Molecular detection of mecA gene in methicillin-resistant Staphylococcus aureus isolated from dairy mastitis in Nineveh governorate, Iraq
Iraqi Journal of Veterinary Sciences,
2022, Volume 36, Issue 4, Pages 939-943
AbstractStaphylococcus (S.) aureus is universally the leading aetiologic cause of dairy mastitis. Additionally, methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogenic bacterium in veterinary medicine and public health. Sixty-six cattleʼs milk samples were collected randomly from different areas of the Nineveh Province from November 2018 to February 2020. In this study, the classical and molecular biology methods had used to identify the MRSA and detect the target genes. The results revealed that S. aureus was isolated and identified based on classical methods such as catalase, clumping factors, and coagulase test. In addition, the nuc gene was detected in all the positive S. aureus isolates 23 (34.8%), while the mecA gene was detected in 12 (52.2%) MRSA isolates by using polymerase chain reaction (PCR) assay. The present work emerged that the results of classical methods and the PCR technique were similar. MRSA is regarded as a significant causative agent of various types of bovine mastitis in Iraq, and it can to resist all types of beta-lactams. MRSA isolated from different regions in Mosul city. PCR assay is a powerful method for detecting the different genes based on the target sequence of the specific gene.
Mastitis is considered a primary distributed disease in dairy herds and it is a frequent infection in dairy cows and ruminants. Mastitis causes a significant economic loss via direct and indirect costs (1). The direct costs of bovine mastitis are veterinary treatment costs and require more of the labor requirements (2). The indirect cost of clinical and subclinical bovine mastitis is the significant economic losses are reduced milk yield and quality due to mastitis (3). More than 100 types of bacteria have been detected in the mammary glands of cows, but a small number of these microorganisms cause mastitis (4). The etiology microorganisms of bovine mastitis are divided into two groups: the first group is a contagious bacterium, and the second group is an environmental bacterium according to their reservoir, source, and mode of transmission (5). Contagious or environmental bacteria infect the mammary gland because of the transmission of this bacteria from the source of contamination such as a contaminated milking machine, towels, the hands of milkers, bedding, soil, and the feces from the udder of cattle (6). Successful programs are applied to control mastitis based on the cleaning and disinfecting of all the machines, and utensils that direct contact with the surface of the teats and udder (5). Staphylococcus is frequently present in various habitats such as humans, animals, and plants (6). Also, it is considered a highly significant cause that triggers clinical and primarily subclinical mastitis in cattle herds (7). Furthermore, S. aureus is considered an essential food-borne pathogen, which is mainly responsible for the cause of food poisoning cases, and outbreaks worldwide (8).The infected cow may be a primary reservoir and source of contamination of the raw milk with S. aureus (9). S. aureus possesses more than 30 different virulence factors (10). S. aureus can produce different types of exotoxins (11). Contaminated milk and dairy products with S. aureus are seen as the primary sources to trigger food poisoning to consumers (12). S. aureus can resist various types of antibiotics and disinfectants (13). This variation is based on the geographical territories, the genetic characteristics of the isolates, and the type of samples (14). Moreover, the S. aureus isolates showed resistance to all types of β-lactam antibiotics. However, yet are applied to cure of bovine mastitis (15). Methicillin-resistant Staphylococcus aureus (MRSA) was documented in the 1970s and isolated from bovine mastitis (16). MRSA has the gene (mecA), which codes penicillin-binding protein (PBP 2a), which has an alow affinity for β-lactam antibiotics (17). Many reports showed that the MRSA strains had been isolated from animals and humans causing infections, and MRSA has been able to transfer between dairy cows and the person milking them (18). The veterinarians, farm workers, and farm animals are exposed to the potential risk by direct contact with the infected cattle by the presence of MRSA in bovine milk (19). The previous studies declared that MRSA had been isolated from domestic animals such as veal calves (20), poultry (21) , horses (22), and camel (23).
This study aimed to detect S. aureus isolates using the traditional and molecular techniques on a specific gene nuc and investigate methicillin-resistant S. aureus by detecting the specific mecA gene for MRSA.
Materials and methods
Sixty-six samples were collected from the sub-clinical mastitis from November 2018 to February 2020 from different areas of the Nineveh Governorate. Milk samples were obtained after cleaning the teat, ant-dipping the teat, and discarding the first few streams of milk. The milk samples collected from subclinical bovine mastitis were 10 ml. All milk was placed into sterile glass vials. After dipping the teat detergent agents such as 70% alcohol were used to disinfect the teat from bacteria (24). The samples were kept on an icebox and transported to the Research Center and Laboratories, College of Veterinary Medicine, Mosul University. The milk samples were streaked onto Blood media (Lab M limited Topley house, Lancashire, United Kingdom) and Mannitol salt media 7.5% plates (118 g/L) (Lab M limited Topley house, Lancashire, United Kingdom). The culture plates were incubated at 37°C for 24 h.
Isolation and Identification of S. aureus
The typical S. aureus colonies were examined by gram staining and the traditional biochemical methods (catalase and coagulase test), and morphology (25).
All the positive S. aureus isolates were grown on mannitol salt media for 24 h at 37°C. Genomics DNA of S. aureus was isolated based on manufacturer's protocol for Gram-positive bacteria, DNA of S. aureus isolated with the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). The amount of DNA extracted was weighted using Nanodrop (Biodrop, United Kingdom), and the DNA was stored at -20°C .
Based on the PCR assay, the specific-species nuc gene of S. aureus and the mecA gene of MRSA was detected. PCR interaction was implemented by the 200μl tube (Biozym, Oldenhorf, Germany) together the whole volume mishmash was 25 μL, consisting of 1 μL of each F and R primer (each ten pmol/μL), (Eurofins Genomics, Ebersberg, Germany) (Table 1). The molecular weight of the nuc gene is 166 bp (27), while the molecular weight of the mecA gene is 533 (28), 12.5 μL of 2×Go Taq Green Master Mix, and eight μL of double-distilled water (Promega). Eventually, a 2.5 μL DNA template of S. aureus or MRSAwas added to each 200 μl tube. The amplicons were determined using gel electrophoresis with a 100 bp ladder in 2% agarose gel (Peqlab, Erlangen, Germany).
Table 1: PCR programs and Primers in detecting of nuc and mecA of S. aureus and MRSA
Sequence (5- 3)
Amplicon Size [bp]
PCR program: I: 35 times (94°C - 30s, 55°C - 30s, 72°C - 30s), II: 35 times (94°C - 30s, 54°C - 30s, 72°C - 30s)
The number of samples used in the current study collected from different regions in the Nineveh Governorate was 66. Regarding the guidelines of the National Mastitis Council (NMC) 2017 (29), S. aureus isolated from the milk samples was 23 (34.8%). All the positive S. aureus isolates had appeared the positive results with Gram, catalase, and coagulase tests. In addition, S. aureus was detected as round, golden-yellow clusters on mannitol salt media and hemolysis on the blood media. Furthermore, the study results demonstrated the nuc gene, which was detected in 23 (34.8%) of S. aureus (Figure 1). PCR assay was similar to the result of the phenotypic determination tests. In terms of the presence and absence, the mecA gene was seen in 12 (52.2%) of the 23 S. aureus isolates (Figure 2).
Figure 1: 2% of Agarose gel electrophoresis shows the product size of the nuc gene of the S. aureus isolates (166 bp).
Figure 2: 2% of Agarose gel electrophoresis shows an amplicon of the mecA gene product of the methicillin-resistant S. aureus isolates (533 bp).
For several decades, S. aureus has been emergency pathogenic bacteria in human and animal importance fields. The current study used classical and molecular biological methods to isolate and identify MRSA in bovine milk. This study showed that the prevalence rate of S. aureus isolated from milk samples was 23 (34.8%). The result of the present study was nearest to the previous studies, which reported that the prevalence rate of S. aureus isolated from dairy mastitis milk was 35.9% and 36.3% in Egypt (30,31). While the result of this study was lower than other studies that reported the prevalence rate of S. aureus isolated from bovine mastitis milk was 74% in Egypt, 70% in Hungary, 43% in the USA (32-34), (33). In addition, the result of the present study was higher than other studies that reported the spread rate of S. aureus isolated from mastitis milk was 21.8% in Germany (35), and 5.6% in Korea (36). The prevalence rate of S. aureus is different based on the geographical distribution and the sanitary conditions in dairy farms, dairy plants. Many previous studies appeared that the S. aureus has isolated from skin of udder, bedding, workers' hands, insects, and dust that played an essential role in the transmission of S. aureus among cattle and contaminated milk (37). In addition, the isolation of S. aureus from the different organs of infected cows such as vagina, muzzle, skin wounds participates in the transmission of S. aureus between body sited in one hand and the environments on the other hand, as well as dairy herds, moreover, from cows to their calves by way of the ventilation or by feeding of milk having S. aureus (38).
Furthermore, the prevalence rate of MRSA isolated from bovine mastitis milk was 12 (52.2%). The result of the current study was higher than other studies that reported the spread rate of MRSA isolate was 28.2% in Egypt (39), 15% in Belgium (40), and 0.18% in Korea (41). MRSA is colonized and proliferated in the udder leading to cause the subclinical and clinical mastitis, which causes enormous economic loss (42). MRSA is transmitted from infected cattle to the calf (20). MRSA has isolated from humans, which contact domestic animals such as veterinarians, workers, owners (19). The molecular biology techniques were used to detect the mecA gene in S. aureus isolate, which was encoded for the synthesis to synthesize Penicillin-binding protein based on the (PCR) assay to detect and identify the target gene in the isolates (43).
- aureus is regarded as a significant pathogenic microorganism to humans and ruminants. It can cause mastitis in mammals. MRSA possesses the mecA gene, causing problems to humans and animals during treatment because MRSA can resist the different types of beta-lactams antibiotics. PCR is an essential method for identifying the bacteria isolates and detecting the specific gene, and PCR is faster, simpler, and more accurate.
The author is grateful to the University of Mosul /College of Veterinary Medicine for all the facilities to achieve this study.
Conflict of interest
There is no conflict of interest.
- The isolation of MRSAfrom dairy mastitis milk.
- Recognition the specific - species aureus nuc gene.
- Detection the specific - species mecA gene in MRSA.
- PCR assay was used as a more modern and accurate technique.
- Data revealed the spread of MRSA in dairy mastitis milk, and the PCR technique has excellent value for the detection of MRSA.
- Kossaibati MA, Esslemont RJ. The costs of production diseases in dairy herds in England. Vet J. 1997;154(1):41-51.DOI: 1016/s1090-0233(05)80004-8
- Lescourret F, Coulon JB. Modeling the impact of mastitis on milk production by dairy cows. J Dairy Sci. 1994;77(8):2289-301. DOI: 3168/jds.S0022-0302(94)77172-1
- Seegers H, Fourichon C, Beaudeau F. Production effects related to mastitis and mastitis economics in dairy cattle herds. Vet Res. 2003;34(5):475-91. DOI: 1051/vetres:2003027
- Owens WE, Watts JL. Antimicrobial susceptibility and beta-lactamase testing of staphylococci isolated from dairy herds. J Dairy Sci. 1988;71(7):1934-9. DOI: 3168/jds.S0022-0302(88)79763-5
- Ruegg PL. Managing cows, milking and environment to minimize mastitis. Adv Dairy Technol. 2012;24:351-9. DOI: 4190/jjlac.5.210
- Hogeveen H, Huijps K, Lam TJ. Economic aspects of mastitis: new developments. N Z Vet J. 2011;59(1):16-23. DOI: 1080/00480169.2011.547165
- Taponen S, Pyorala S. Coagulase-negative staphylococci as a cause of bovine mastitis- not so different from Staphylococcus aureus? Vet. Microbiol. 2009;134(1-2):29-36. DOI: 1016/j.vetmic.2008.09.011
- Jacques-Antoine Hennekinne , Annick Ostyn, Florence Guillier, Sabine Herbin, Anne-Laure Prufer, Dragacci S. How Should Staphylococcal Food Poisoning Outbreaks Be Characterized. Toxins. 2010;2(8):2106-16. DOI: 3390/toxins2082106
- Jayarao BM, Pillai SR, Sawant AA, Wolfgang DR, Hegde NV. Guidelines for monitoring bulk tank milk somatic cell and bacterial counts. J Dairy Sci. 2004;87(10):3561-73. DOI: 3168/jds.S0022-0302(04)73493-1
- Ote I, Taminiau B, Duprez JN, Dizier I, Mainil JG. Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis. Vet. Microbiol. 2011;153(3-4):285-92. DOI: 1016/j.vetmic.2011.05.042
- Francis JS, Doherty MC, Lopatin U, Johnston CP, Sinha G, Ross T. Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin Infect Dis. 2005;40(1):100-7. DOI: 1086/427148
- Silva NCC, Guimar~aes FF, Manzi MP, ́nior AFJ, ́mez-Sanz EG, P.G omez. Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows. Lett. Appl. Microbiol. 2014;59:665-9. DOI: 1111/lam.12329
- Bjorland J, Sunde M, Waage S. Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus. Clin. Microbiol. Rev. 2001;39(11):3999-4004. DOI: 1128/JCM.39.11.3999-4004.2001
- Vintov J, Aarestrup FM, Zinn CE, Olsen JE. Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries. Vet. Microbiol. 2003;95(1-2):133-47. DOI: 1016/s0378-1135(03)00156-1
- Sawant AA, Sordillo LM, Jayarao BM. A survey on antibiotic usage in dairy herds in Pennsylvania. J. Dairy Sci. 2005;88(8):2991-9. DOI: 3168/jds.S0022-0302(05)72979-9
- Devriese LA, Van Damme LR, Fameree L. Methicillin (cloxacillin)-resistant Staphylococcus aureus strains isolated from bovine mastitis cases. J. Vet. Med. Series B. 1972;19(7):598-605. DOI: 1111/j.1439-0450.1972.tb00439.x
- Utsui Y, Yokota T. Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 1985;28(3):397-403. DOI: 1128/AAC.28.3.397
- Garcia-Alvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 2011;11(8):595-603. DOI: 1016/S1473-3099(11)70126-8
- Lee JH. Methicillin (Oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans. Appl. Environ. Microbiol. 2003;69(11):6489-94. DOI: 1128/AEM.69.11.6489-6494.2003
- Graveland H, Wagenaar JA, Heesterbeek H, Mevius D, van Duijkeren E, Heederik D. Methicillin-resistant Staphylococcus aureus ST398 in veal calf farming: human MRSA carriage related with animal antimicrobial usage and farm hygiene. PLoS One. 2010;5(6):e10990. DOI: 1371/journal.pone.0010990
- Sheet O.H., S.A. Hussien, Alchalaby AY. Detection of methicillin-resistant Staphylococcus aureus from broiler carcasses in Mosul city. Iraqi J. Vet. Sci. 2021;35(3):489-93. DOI: 33899/ijvs.2020.127052.1451
- Van den Eede A, Martens A, Lipinska U, Struelens M, Deplano A, Denis O. High occurrence of methicillin-resistant Staphylococcus aureus ST398 in equine nasal samples. Vet. Microbiol. 2009;133(1-2):138-44.DOI: 1016/j. vetmic. 2008.06.021
- Sheet O.H., Dh. M. Jwher, R.A. Al-Sanjary, Alajami AD. Direct Detection of Staphylococcus aureus in camel milk in the Nineveh governorate by using the PCR technique. Iraqi J. Vet. Sci. 2021;35(4):669-72.DOI: 33899/ijvs.2020.127725.1524
- Hatem ME, Arab RH, Ata SN, El-Moez SIA, Khairy EA, EA F. Bacterial Abscessationin in sheep and goat in Giza governorate with full antibiogram screening. Glob Vet. 2013;10(4):372-81. DOI: 5829/idosi.gv.2013.10.4.72112
- Quinn PJ, Markey BK, Carter ME, Donnelly WJC, Leonard FC, Maguire D. Veterinary Microbiology and Microbial Diseases. 1st ed Blackwell Science Ltd, Chichester, West Sussex, UK 2002. [available at]
- Strube ML, Hansen JE, Rasmussen S, Pedersen K. A detailed investigation of the porcine skin and nose microbiome using universal and Staphylococcus specific primers. Sci. Rep. 2018;8(1):12751. DOI: 1038/s41598-018-30689-y
- Graber HU, Casey MG, Naskova J, Steiner A, Schaeren W. Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk. J Dairy Sci. 2007;90(10):4661-9. DOI: 3168/jds.2006-902
- Kazuhisa M, Wakio M, Koji W, Etuo N, Hiroshi T, Watanabe S. Identification of Methicillin-Resistant Strains of Staphylococci by Polymerase Chain Reaction. J. Clin. Microbiol. 1991;29(10):2240-4. DOI: [available at]
- Laboratory handbook on bovine mastitis, 3rd edn. National Mastitis Council, New Prague. 2017. [available at]
- Algammal AM, Hetta HF, Elkelish A, Alkhalifah DHH, Hozzein WN, Batiha GE. Methicillin-Resistant Staphylococcus aureus (MRSA): One Health Perspective Approach to the Bacterium Epidemiology, Virulence Factors, Antibiotic-Resistance, and Zoonotic Impact. Infect Drug Resist. 2020;13:3255-65. DOI: 2147/IDR.S272733
- Algammal AM, Enany ME, El-Tarabili RM, Ghobashy MOI, Helmy YA. Prevalence, Antimicrobial Resistance Profiles, Virulence and Enterotoxins-Determinant Genes of MRSA Isolated from Subclinical Bovine Mastitis in Egypt. J. Pathog. 2020;9(5). DOI: 3390/pathogens9050362
- Hala Ali, Nagla Koraney, Esraa Hefny, Samah Ali, Shymaa Abdel Mawgoud, Eltokhy E. Phenotypic and genotypic profiling of Methicillin-resistant Staphylococcus aureus isolates from human and bovine milk. Egypt. J. Agric. Res. 2021;99(2):190-6. DOI: 21608/EJAR.2021.79959.1115
- Peles F, Wagner M, Varga L, Hein I, Rieck P, Gutser K. Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary. Int J Food Microbiol. 2007;118(2):186-93. DOI: 1016/j.ijfoodmicro.2007.07.010
- Lombard J, Slyke T V, Welcome F, Schukken Y, C. K. Prevalence of contagious mastitis pathogens on US dairy operation. NMC 47th Annual Meeting Proceedings, New Orleans LA. 2008:170-1. [available at]
- Tenhagen BA, Koster G, Wallmann J, Heuwieser W. Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany. J Dairy Sci. 2006;89(7):2542-51. DOI: 3168/jds.S0022-0302(06)72330-X
- Moon JS, Lee AR, Kang HM, Lee ES, Kim MN, Paik YH. Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea. J Dairy Sci. 2007;90(3):1176-85. DOI: 3168/jds.S0022-0302(07)71604-1
- Piccinini R, Tassi R, Dapra V, Pilla R, Fenner J, Carter B. Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis. J Dairy Res. 2012;79(2):249-55. DOI: 1017/S002202991200009X
- Mork T, Kvitle B, Jorgensen HJ. Reservoirs of Staphylococcus aureus in meat sheep and dairy cattle. Vet Microbiol. 2012;155(1):81-7. DOI: 1016 /j. vetmic. 2011.08.010
- Enany ME, Younes S, Al-Gammal A M, Salem M, A. Prevalence of coagulase (coa) gene and mec A gene of aureus isolated from bovine clinical mastitis. Suez Canal Vet Med J. 2013;18:149-57. [available at]
- Rene S Hendriksen, Dik J Mevius, Andreas Schroeter, Christopher Teale, Danièle Meunier, Patrick Butaye. Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002-2004. Acta Vet. Scand. 2008;50:28. DOI: 1186/1751-0147-50-28
- Kwon NH, Park KT, Moon JS, Jung WK, Kim SH, Kim JM. Staphylococcal cassette chromosome mec (SCCmec) characterization and molecular analysis for methicillin-resistant Staphylococcus aureus and novel SCCmec subtype IVg isolated from bovine milk in Korea. J Antimicrob Chemother. 2005;56(4):624-32. DOI: 1093/jac/dki306
- Tenhagen B-A, Vossenkuhl B, Käsbohrer A, Alt K, Kraushaar B, Guerra B. Methicillin-resistant Staphylococcus aureus in cattle food chains - prevalence, diversity, and antimicrobial resistance in Germany. J Anim Sci. 2014;92(6):2741-51. DOI: 2527/jas.2014-7665
- Fournier C, Kuhnert P, Frey J, Miserez R, Kirchhofer M, Kaufmann T. Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome. Res Vet Sci. 2008;85(3):439-48. DOI: 1016/j.rvsc.2008.01.010
- Article View: 73
- PDF Download: 7