Buck J, Nobrega D B, Carson D A, Roy J P, Keefe G P, DeVries T J, Middleton J R, Dufour S and Barkema H W. 2017. Distribution of non-aureus staphylococci species in udder quarters with low and high somatic cell count, and clinical mastitis. Journal of Dairy Science 100: 5613–27. DOI: https://doi.org/10.3168/jds.2016-12479

Cunha M L, Sinzato Y K and Silveira L V A. 2004. Comparison of methods for the identification of coagulase-negative staphylococci. Memorias do Institutes Oswaldo Cruz 99: 855– 860. DOI: https://doi.org/10.1590/S0074-02762004000800012

Fry P R, Middleton J R, Dufour S, Perry J, Scholl D and DohooI. 2014. Association of coagulase-negative staphylococcal species, mammary quarter milk somatic cell count, and persistence of intramammary infection in dairy cattle. Journal of Dairy Science 97(8): 4876–85. DOI: https://doi.org/10.3168/jds.2013-7657

Gillespie B E, Headrick S I, Boonyayatra S and Oliver S P. Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds. Veterinary Microbiology 134(1–2): 65–72. DOI: https://doi.org/10.1016/j.vetmic.2008.09.007

Hauschild T and Stepanovic S. 2008. Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene. Journal of Clinical Microbiology 46(12): 3875–79. DOI: https://doi.org/10.1128/JCM.00810-08

Hirotaki S, Sasaki T, Kuwahara-Arai K and Hiramatsu K. 2011. Rapid and accurate identification of human-associated staphylococci by use of multiplex PCR. Journal of Clinical Microbiology 49(10): 3627–31. DOI: https://doi.org/10.1128/JCM.00488-11

Hogan J S, Gonzales R N, Harmon R J, Nickerson S P, Oliver J W, Pankey J W and Smith K L. 2009. Laboratory Handbook on Bovine Mastitis. National Mastitis Council, Madison, WI.

Luini M, Cremonesi P, Magro G, Bianchini V, Minozzi G, Castiglioni B and Piccinini R. 2015. Methicillin-resistant Staphylococcus aureus (MRSA) is associated with low within- herd prevalence of intra-mammary infections in dairy cows: Genotyping of isolates. Veterinary Microbiology 178(3–4): 270–74. DOI: https://doi.org/10.1016/j.vetmic.2015.05.010

MacDonald J S. 1997. Streptococcal and staphylococcal mastitis. Journal of American Veterinary Medical Association 170: 1157.

Mir A Q, Bansal B K and Gupta D K. 2014. Subclinical mastitis in machine milked dairy farms in Punjab: Prevalence, distribution of bacteria and current antibiogram. Veterinary World 7(5): 291–94. DOI: https://doi.org/10.14202/vetworld.2014.291-294

Morot-Bizot S C, Talon R and Leroy S. 2004. Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food. Journal of Applied Microbiology 97(5): 1087–94. DOI: https://doi.org/10.1111/j.1365-2672.2004.02399.x

Persson Waller K, Aspán A, Nyman A, Persson Y and Grönlund Andersson U. 2011. CNS species and antimicrobial resistance in clinical and subclinical bovine mastitis. Veterinary Microbiology 152(1–2): 112–16. DOI: https://doi.org/10.1016/j.vetmic.2011.04.006

Piepers S, Schukken Y H, Passchyn P and De Vliegher S. 2013. The effect of intramammary infection with coagulase-negative staphylococci in early lactating heifers on milk yield throughout first lactation revisited. Journal of Dairy Science 96(8): 5095–5105. DOI: https://doi.org/10.3168/jds.2013-6644

Qu Y, Zhao H, Nobrega D B, Cobo E R, Han B, Zhao Z, Li S, Li M, Barkema H W and Gao J. 2019. Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis. Journal of Dairy Science 102(2): 1571–83. DOI: https://doi.org/10.3168/jds.2018-15136

Sampimon O C, Barkema H W, Berends I M G A, Sol J and Lam T J G M. 2009. Prevalence and herd-level risk factors for intramammary infection with coagulase-negative staphylococci in Dutch dairy herds. Veterinary Microbiology 134(1–2): 37–44. DOI: https://doi.org/10.1016/j.vetmic.2008.09.010

Sasaki T, Tsubakishita S, Tanaka Y, Sakusabe A, Ohtsuka M, Hirotaki S, Kawakami T, Fukata T and Hiramatsu K. 2010. Multiplex-PCR method for species identification of coagulase- positive staphylococci. Journal of Clinical Microbiology 48(3): 765–69. DOI: https://doi.org/10.1128/JCM.01232-09

Shome B R, Mitra S D, Bhuvana M, Krithiga N, Velu D, Shome R, Isloor S, Barbuddhe SB and Rahman H. 2011. Multiplex PCR assay for species identification of bovine mastitis pathogens. Journal of Applied Microbiology 111(6): 1349–56. DOI: https://doi.org/10.1111/j.1365-2672.2011.05169.x

Srednik M E, Grieben M A, Bentancor A and Gentilini E R. 2015. Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of ß-lactam resistance. Journal of Infection in Developing Countries. 9(9): 1022–27. DOI: https://doi.org/10.3855/jidc.5871

Strommenger B, Kettlitz C, Werner G and Witte W. 2003. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. Journal of Clinical Microbiology 41(9): 4089–94. DOI: https://doi.org/10.1128/JCM.41.9.4089-4094.2003

Thorberg B M, Danielsson-Tham M L, Emanuelson U and Persson Waller K. 2009. Bovine subclinical mastitis caused by different types of coagulase-negative staphylococci. Journal of Dairy Science 92(10): 4962–70. DOI: https://doi.org/10.3168/jds.2009-2184

Traversari J, Van Den Borne B H P, Dolder C, Thomann A, Perreten V and Bodmer M. 2019. Non-aureus staphylococci species in the teat canal and milk in four commercial swiss dairy herds. Frontiers in Veterinary Science 6(Jun): 11–14. DOI: https://doi.org/10.3389/fvets.2019.00186

Veerle P, Van Coillie E, Verbist B, Supré K, Braem G, Van Nuffel A, De Vuyst L, Heyndrickx M and De Vliegher S. 2011. Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds. Journal of Dairy Science 94: 2933–44. DOI: https://doi.org/10.3168/jds.2010-3956