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. 2020 May 9;9(5):362. doi: 10.3390/pathogens9050362

Prevalence, Antimicrobial Resistance Profiles, Virulence and Enterotoxins-Determinant Genes of MRSA Isolated from Subclinical Bovine Mastitis in Egypt

Abdelazeem M Algammal 1,*, Mohamed E Enany 1, Reham M El-Tarabili 1, Madeha O I Ghobashy 2,3, Yosra A Helmy 4,5,*
PMCID: PMC7281566  PMID: 32397408

Abstract

Subclinical mastitis caused by Staphylococcus aureus has worldwide public health significance. Here, we aimed to determine the prevalence of S. aureus, antimicrobial resistance profiles, and the virulence and enterotoxins determinant genes of MRSA strains that caused subclinical bovine mastitis. Milk samples were collected from 120 lactating animals (50 buffaloes and 70 dairy cattle) from different farms located in Ismailia Province (Egypt). The collected samples were investigated for subclinical mastitis using a California mastitis test. The total prevalence of S. aureus was 35.9% (84/234) with 36.3% (53/146) in cattle and 31% (31/88) in buffaloes. Antimicrobial susceptibility testing showed that 35.7% (30/84) of the isolated strains were resistant to cefoxitin, defined as methicillin-resistant S. aureus (MRSA), with 37.7% (20/53) in cattle and 32.2% (10/31) in buffaloes. Using PCR, 100% of the tested strains harbored coa and mecA genes, while 86.6% were positive for spa gene, with remarkable gene size polymorphism. Additionally, 10% of the tested strains contained the pvl gene. Further, using multiplex PCR, 26.6% of the tested samples had sea gene, two strains had sec gene and only one strain had sea and sec genes. The seb and sed genes were absent in the tested strains. In conclusion, mecA, coa and spa virulence genes were widely distributed in MRSA strains isolated from bovine milk, whereas the sea gene was the most predominant enterotoxin gene. Notably, this is the first report that emphasizes the prevalence of pvl gene of MRSA isolated from bovine milk in Egypt.

Keywords: MRSA, subclinical bovine mastitis, prevalence, antimicrobial resistance, virulence genes, enterotoxins genes

1. Introduction

Staphylococcus aureus is a significant public health bacterial pathogen, causing mastitis in dairy animals including cattle, buffalo, sheep and goats [1]. S. aureus mastitis and its produced toxins lead to great economic losses in dairy farms due to: (1) reduction in the milk production, (2) alteration in the composition and quality of the produced milk, (3) the need to discard the produced milk, (4) early culling of infected animals, and (5) high cost of treatment and control [2]. Resistance of S. aureus to several antimicrobials complicates the treatment of these pathogenic bacteria, which is considered an increasing challenge. Methicillin-resistant S. aureus (MRSA) strains can cause nosocomial infections and consequently high mortality in humans [3]. In Egypt, there was high prevalence of resistance among S. aureus in bovine species to antimicrobial agents such as β-lactams, which are used to treat mastitis [4,5]. This high prevalence is caused by the uncontrolled widespread use of antibiotics. Therefore, MRSA has high clinical significance and poses a potential public health hazard.

The ability of S. aureus to cause infections is due to virulence factors, such as the secretion of several toxins and presence of cell wall adhesion proteins. Thus the bacteria can survive in the udder, causing chronic inflammation [6]. Coagulase is one of the S. aureus virulence factors that stimulates prothrombin, resulting in blood clotting [7,8]. Additionally, protein A is a cell wall component that hinders phagocytosis by neutrophils and contains the Fc-portion, X-segment, and C-terminal portion. This X-region of spa gene usually undergoes repetition (up to 24 repeats) and differs from one strain to another [9,10]. Another virulence factor of S. aureus is leukotoxin which is very toxic to WBCs, especially neutrophils. The most important S. aureus leukotoxin is Panton-Valentine leukocidin (PVL) which is composed of S and F proteins and destroys the neutrophil’s cell membrane. PVL and SEs are the most potent virulence determinants of S. aureus with a significant role in the initiation and pathogenesis of the disease [11,12].

Milk ingredients enhance the growth of S. aureus and subsequently the production of enterotoxins which are heat stable and resist pasteurization. Therefore, raw milk with improper storage standards has an increased rate of food intoxication. For example, S. aureus enterotoxin A, considered as a potent virulence markers, can resist heating temperature up to 121 °C for 20 min [13]. Further, staphylococcal enterotoxins (A, B, C, D, and E) are the primary cause of food poisoning outbreaks, while the other types are responsible for sporadic cases [8,14].

The estimated population of cattle and buffaloes in Egypt by 2019 was 9.3 million head, with more than 7.2 million tons of milk production [15]. Therefore, in this study, we aimed to investigate the prevalence and antimicrobial resistance of S. aureus in tested milk samples, and investigate the prevalence virulence determinant (coa, spa, pvl and mecA) and enterotoxins (sea, seb, sec and sed) genes of MRSA isolated from bovine species milk in Egypt.

2. Materials and Methods

2.1. Collection of Milk Samples

Four hundred eighty milk samples were collected in the period between December 2018 and February 2019 under aseptic conditions. Milk samples were collected from 120 clinically healthy lactating animals from two farms (50 local breed buffaloes from one farm and 70 dairy cattle from the other farm) located in Ismailia Province, Egypt. The selected farms use manual milking regimes and practice intensive management systems. Four milk samples were collected from each animal (one sample per quarter of the udder).

Subclinical mastitis animals were selected based on specific characteristics including: (1) being apparently unaffected by the illness, and (2) exhibiting a reduction in milk yield, which might result in high somatic cell count.

The handling of animals was done as described by the Animal Ethics Board Committee of Suez Canal University, Egypt. Before sample collection, the udder of each animal was palpated for the detection of any abnormalities such as swelling, hotness, asymmetry, and any physical changes. Animal’s udder, teats, and hands of the examiner were washed using running water and soap and were dried with a clean towel. The udder, teats, and tester hands were then sterilized with 70% ethyl alcohol to ensure that there was no external contamination. The first strips of milk were excluded and thrown away as they may be contaminated from the teat orifice, then 15–20 mL of milk samples were collected from each quarter into sterile screw-capped McCartney bottles (Thermo Fisher Scientific, Waltham, MA, USA). Milk samples were immediately transported to the laboratory in an ice container [16].

2.2. California Mastitis Test (CMT)

In order to determine the milk samples infected with subclinical mastitis, CMT (screening test) was used. The test procedures were conducted as previously mentioned [17,18]. The test depends on the interaction of the reagent with DNA of somatic cells present in milk. Briefly, in a cup of the white plastic paddle, 2 mL of scam reagent was added to an equal volume of milk sample from each quarter of the udder and mixed by a gentle circular motion. The test results were evaluated visually within 20 s and interpreted as; − (0), ± (T), + (1), + + (2), and + + + (3) based on the amount of gel formation. Milk samples from individual quarters with positive CMT scores were subjected to bacteriological examination.

2.3. Isolation and Identification of S. aureus

The screened positive milk samples were incubated at 37 °C for 24 h, and then centrifuged at 3000 rpm for 5 min. The cream layer was discarded and sediments were streaked onto blood agar, nutrient agar, and mannitol salt agar plates (Oxoid, Hampshire, UK). The streaked plates were then incubated at 37 °C for 24–48 h. The suspected grown colonies were identified morphologically and biochemically as previously mentioned [17]. S. aureus circular convex golden-yellow colonies were collected and preserved at −80 °C in media containing 10% glycerol (v/v) for further analysis. The confirmation of the retrieved colonies was performed using PCR for 16Sr RNA gene identification as previously described [19].

2.4. Antimicrobial Susceptibility Testing of S. aureus

Antimicrobial susceptibility testing was carried out using disc diffusion technique [20]. The isolated strains were tested for their susceptibility to cefoxitin (Cef; indicative for MRSA), penicillin (Pen), ampicillin-sulbactam (Amp-Sul), amoxicillin-clavulanic acid (Amo-Cla), tetracycline (Tet), cefotaxime (Ceft), and erythromycine (Ery) (Oxoid).

The selected antimicrobials are representative of the drugs used for humans and in the animal industry and were chosen according to the National Antimicrobial Resistance Monitoring System (NARMS) records. The reference strain (S. aureus ATCC 25923) was used as a control for the disc diffusion technique. The test was conducted on Muller Hinton agar plates (MH, Oxoid) and the plates were incubated at 37 °C for 24 h. The test was performed in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI) criteria using the available CLSI interpretive criteria (Table 1).

Table 1.

Interpretive criteria for inhibition zone diameter [20,21].

Antimicrobial Agent Disc Conc. Diameter of Inhibition Zone (mm)
R I S
Pen 10 units 28 or less - 29 or more
Amo-Cla 10–20 µg 19 or less - 20 or more
Amp-Sul 10 µg 11or less 12–14 15 or more
Tet 30 µg 14 or less 15–18 19 or more
Ceft 30 µg 14 or less 15–22 23 or more
Cef 30 µg ≤21 mm - -
Ery 15 µg 13 or less 14–17 18 or more
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R: Resistant, I: Intermediate, S: Sensitive. According to the National Committee for Clinical Laboratory standards. Cefoxitin (Cef); Penicillin (Pen); Ampicillin-sulbactam (Amp-Sul); Amoxicillin-clavulanic acid (Amo-Cla); Tetracycline (Tet); Cefotaxime (Ceft); Erythromycine (Ery).

2.5. Detection of Virulence and Enterotoxins Genes of MRSA Strains Using PCR

2.5.1. Genomic DNA Extraction

DNA of MRSA strains was extracted using the boiling method. Briefly, a half loopfull from S. aureus plate cultures were suspended in 100 μL of DNase-free water, heated at 95 °C for 10 min, cooled, and then centrifuged at 5000× g for 10 min. The supernatant containing the genomic DNA was collected in a new tube and stored at −20 °C for further use. DNA was quantified using a Nanodrop 1000 instrument (Thermo Scientific, Loughborough, UK).

2.5.2. Polymerase Chain Reaction (PCR)

The extracted DNA from MRSA strains were screened for virulence genes (coa, spa, pvl and mecA) and enterotoxins genes (sea, seb, sec and sed) detection. Amplification was performed in PCR tubes, in a 25 µL reaction volume, containing 200 µM of dNTPS buffer (dATP, dGTP, dCTP and dTTP), 50 picomol of each forward and reverse primers, and 0.5 units of taq DNA polymerase (NZYtech, Lisbon, Portugal). The PCR reaction mixtures were amplified in the MJ MiniTM Gradient Thermocycler apparatus (Biometra, Göttingen, Germany). Primers sequence, expected amplicon size, and annealing temperature are described in Table 2. Nuclease-free water was used as a negative control. Positive controls DNA were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Suez Canal University, Egypt. PCR products were visualized on a 1.5% agarose gel containing ethidium bromide under UV light and 100 bp ladder (Fermentas, Thermo Scientific, Darmstadt, Germany) was used.

Table 2.

List of primers and recycling conditions of PCR assay.

Primer Primer Sequence. Annealing Temperature Recycling Conditions References
coa1 ATA GAG ATG CTG GTA CAG G 58 °C 39 cycles; 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min [22]
coa 2 GCT TCC GAT TGT TCG ATG C
sea-3b CCT TTG GAA ACG GTT AAA ACG 55 °C 30 cycles; 95 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min [23]
sea-4b TCT GAA CCT TCC CAT CAA AAA C
seb-1c TCG CAT CAA ACT GAC AAA CG 55 °C
seb-4b GCA GGT ACT CTA TAA GTG CCT GC
sec-3b CTC AAG AAC TAG ACA TAA AAG CTA GG 55 °C
sec-4b TCA AAA TCG GAT TAA CAT TAT CC
sed-3b CTA GTT TGG TAA TAT CTC CTT TAA ACG 55 °C
sed-4b TTA ATG CTA TAT CTT ATA GGG TAA ACA TC
spa-III CAA GCA CCA AAA GAG GAA 60 °C 30 cycles; 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min [24]
spa-IV CAC CAG GTT TAA CGA CAT
luk-PV-1 ATCATTAGGTAAAATGTCTGGACATGATCCA 66 °C 34 cycles; 94 °C for 30 s, 66 °C for 30 s, 72 °C for 1 min 30 s [25]
luk-PV-2 GCATCAACTGTATTGGATAGCAAAAGC
mecA-1 TGGCATTCGTGTCACAATCG 53 °C 34 cycles; 94 °C for 1 min, 53 °C for 50 s, 72 °C for 1 min [25]
mecA-2 CTGGAACTTGTTGAGCAGAG
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2.6. Statistical Analysis

The Chi-square test was used for the analysis of the recovered frequencies using SAS® software (version 9.4, SAS Institute, Cary, NC, USA) to test the null hypothesis of various treatments. A p-value of <0.05 was considered statistically significant.

3. Results

3.1. Prevalence of S. aureus Subclinical Bovine Mastitis

The prevalence of subclinical mastitis in the collected milk samples from individual quarters using CMT was 44% (88/200) and 52.1% (146/280) in buffaloes and cattle, respectively (Table 3). There was no significant differences between the prevalence of subclinical mastitis between buffaloes and cattle (p > 0.05). Additionally, the total prevalence of S. aureus in the collected milk samples from individual quarters using CMT was 35.9% (84/234) with 36.3%% (53/146) in cattle and 31% (31/88) in buffaloes and no significant difference between cattle and buffaloes (p = 0.8654; X2 = 0.029). Out of them, the prevalence of MRSA was 35.7% (30/84) with 37.7% (20/53) in cattle and 32.2% (10/31) in buffaloes and no significant difference between cattle and buffaloes (p = 0.6203; X2 = 0.245). Detailed results of CMT screening in the collected milk samples are shown in Table 4.

Table 3.

Prevalence of subclinical mastitis in buffaloes and cattle (based on CMT).

Animal Species Total Animals (No.) Total Samples (No.) Negative Samples (No.) Positive Samples (No.) Positive Samples (%) Chi-Square Value
Buffaloes 50 200 112 88 44 3.057
NS *
p = 0.0804
Cows 70 280 134 146 52.1
Total 120 480 246 234 48.75
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* NS = non-significant.

Table 4.

Results of CMT screening in the collected milk samples.

CMT Grads Examined Quarters (No.) Positive S. aureus Isolates (No.) S. aureus
Isolates (%)
+++ 57 43 75.4
++ 39 32 82
+ 138 9 6.5
Total 234 84 35.9
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Percentages were calculated in comparison to the total number of examined samples of each grade.

3.2. Antimicrobial Susceptibility Phenotypic Profiles of the S. aureus Isolates

The antimicrobials susceptibility test for S. aureus strains showed that 64.3% (54/84) of the samples were resistant to penicillin, 59.5% (50/84) were resistant to tetracycline and 35.7% (30/84) of the strains were resistant to cefoxitin (defined as MRSA). In addition, 58.3% (49/84) of the tested strains exhibited intermediate sensitivity to cefotaxime, whereas 64.3% (54/84) of the tested strains were sensitive to cefoxitin. Our results also showed that 78.6% (66/84) of the strains were sensitive to amoxicillin-clavulanic acid, 72.6% (61/84) to ampicillin-sulbactam, and 63.1% (53/84) were sensitive to erythromycin. Details about the antimicrobial susceptibility phenotypic profiles of the S. aureus strains are shown in Table 5.

Table 5.

Antimicrobial sensitivity test of the isolated S. aureus strains.

Antimicrobial Agents Resistant Intermediate Sensitive
No. % No. % No. %
Cef 30 (MRSA) 35.7 - - 54 64.3
Pen 54 64.3 - - 30 35.7
Amo- Cla 18 21.4 - - 66 78.6
Amp-Sul 11 13.1 12 14.3 61 72.6
Tet 50 59.5 15 17.9 19 22.6
Ceft 13 15.5 49 58.3 22 26.2
Ery 26 30.9 5 5.9 53 63.1
Chi-square value 94.7860 *
p < 0.0001		 186.19 *
p < 0.0001		 104.25 *
p < 0.0001
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* Significant differences in the prevalence between the antimicrobial agents. Cefoxitin (Cef); Penicillin (Pen); Ampicillin-sulbactam (Amp-Sul); Amoxicillin-clavulanic acid (Amo-Cla); Tetracycline (Tet); Cefotaxime (Ceft); Erythromycin (Ery). There was significant differences between Cef and Pen: X2 = 13.660, p = 0.0002, Cef and Amp-Sul: X2 = 11.560, p = 0.0007; Cef and Tet: X2 = 9.481, p = 0.0021; Pen and Amo-Cla: X2 = 31.376, p < 0.0001; Pen and Amp-Sul: X2 = 46.134, p < 0.0001; Pen and Ceft: X2 = 41.462, p < 0.0001; Pen and Ery: X2 =18.673, p < 0.0001; Amo-Cla and Tet: X2 = 25.160, p < 0.0001; Amp-Sul and Tet: X2 = 38.873, p < 0.0001; Tet and Ceft: X2 = 34.487, p < 0.0001; and Tet and Ery: X2 = 13.787, p = 0.0002.

3.3. Virulence Determinant Genes of MRSA Strains

Thirty isolates of MRSA strains were subjected to PCR for the detection of coa, spa, pvl and mecA genes. All the 30 tested strains (100%) were positive for coa gene that showed no gene polymorphism, while 26 out of 30 strains (86.6%) were positive for spa gene and showed a remarkable gene polymorphism with different amplicons size (140 bp, 270 bp and 290 bp). Only 3 out of 30 strains (10%) were positive for pvl gene. Furthermore, all the examined strains (100%) were positive for mecA gene. There is a significant difference in the prevalence of virulence determinant genes (p < 0.0001) among the examined MRSA strains (Table 6).

Table 6.

Prevalence of virulence and enterotoxins determinant genes of MRSA strains isolated from bovine milk.

Genes No % Chi-Square Value
Virulence Genes coa 30 100 62.6900 *
p < 0.0001
spa 26 86.6
pvl 3 10
mecA 30 100
Enterotoxins Genes sea 8 26.6 21.9751 *
p < 0.001
sea+sec 1 3.3
sec 2 6.6
seb 0 0
sed 0 0
Chi-square value 168.0403 *
p < 0.0001
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No of the examined MRSA strains= 30. There were significant differences between spa and pvl at X2 = 34.659, between coa and pvl at X2 = 48.273; and between mecA and pvl virulence genes at X2 = 48.273 (p < 0.0001). There were significant differences between sea and seb/sed enterotoxins genes at X2 = 9.051 and p = 0.0026. * Significant difference in the prevalence between different virulence genes and between different enterotoxins genes.

3.4. Prevalence of Enterotoxins Genes among MRSA Strains

The prevalence of enterotoxins genes (sea, seb, sec and sed) among MRSA strains were detected using multiplex PCR. Our results showed that eight out of 30 strains (26.6%) were positive for sea gene, two out of 30 strains were positive for sec gene, one out of 30 strains (3.3%) was positive for both sea and sec genes, and none of the tested strains harbored seb and sed genes. Hence, 11 out of 30 strains (36.6%) were enterotoxignic (Table 6). The prevalence of the enterotoxins genes showed a significant difference (p < 0.0001) among the examined MRSA strains.

4. Discussion

Subclinical mastitis is a significantly important disease, causing economic losses in the livestock industry, not only in Egypt, but also worldwide. Antibiotic resistance has increased among various bacterial pathogens, which is considered an emerging problem with a major public health concern due to the risk of resistance transmission to human as well as its influence on the effectiveness of the current antibiotic therapy [26,27,28,29]. Further, MRSA strains can cause nosocomial infections and high mortality in humans [3]. In this study, the detected prevalence of subclinical mastitis was 44% and 52.1% in buffaloes and cattle, respectively, with a total prevalence of 48.6% and no significant difference in the prevalence between both animal species (Table 3). This high prevalence of bovine subclinical mastitis was similar to the results obtained in other previous studies [16,30]. The increased incidence of subclinical mastitis in dairy livestock is attributed to multiple predisposing factors including: (1) contaminated milking machines, (2) improper housing, (3) bad sanitation, and (4) bad handling of animals. Furthermore, the failure in the treatment always occurred due to: (1) chronic infection accompanied with fibrosis, (2) inadequate dose of antibiotics, and (3) emergence of multidrug-resistant bacterial pathogens [31].

The prevalence of S. aureus recovered from subclinical mastitis infected animals was 36% (p < 0.0001). This result was similar to previous studies [16,32] and lower than the prevalence (6.5%) reported by Haltia, et al. [33]. S. aureus transmission between animals is due to using of contaminated milk utensils and is also due to contaminated milker’s hands [34]. Additionally, cefoxitin resistance (MRSA) was used determine the methicillin-resistant S. aureus isolates, and antimicrobial susceptibility testing showed that 35.7% of the recovered S. aureus strains were resistant to cefoxitin (Table 5). Further, the identified MRSA strains were confirmed using PCR for detection of mecA gene, where all the tested strains (100%) harbored this gene (Table 6) conferred marked resistance to various antimicrobial agents such as cefoxitin, penicillins, cephalosporins, macrolides, aminoglycosides and tetracyclines. Since the 1990s, most MRSA isolates possessed a multidrug-resistant phenotype and carried many resistant determinants in chromosome and plasmids. Resistance to methicillin is attributed to the existence of mecA gene on the S. aureus chromosome, which is encoded for the synthesis of PBP2a. Methicillin is stable in the presence of β-lactamase enzymes and is effective in the treatment of S. aureus infection, but not against MRSA that resist methicillin [35,36,37].

In this study, 64.3% S. aureus strains were resistant to penicillin which was in agreement with other studies (more than 50%) [38] and lower than resistance rate (7.1%) determined by Bengtsson, et al. [39]. The failure of treatment of subclinical mastitis with penicillin was mainly due to the release of β-lactamase by S. aureus, which causes hydrolysis of β-lactam rings. The majority of S. aureus strains release penicillinases which consequently make S. aureus resistant to the β-lactam group of antibiotics [40]. Furthermore, 78.57% S. aureus strains were sensitive to amoxicillin-clavulanic acid, while 72.62% was sensitive to ampicillin-sulbactam. The isolation of a naturally occurring β-lactamase inhibitor (clavulanic acid) from Streptomyces clavuligerus was a major step in the formulation of new antibiotic combinations. Sulbactam and clavulanic acid (β-lactam antibiotics) have very poor bactericidal ability, however, they are a very strong β -lactamase inhibitors [41].

We observed that 59.5% of the strains were resistant to tetracyclin, a broad-spectrum antibiotic groups that, due to its extensive use, resulted in the development of resistant strains. Resistance to tetracyclines resulted from the production of ribosomal protection protein by S. aureus that makes a competitive binding to tetracycline. The tet gene that is carried on conjugative plasmids of S. aureus is responsible for their production [42].

In addition, 58.3% of the S. aureus strains were moderately sensitive to cefotaxime, while 63.1% were sensitive to erythromycin. In general, third-generation cephalosporins have strong activity against Gram-negative bacteria and moderate activity against Gram-positives, such as S. aureus and Streptococci. Unlike penicillin, third-generation cephalosporins have stable activity in presence of β-lactamase enzyme. Erythromycin belongs to the macrolide class and showed a potent antibacterial activity against both Gram-positive and Gram-negative bacteria, including Staphylococci, Streptococci and E. coli [41,43].

The coa gene was 100% prevalent in the tested MRSA strains. These results are similar to those reported by Akineden, Annemüller, Hassan, Lämmler, Wolter and Zschöck [24]. This gene exhibited no size polymorphisms [44]. However, in other studies, coagulase gene amplification resulted in different amplicons, indicating coagulase gene size polymorphism [34]. The spa gene displayed remarkable gene polymorphisms where different sized amplicons were found (140, 270, and 290 bp). The X region of spa gene usually undergoes variable repetitions (up to 24 repeats) which might be different in different strains [10]. Number of repeats is associated with the dissemination potential of S. aureus, where strains that have more than seven repeats in the X region were considered as epidemic, whereas the presence of seven or less repeats were considered as non-epidemic MRSA [14]. Further, the pvl gene was detected in 10% of the tested strains. The presence of pvl gene in S. aureus was similar to that obtained previously [45] and disagrees with the results obtained by Ikawaty, et al. [46]. The pvl gene is considered as the most powerful staphylococcal leukotoxin that could resist bovine neutrophils [47]. Therefore, pvl may contribute to resistance by attacking the bovine polymorph-nuclear cells and increase pathogenicity against the host [46]. Interestingly, the sea gene was detected in 26.6% of the tested strains followed by sec gene (6.6%) then mixed (sea and sec) genes in 3.3%, while none of MRSA strains harbored seb and sed genes (Table 6). The high prevalence of sea and sec gene was previously reported by Rall, et al. [48]. The sea gene was the most predominant enterotoxins gene isolated in other studies conducted on S. aureus [49]. This gene is very resistant to pasteurization heat and maintain some biological activity after 28 min at 121 °C [50]. It’s also considered as the most frequently detected gene in the US food poisoning outbreaks followed by sed and seb and 95% of these outbreaks have sea and see enterotoxins [51].

5. Conclusions

In conclusion, subclinical mastitis caused by S. aureus is considered as one of the major economically important diseases with public health significance. The most predominant virulence genes associated with MRSA strains in bovine milk were coa, mecA and spa, whereas, the most predominant enterotoxin gene is sea that causes food poisoning in human consumers after ingestion of contaminated milk. Based on our knowledge, this is the first study that emphasizes the occurrence of pvl in MRSA strains originating from bovine species milk in Egypt. The continuous application of the antimicrobial susceptibility testing of S. aureus in the future will be necessary to determine the drug choice for disease control.

Acknowledgments

We thank Geoffrey Carney-Knisely, The Ohio State University, College of Medicine for English proofreading and confirming the statistical analysis of the manuscript.

Author Contributions

A.M.A. prepared and conducted the experiments. A.M.A., M.E.E., Y.A.H., R.M.E.-T., and M.O.I.G. did the Data analysis, statistical analysis and Data accuracy. A.M.A. and Y.A.H. wrote and revised the manuscript. All authors have revised and approved the final manuscript.

Funding

This research received no external funding.

Conflicts of Interest

Authors declare that there is no conflict of interest.

References

  • 1.Abdel-Moein K.A., Zaher H.M. Occurrence of multidrug-resistant methicillin-resistant Staphylococcus aureus among healthy farm animals: A public health concern. Int. J. Veter. Sci. Med. 2019;7:55–60. doi: 10.1080/23144599.2019.1689630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Martins S., Martins V.C., Cardoso F.A., Germano J., Rodrigues M., Duarte C., Bexiga R., Cardoso S., Freitas P.P. Biosensors for On-Farm Diagnosis of Mastitis. Front. Bioeng. Biotechnol. 2019;7:186. doi: 10.3389/fbioe.2019.00186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gordon R.J., Lowy F.D. Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection. Clin. Infect. Dis. 2008;46:S350–S359. doi: 10.1086/533591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ameen F., Reda S.A., El Shatoury S., Riad E.M., Enany M.E., Alarfaj A.A. Prevalence of antibiotic resistant mastitis pathogens in dairy cows in Egypt and potential biological control agents produced from plant endophytic actinobacteria. Saudi J. Biol. Sci. 2019;26:1492–1498. doi: 10.1016/j.sjbs.2019.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.El-Jakee J., Atta N.S., Samy A., Bakry M., Elgabry E., Kandil M.M., El-Said W.G. Antimicrobial resistance in clinical isolates of Staphylococcus aureus from bovine and human sources in Egypt. Glob. Vet. 2011;7:581–586. [Google Scholar]
  • 6.Bień J., Sokolova O., Bozko P. Characterization of Virulence Factors of Staphylococcus aureus: Novel Function of Known Virulence Factors That Are Implicated in Activation of Airway Epithelial Proinflammatory Response. J. Pathog. 2011;2011:1–13. doi: 10.4061/2011/601905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Panizzi P., Friedrich R., Fuentes-Prior P., Bode W., Bock P.E. The staphylocoagulase family of zymogen activator and adhesion proteins. Cell. Mol. Life Sci. 2004;61:2793–2798. doi: 10.1007/s00018-004-4285-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Mubarack H., Doss A., Vijayasanthi M., Venkataswamy R. Antimicrobial drug susceptibility of Staphylococcus aureus from subclinical bovine mastitis in Coimbatore, Tamilnadu, South India. Vet. World. 2012;5:352. doi: 10.5455/vetworld.2012.352-355. [DOI] [Google Scholar]
  • 9.Mathema B., Mediavilla J., Kreiswirth B.N. Methods in Molecular Biology. Springer Science and Business Media LLC; Totowa, NJ, USA: 2008. Sequence Analysis of the Variable Number Tandem Repeat in Staphylococcus aureus Protein A Gene; pp. 285–305. [DOI] [PubMed] [Google Scholar]
  • 10.Enany M.E., Algammal A.M., Shagar G.I., Hanora A.M., Elfeil W.K., Elshaffy N.M. Molecular typing and evaluation of Sidr honey inhibitory effect on virulence genes of MRSA strains isolated from catfish in Egypt. Pak. J. Pharm. Sci. 2018;31:5. [PubMed] [Google Scholar]
  • 11.Younis A., Krifucks O., Fleminger G., Heller E.D., Gollop N., Saran A., Leitner G. Staphylococcus aureus leucocidin, a virulence factor in bovine mastitis. J. Dairy Res. 2005;72:188–194. doi: 10.1017/S002202990500083X. [DOI] [PubMed] [Google Scholar]
  • 12.Holzinger D., Gieldon L., Mysore V., Nippe N., Taxman D.J., Duncan J.A., Broglie P.M., Marketon K., Austermann J., Vogl T., et al. Staphylococcus aureus Panton-Valentine leukocidin induces an inflammatory response in human phagocytes via the NLRP3 inflammasome. J. Leukoc. Biol. 2012;92:1069–1081. doi: 10.1189/jlb.0112014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chang B.S., Bohach G.A., Lee S.U., Davis W.C., Fox L.K., Ferens W.A., Seo K.S., Koo H.C., Kwon N.H., Park Y.H. Immunosuppression by T regulatory cells in cows infected with Staphylococcal superantigen. J. Vet. Sci. 2005;6:247–250. doi: 10.4142/jvs.2005.6.3.247. [DOI] [PubMed] [Google Scholar]
  • 14.Burton J., Erskine R.J. Immunity and mastitis Some new ideas for an old disease. Vet. Clin. N. Am. Food Anim. Pr. 2003;19:1–45. doi: 10.1016/S0749-0720(02)00073-7. [DOI] [PubMed] [Google Scholar]
  • 15.GAIN Egypt Livestock and Products Annual 2018. [(accessed on 19 September 2018)]; Available online: https://apps.fas.usda.gov/newgainapi/api/report/downloadreportbyfilename?filename=Livestock%20and%20%E2%80%8EProducts%20Annual_Cairo_Egypt_9-19-2018.pdf%E2%80%8E.
  • 16.Dego O.K., Tareke F. Bovine mastitis in selected areas of southern Ethiopia. Trop. Anim. Health Prod. 2003;35:197–205. doi: 10.1023/A:1023352811751. [DOI] [PubMed] [Google Scholar]
  • 17.Quinn P.J., Markey B.K., Leonard F.C., Hartigan P., Fanning S., Fitzpatrick E. Veterinary Microbiology and Microbial Disease. 2nd ed. John Wiley & Sons; Hoboken, NJ, USA: 2011. pp. 1–928. [Google Scholar]
  • 18.Clements A., Taylor D.J., Fitzpatrick J.L. Evaluation of diagnostic procedures for subclinical mastitis in meat-producing sheep. J. Dairy Res. 2003;70:139–148. doi: 10.1017/S0022029903006022. [DOI] [PubMed] [Google Scholar]
  • 19.Monday S.R., Bohach G.A. Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates. J. Clin. Microbiol. 1999;37:3411–3414. doi: 10.1128/JCM.37.10.3411-3414.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.CLSI C. Performance Standards for Antimicrobial Susceptibility Testing. Volume 26. Clinical Lab Standards Institute; Wayne, PA, USA: 2016. pp. 1–251. [Google Scholar]
  • 21.Abbey T.C., Deak E. What’s New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition. Clin. Microbiol. Newsl. 2019;41:203–209. doi: 10.1016/j.clinmicnews.2019.11.002. [DOI] [Google Scholar]
  • 22.Hookey J.V., Richardson J.F., Cookson B.D. Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene. J. Clin. Microbiol. 1998;36:1083–1089. doi: 10.1128/JCM.36.4.1083-1089.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Becker K., Roth R., Peters G. Rapid and Specific Detection of Toxigenic Staphylococcus aureus: Use of Two Multiplex PCR Enzyme Immunoassays for Amplification and Hybridization of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene. J. Clin. Microbiol. 1998;36:2548–2553. doi: 10.1128/JCM.36.9.2548-2553.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Akineden O., Annemüller C., Hassan A.A., Lämmler C., Wolter W., Zschöck M. Toxin Genes and Other Characteristics ofStaphylococcus aureus Isolates from Milk of Cows with Mastitis. Clin. Diagn. Lab. Immunol. 2001;8:959–964. doi: 10.1128/CDLI.8.5.959-964.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Bonnstetter K.K., Wolter D.J., Tenover F.C., McDougal L.K., Goering R. Rapid Multiplex PCR Assay for Identification of USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates. J. Clin. Microbiol. 2006;45:141–146. doi: 10.1128/JCM.01228-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.El-Sayed M., Algammal A., Abouel-Atta M., Mabrok M., Emam A. Pathogenicity, genetic typing, and antibiotic sensitivity of Vibrio alginolyticus isolated from Oreochromis niloticus and Tilapia zillii. Rev. Med. Vet. 2019;170:80–86. [Google Scholar]
  • 27.Algammal A.M., Wahdan A., Elhaig M. Potential efficiency of conventional and advanced approaches used to detect Mycobacterium bovis in cattle. Microb. Pathog. 2019;134:103574. doi: 10.1016/j.micpath.2019.103574. [DOI] [PubMed] [Google Scholar]
  • 28.Helmy Y.A., Kassem I.I., Kumar A., Rajashekara G. In Vitro Evaluation of the Impact of the Probiotic E. coli Nissle 1917 on Campylobacter jejuni’s Invasion and Intracellular Survival in Human Colonic Cells. Front. Microbiol. 2017;8:1588. doi: 10.3389/fmicb.2017.01588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Helmy Y.A., Deblais L., Kassem I.I., Kathayat D., Rajashekara G. Novel small molecule modulators of quorum sensing in avian pathogenic Escherichia coli (APEC) Virulence. 2018;9:1640–1657. doi: 10.1080/21505594.2018.1528844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Sudhan N., Singh R., Singh M., Soodan J. Studies on prevalence, etiology and diagnosis of subclinical mastitis among crossbred cows. Indian J. Anim. Res. 2005;39:127–130. [Google Scholar]
  • 31.Seegers H., Fourichon C. Production effects related to mastitis and mastitis economics in dairy cattle herds. Vet. Res. 2003;34:475–491. doi: 10.1051/vetres:2003027. [DOI] [PubMed] [Google Scholar]
  • 32.Whist A., Østerås O., Sølverød L. Staphylococcus aureus and Streptococcus dysgalactiae in Norwegian herds after introduction of selective dry cow therapy and teat dipping. J. Dairy Res. 2006;74:1–8. doi: 10.1017/S0022029906002135. [DOI] [PubMed] [Google Scholar]
  • 33.Haltia L., Honkanen-Buzalski T., Spiridonova I., Olkonen A., Myllys V. A study of bovine mastitis, milking procedures and management practices on 25 Estonian dairy herds. Acta Vet. Scand. 2006;48:22. doi: 10.1186/1751-0147-48-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Scherrer D., Corti S., Muehlherr J., Zweifel C., Stephan R. Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep. Vet. Microbiol. 2004;101:101–107. doi: 10.1016/j.vetmic.2004.03.016. [DOI] [PubMed] [Google Scholar]
  • 35.Ito T., Okuma K., Ma X.X., Yuzawa H., Hiramatsu K. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: Genomic island SCC. Drug Resist. Updat. 2003;6:41–52. doi: 10.1016/S1368-7646(03)00003-7. [DOI] [PubMed] [Google Scholar]
  • 36.Leonard F., Markey B. Meticillin-resistant Staphylococcus aureus in animals: A review. Vet. J. 2008;175:27–36. doi: 10.1016/j.tvjl.2006.11.008. [DOI] [PubMed] [Google Scholar]
  • 37.Eid H.M., Algammal A.M., Elfeil W.K., Youssef F.M., Harb S.M., Abd-Allah E.M. Prevalence, molecular typing, and antimicrobial resistance of bacterial pathogens isolated from ducks. Vet. World. 2019;12:677–683. doi: 10.14202/vetworld.2019.677-683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Pitkälä A., Haveri M., Pyörälä S., Myllys V., Honkanen-Buzalski T. Bovine Mastitis in Finland 2001—Prevalence, Distribution of Bacteria, and Antimicrobial Resistance. J. Dairy Sci. 2004;87:2433–2441. doi: 10.3168/jds.S0022-0302(04)73366-4. [DOI] [PubMed] [Google Scholar]
  • 39.Bengtsson B., Unnerstad H.E., Ekman T., Artursson K., Nilsson-Öst M., Waller K.P. Antimicrobial susceptibility of udder pathogens from cases of acute clinical mastitis in dairy cows. Vet. Microbiol. 2009;136:142–149. doi: 10.1016/j.vetmic.2008.10.024. [DOI] [PubMed] [Google Scholar]
  • 40.Weese J.S. Methicillin-resistant Staphylococcus aureus in animals. ILAR J. 2010;51:233–244. doi: 10.1093/ilar.51.3.233. [DOI] [PubMed] [Google Scholar]
  • 41.Feucht C., Patel D.R. Principles of pharmacology. Pediatric Clin. 2011;58:11–19. doi: 10.1016/j.pcl.2010.10.005. [DOI] [PubMed] [Google Scholar]
  • 42.Chopra I., Roberts M.C. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol. Mol. Biol. Rev. 2001;65:232–260. doi: 10.1128/MMBR.65.2.232-260.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Enany M.E., Algammal A.M., Nasef S.A., Abo-Eillil S.A.M., Bin-Jumah M.N., Taha A.E., Allam A. The occurrence of the multidrug resistance (MDR) and the prevalence of virulence genes and QACs resistance genes in E. coli isolated from environmental and avian sources. AMB Express. 2019;9:1–9. doi: 10.1186/s13568-019-0920-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Frénay H.M., Theelen J.P., Schouls L.M., Vandenbroucke-Grauls C.M., Verhoef J., Van Leeuwen W.J., Mooi F.R. Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. J. Clin. Microbiol. 1994;32:846–847. doi: 10.1128/JCM.32.3.846-847.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Hata E., Katsuda K., Kobayashi H., Uchida I., Tanaka K., Eguchi M. Genetic Variation among Staphylococcus aureus Strains from Bovine Milk and Their Relevance to Methicillin-Resistant Isolates from Humans. J. Clin. Microbiol. 2010;48:2130–2139. doi: 10.1128/JCM.01940-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ikawaty R., Brouwer E., Mevius D., Fluit A.C., Van Duijkeren E., Verhoef J. Virulence Factors of Genotyped Bovine Mastitis Staphylococcus aureus Isolates in The Netherlands. Int. J. Dairy Sci. 2010;5:60–70. doi: 10.3923/ijds.2010.60.70. [DOI] [Google Scholar]
  • 47.Barrio M.B., Rainard P., Prévost G. LukM/LukF′-PV is the most active Staphylococcus aureus leukotoxin on bovine neutrophils. Microbes Infect. 2006;8:2068–2074. doi: 10.1016/j.micinf.2006.03.004. [DOI] [PubMed] [Google Scholar]
  • 48.Rall V.L.M., Vieira F., Rall R., Vieitis R., Fernandes A., Candeias J.M.G., Cardoso K., Araujo J. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet. Microbiol. 2008;132:408–413. doi: 10.1016/j.vetmic.2008.05.011. [DOI] [PubMed] [Google Scholar]
  • 49.Normanno G., Firinu A., Virgilio S., Mula G., Dambrosio A., Poggiu A., Decastelli L., Mioni R., Scuota S., Bolzoni G. Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy. Int. J. Food Microbiol. 2005;98:73–79. doi: 10.1016/j.ijfoodmicro.2004.05.008. [DOI] [PubMed] [Google Scholar]
  • 50.Asao T., Kumeda Y., Kawai T., Shibata T., Oda H., Haruki K., Nakazawa H., Kozaki S. An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: Estimation of enterotoxin A in the incriminated milk and powdered skim milk. Epidemiol. Infect. 2003;130:33–40. doi: 10.1017/S0950268802007951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Omoe K., Ishikawa M., Shimoda Y., Hu D.-L., Ueda S., Shinagawa K. Detection of seg, seh, and sei genes in Staphylococcus aureus Isolates and Determination of the Enterotoxin Productivities of S. aureus Isolates Harboring seg, seh, or sei Genes. J. Clin. Microbiol. 2002;40:857–862. doi: 10.1128/JCM.40.3.857-862.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

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