Coagulase gene polymorphism of Staphylococcus aureus isolated from clinical and sub-clinical bovine mastitis in Isfahan and Chaharmahal va Bakhtiari provinces of Iran.

Mastitis is a common disease in dairy cattle and is an inflammatory response of the breast tissue to bacterial attack to this tissue. Mastitis causes considerable loss to the dairy industry, among the several bacterial pathogens that can cause mastitis; Staphylococcus aureus is probably the most lethal agent because it causes chronic and deep infection in the mammary glands that is extremely difficult to cure. Several virulence factors including coagulase gene are produced by S. aureus and may contribute to its pathogenicity. This study was conducted to investigate the coagulase gene polymorphism of S. aureus isolated from clinical and sub-clinical bovine mastitis milk samples in Isfahan and Chaharmahal va Bakhtiari provinces of Iran. Amplification of the coagulase gene from 86 S. aureus strains isolates by specific primers showed 31 specimens contained 970 bp fragment, and 11 strains contained 730 bp fragment relevant to coa gene (coagulase) in PCR. After enzymatic digestion with AluI, 31 specimens contained three bands: 320, 490, and 160 bp (genotype I) and 11 specimens contained two bands: 490 and 240 bp (genotype VIII) in the RFLP.

Introduction

Bovine mastitis is a major disease that affects the dairy industry, and Staphylococcus aureus is one of the most frequently isolated pathogens from both sub-clinical and chronic infections (Watts 1988). Currently used comprehensive mastitis control protocols have decreased the incidence, but S. aureus still remains one of the most significant organisms associated with clinical and sub-clinical bovine mastitis, not only in the USA but worldwide. A better understanding of the epidemiology of S. aureus is needed for the improvement of current mastitis control protocols. In the past decade, numerous molecular techniques have been developed and used for the identification and comparison of S. aureus isolates in epidemiological studies. Among these methods, coagulase gene typing has proven to be a simple and effective means to identify coagulase-positive S. aureus isolates from both human and animal sources (Aarestrup et al. 1995; Goh et al. 1992; Hookey et al. 1998; Kobayashi et al. 1995; Lawrence et al. 1996; Nada et al. 1996; Schwarzkopf and Karch 1994; Su et al. 1999; Tenover et al. 1994).

In Iran, little information is available on the genetic diversity of S. aureus isolated from cows with clinical and sub-clinical bovine mastitis. In this study, coa gene polymorphism was used for typing and differentiation of S. aureus strains isolated from bovine mastitic milk samples from two different provinces in Iran.

Materials and methods

Bacterial isolates

A total of 86 S. aureus isolates from Chaharmahal va Bakhtiari (n = 30) and Isfahan (n = 56) were isolated from 360 clinical and sub-clinical bovine mastitic milk samples (140 samples from Chaharmahal va Bakhtiari and 220 samples from Isfahan). Milk samples were inoculated onto blood agar base (Merck) supplemented with 5% defibrinated sheep blood. Isolates were identified by conventional methods, including Gram staining, colony morphology, hemolysis, tests for catalase, clumping factor, DNase, acetoin, and anaerobic fermentation of mannitol.

Extraction and purification of DNA

Bacterial DNA extraction was carried out according to Rodrigues da Silva and da Silva (2005). Bacterial cell lysate was prepared from 0.5 ml of overnight trypticase soy broth cultures. After centrifugation at 12,000×g for 10 min, bacterial pellets were washed with 500 μl of Tris–hydrochloride–ethylene diamine tetra acetic acid (TE) buffer (10 mM Tris–HCl, pH 7.5, and 1 mM EDTA) and centrifuged again. The pellets were re-suspended in 200 μl of TE buffer (pH 7.5) with 15 U of lysostaphin (2 mg, Sigma) per milliliter and incubated at 37°C for 1 h. Next, 15 μl of proteinase K (20 mg/ml; Fermentas) was added, and the suspension was incubated at 56°C for 1 h. The suspension was then heated at 95°C for 15 min to inactivate the proteinase K. An equal volume of phenol–chloroform was added and the mixture centrifuged at 12,000×g for 10 min. The upper phase was carefully transferred into another Eppendorf tube and mixed with two volumes of 95% ethanol and stored overnight at –20°C. The mixture was then centrifuged at 12,000×g for 5 min. The DNA pellet was washed with ice-cold 70% ethanol, re-centrifuged, and dried by tube inversion. The DNA was suspended in 100 μl of sterile TE, quantified in a spectrophotometer (at 260 nm), and kept frozen at –20°C.

Polymerase chain reaction (PCR) amplification

PCR was performed in a 50-μl reaction mixture containing 2 μl of template DNA (approximately 500 ng/μl), 5 μl of 10 × PCR buffer (750 mM Tris–HCl (pH 8.8), 200 mM (NH4)2SO4, and 0.1% Tween 20), 200 μM of each of the four deoxynucleotide triphosphates, 1 U of Taq DNA polymerase (Roch Applied Science), and 50 pmol of each primer (COAG2: CGA GAC CAA GAT TCA ACA AG; COAG3-AAA GAA AAC CAC TCA CAT CA). The PCR reaction was performed in a thermocycler (Eppendorf, Mastercycler® 5330, Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) using the following cyclic conditions: initial denaturation at 95°C for 2 min, 30 cycles of 30 s each with denaturation at 95°C, 2 min annealing at 58°C, 4 min extension at 72°C, and a final 7 min extension at 72°C (Goh et al. 1992).

Restriction enzyme digestion

The PCR products were digested with AluI (Fermentas) for restriction analysis. For this aim, 12.5 μl of PCR products was mixed with 10 U of enzyme and 10 × 1.5 μl restriction buffer and then incubated at 37°C overnight (Aslantas et al. 2007).

Agarose gel electrophoresis

The PCR products and the digested fragments were separated in 1% and 3% agarose gel (Fermentas), respectively, with 10 mg/ml aqueous solution of ethidium bromide (Fermentas) and then were photographed under ultraviolet illumination. The 100-bp marker (Fermentas) was used as a size standard for the calculation of the sizes of the coa and AluI-generated coa fragments.

Results

A total of 360 clinical and sub-clinical bovine mastitic milk samples from several major herds in the Chaharmahal va Bakhtiari and Isfahan provinces were used in this study. Eighty-six specimens (23.88%) from these samples were identified as infected with S. aureus in microbiological studies.

The PCR assay was able to detect S. aureus DNA from 86 samples of milk by using primers mentioned in the Materials and methods above. Eighty-six specimens contained 1,250-bp DNA fragment of the 23srRNA for S. aureus.

From the total of 86 bacterial strains isolated in this study, 42 of them contained the coa gene. The results are shown in Fig. 1:

Fig. 1

Agarose gel electrophoresis of coa gene. Line M 1 kb DNA ladder, line 1 negative control, lines 2–4 positive samples with 970-bp fragment; lines 5–7 positive samples with 730-bp fragment

S. aureus strains isolated in this study are relevant to the clinical and sub-clinical mastitis which in the California mastitis test (CMT) test had shown positive reactions. From the total of 86 strains isolated in this study, 20 specimens were 1+ mastitis (in CMT test); 45 specimens were 2+ mastitis, and 21 specimens were 3+ mastitis. The number of coa gene of S. aureus are shown in Table 1.

Table 1

Relationship between severity of mastitis (from the viewpoint of CMT test) and presence of the coa gene in the Staphylococcus aureus strain isolated from the cases of bovine mastitis

Specimens	coa	Degree of CMT
20	7	CMT+
45	12	CMT++
21	23	CMT+++

Restriction fragment length polymorphism (RFLP) was used to determine the polymorphism of the coagulase gene from the 42 strains of S. aureus isolated in this study. Thirty-one strains contained 970 bp, and 11 strains contained 730-bp fragment relevant to coa gene in the PCR. After enzymatic digestion with AluI in the PCR, products indicated that 31 strains contained three bands of 490, 320, and 160 bp and 11 strains contained two bands of 490 and 240 bp. The results are shown in Fig. 2.

Fig. 2

Agarose gel electrophoresis, RFLP of the coa gene S. aureus strains isolated from bovine mastitis milk. Line M 100 bp DNA ladder, line 1 negative control, lines 2–5 genotype I coa gene, lines 6–7 genotype VIII coa gene

Discussion

The production of coagulase is an important feature used worldwide for the identification of S. aureus. The 3′ end of the coa gene contains a series of 81-bp tandem repeats, which differ among S. aureus isolates, both in their number and in the location of AluI restriction sites (Goh et al. 1992). Classification based on the coa gene of S. aureus isolated has been considered a simple and accurate method for molecular typing (Goh et al. 1992; Rodrigues da Silva and da Silva 2005). Raimundo et al. (1999) reported that this technique could be used in epidemiological investigations of S. aureus isolates from bovine mastitis because of its high reproducibility and good discriminatory power, it is the easiest with which to analyze coa gene polymorphism among a large number of bacterial isolates, and it generates multiple distinct polymorphism patterns (Raimundo et al. 1999).

Among the 86 S. aureus strains, 31 strains contained 970 bp fragment, and 11 strains contained 730 bp fragments relevant to the coa gene. Following enzymatic digestion with the AluI, five different models were observed in the RFLP test such that 31 strains of genotype I contained three separate bands of 490, 320, and 160 bp, and 11 strains relevant to genotype VIII contained two bands of 490 and 240 bp (Fig. 2). These results indicate a considerable heterogeneity in the coa gene of the strains isolated in the Chaharmahal va Bakhtiari and Isfahan provinces, and as shown by Su et al. and Aarestrup et al., in each country, a special genotype is dominant (Aarestrup et al. 1995; Su et al. 2000).

Results of similar studies correspond with ours. In two studies performed by Su et al. (1999) and Aarestrup et al. (1995), 83.3% of the isolated strains in the Hatay region were relevant to type I, and 65.4% were relevant to type VIII of coagulase.

A study by Aslantas et al. (2007) from Turkey indicated greater variability of RFLP models of coa gene of the S. aureus strains isolated from bovine mastitis cases.

This variation is relevant to the bacterial pool, hard management, and environmental conditions in each geographical region. In this study, polymorphism model of the coa gene, as individual bands measuring 730 to 970 bp, was observed (Aslantas et al. 2007; Goh et al. 1992; Rodrigues da Silva and da Silva 2005). In samples from Brazil, a double bond from the reproduced products of coa gene in the strains isolated from the bovine mastitis cases has been reported (Rodrigues da Silva and da Silva 2005).

In the study performed by Karahan and Cetinkaya (2007) in Turkey, of the 200 strains of S. aureus isolated from 700 milk samples of cows bovine mastitis milk samples, 16 samples contained coa gene, which were positive following RFLP with the AluI and HinGI enzymes; 23 and 22 bands were observed in the electrophoresis model, respectively.

Saei et al. (2009) in 58 S. aureus strains isolated from 370 bovine mastitis milk samples reported 490 to 850 bp fragment from PCR product after digestion with HaeIII enzyme, 39.66% of the strains were relevant to genotype I, and 24.14% of strains were relevant to genotype VIII coagulase.

As a whole, results obtained from this study indicate that cases of mastitis in cattle husbandries in the Chaharmahal va Bakhtiari and Isfahan provinces in Iran are caused by certain strains of S. aureus mostly carrying a coagulase genotype and/or 1 or 2 genotypes of the coa gene is predominant in them. This information might help better control and managing of cases of mastitis caused by S. aureus.