Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds

doi:10.1016/j.vetmic.2004.12.005

Veterinary Microbiology

Volume 106, Issues 1–2, 20 March 2005, Pages 103-107

https://doi.org/10.1016/j.vetmic.2004.12.005 Get rights and content

Abstract

To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis.

Introduction

The staphylococcal enterotoxins (SEs) are distinguished for their role in the pathogenesis of human and animal illnesses. These toxins are responsible for food poisoning outbreaks and toxigenic syndrome in humans, and may contribute to the persistence of S. aureus in bovine mammary glands (Ferens et al., 1998, Llewelyn and Cohen, 2002).

Several reports described the production of SEs by S. aureus isolated from bovine mastitis with a great geographical variation in the distribution of enterotoxigenic strains (Magalhães Lopes et al., 1990, Larsen et al., 2000, Stephan et al., 2001). In addition, there were differences between S. aureus strains from bovine and goat mastitis regarding their ability to produce enterotoxins (Orden et al., 1992).

Thus, the purpose of this study was to analyze the distribution of the genes that encode SEA, SEB, and SEC in S. aureus isolated from bovine and goat mastitis. Also, the production in vitro of the SEs by isolates harboring the respective gene was analyzed.

Section snippets

Sources and strains of S. aureus

One hundred S. aureus strains isolated from goat (n = 36) and bovine mastitis (n = 64) were studied. The goats strains originated from Brazilian dairy herds located in Ceara state and in Rio de Janeiro state, and were previously isolated and identified (da Silva et al., 2004). The bovine strains originated from dairy herds located in Minas Gerais state and were also previously isolated and identified (Cardoso et al., 1999).

Production and detection of SEA, SEB, and SEC

All strains were tested for enterotoxins A to C (SEA-C) by the

Specificity and reproducibility testing

The reaction with each individual primer pair resulted in the amplification of single products when DNA from each reference strain was used as a template. In the Multiplex, the amplification of three bands in set A (sea, seb, and FemA) and two bands in set B (sec and FemA) were successfully obtained (Fig. 1). The sizes of the products obtained from control strains in both PCR designs corresponded to the predicted sizes. Reproducibility was observed in all tested strains. Although there were

Discussion

Some S. aureus strains produce one or more enterotoxigenic toxins including SEA, B, and C. These SEs represent the main cause of staphylococcal food poisoning and are potential virulence factors that contribute toward mastitis pathogenesis (Ferens et al., 1998). Furthermore, the SEs could be able to indicate the origin of the S. aureus strains because it was observed that a higher ratio of isolates from animals produced SEC and those from humans produced mainly SEA (Bergdoll, 1990, Orden et

References (15)

T.-R. Chen et al.
Development and use of PCR primers for the investigation of C1, C2 and C3 enterotoxin types of Staphylococcus aureus strains isolated from food-borne outbreaks
Int. J. Food Microbiol.
(2001)
E.R. da Silva et al.
Hemolysin production by Staphylococcus species isolated from mastitic goat milk
Small Rumin. Res.
(2004)
H.D. Larsen et al.
Differences between Danish bovine and human Staphylococcus aureus isolates in possession of superantigens
Vet. Microbiol.
(2000)
M. Llewelyn et al.
Superantigens: microbial agents that corrupt immunity
Lancet Infect. Dis.
(2002)
J.P. Rosec et al.
Staphylococcal enterotoxin genes of classical and new types detected by PCR in France
Int. J. Food Microbiol.
(2002)
H-J. Schuberth et al.
Characterization of leukocytotoxic and superantin-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis
Vet. Microbiol.
(2001)
R. Stephan et al.
Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland
Vet. Microbiol.
(2001)

There are more references available in the full text version of this article.

Cited by (57)

Relationship between virulence factors and antimicrobial resistance in Staphylococcus aureus from bovine mastitis
2020, Journal of Global Antimicrobial Resistance
Citation Excerpt :
Thus, due to the great importance of this toxin in regard to animal and public health, it is essential to obtain more information about its frequency, distribution and role in pathogenesis among S. aureus strains involved in bovine mastitis, as well as the development of useful tools for the routine detection of this toxin in milk and milk products. Hemolysins are considered important virulence factors of S. aureus that contribute to bacterial invasion and escape from the host immune response [48]; they are of crucial importance in cases of chronic mammary gland infections. S. aureus mainly produce α and β hemolysins, encoded by hla and hlb genes, respectively [66,67], with α-hemolysin being the most frequent cytotoxin produced by S. aureus.
This review summarizes the literature on the role of virulence and antimicrobial resistance genes of Staphylococcus aureus in bovine mastitis, focusing on the association between these characteristics and their implications for public and animal health.
There is the possibility of antimicrobial resistance gene exchange among different bacteria, which is of serious concern in livestock husbandry, as well as in the treatment of human staphylococcal infections.
Staphylococcus aureus isolated from handmade sweets: Biofilm formation, enterotoxigenicity and antimicrobial resistance
2016, Food Microbiology
Citation Excerpt :
However, this is not the only mechanism involved as there were some isolates that did not present those genes and they were still able to form biofilms on SS, e.g. isolate SAS8. This isolate had both the enterotoxin A and B genes, and according to Da Silva et al. (2005), the presence of these two genes in the same isolate increases its toxigenic capacity. Moreover, it was able to form biofilms on both PS and SS, even without having the icaA and icaD genes.
Staphylococcus aureus is the second most important pathogen involved in foodborne outbreaks in Brazil. Because of their widespread distribution and biofilm forming ability, handmade sweets are easily contaminated with S. aureus. The aim of this study was to isolate and identify coagulase-positive staphylococci (CPS) from handmade sweets produced in Pelotas City/Brazil. The virulence potential was checked by evaluating the presence of the staphylococcal enterotoxin genes, icaA and icaD genes, the biofilm forming potential and antimicrobial resistance of the isolates. It was find just S. aureus among the CPS isolates. All the S. aureus isolates had biofilm forming ability on stainless steel and more than half of them on polystyrene surfaces. The majority of the isolates carried the icaA (66.6%) and icaD (58.4%) genes and some of them had the genes encoding enterotoxins A (33.4%) and B (16.6%). Furthermore, the majority of the isolates (83%) were resistant to at least one of the tested antimicrobials and multidrug resistance was observed in 8.4% of the isolates. The isolates had virulence potential, and half of them were enterotoxigenic. In addition, the ability of all the isolates to produce biofilms highlights the danger posed by these potentially virulent microorganisms persisting in food manufacturing environments.
Detection of biofilm related genes, classical enterotoxin genes and agr typing among Staphylococcus aureus isolated from bovine with subclinical mastitis in southwest of Iran
2016, Microbial Pathogenesis
Citation Excerpt :
In a study by Karahan et al. on S. aureus isolated from cow with subclinical mastitis the sea gene was not detected and the seb, sec and sec was detected in 3.26, 3.26 and 2.17% of isolates respectively [51]. Da Silva detected the sea and seb genes in 6.3% and the sec in 3.1% of mastitis causing S. aureus isolates [52]. Similar to our results in studies by Rall et al. and Boynukara et al. on S. aureus isolated from bovine with subclinical mastitis, the sea gene was the prominent genes but with much higher rate than our result (52.25, 23.6% Vs 10%) [17,53].
Staphylococcus aureus by producing biofilm and facilitating chronic infection is a common cause of mastitis in cows and thereby can cause food poisoning by production of enterotoxins in milk. The agr typing method is an important tool for epidemiological investigation about S. aureus. The aims of the present study were to detect biofilm related genes, 5 classical enterotoxin genes and the agr types among S. aureus isolates. The ability of S. aureus isolates to produce biofilm was evaluated by modified CRA plate. Six biofilm related adhesion genes (icaD, icaA, fnbA, bap, clfA and cna), five classical enterotoxin genes (sea, seb, sec, sed and see) and tst-1 gene were detected by PCR methods. Multiplex-PCR was used to determination of the agr groups. 55 out of 80(68.8%) S. aureus isolates were biofilm producer. The icaD gene was detected in 70 (87.5%) of isolates. The prevalence rates of fnbA, icaA, clfA, cna and bap were 72.5, 56.25, 50, 22.5, and 5% respectively. The agr group I and III were detected in 57.5% 25% of studied isolates. The sea, sed and tst-1 genes were found in 10%, 7.5% and 1.25% of isolates respectively. The majority of S. aureus were able to produce biofilm. Significant associations were observed between presence of the icaD, icaA, fnbA, clfA and the cna genes as well as biofilm formation. The present study revealed that isolates with the agr type III are more potent for biofilm production. Our data supported a possible link between the agr types and certain SE genes.
Antimicrobial activity of essential oils of Origanum vulgare L. and Origanum majorana L. against Staphylococcus aureus isolated from poultry meat
2015, Industrial Crops and Products
Citation Excerpt :
These results emphasize that three isolates (L1, L5 and L18) are carriers of both sea and seb genes, and can be considered potential enterotoxigens. Silva et al. (2005) highlight that the presence of two genes of staphylococcal enterotoxins in the same isolate maximizes their toxigenic capacity. When they are present in food the expression of two genes can occur at the same time, and thus they are involved in cases and/or food poisoning outbreaks.
One of the microorganisms present in poultry meat products responsible for the most usual foodborne intoxication is Staphylococcus aureus. Essential oil of spices has been used as a bio-preservative due to its antimicrobial activity against foodborne pathogens. The aim of this study was to evaluate the antimicrobial activity in vitro of the essential oils of oregano and marjoram against S. aureus isolated from poultry meat marketed in Pelotas/RS—Brazil, and to carry out the phenotypic and molecular characterization of these oils. For this, twenty samples of poultry meat were analyzed. Staphylococcus spp. was isolated from Baird Parker agar, and its molecular characterization was performed by PCR. All isolates of S. aureus were tested for the presence of genes coding for staphylococcal enterotoxins. Antimicrobial activity of essential oils of oregano and marjoram was evaluated by disk diffusion, minimal inhibitory concentration and minimal bactericidal concentration. The results showed that seventeen samples were contaminated by Staphylococcus positive-coagulase and nine were positive to acriflavine resistance and thermonuclease tests, and in the last ones S. aureus was confirmed. The genes coding for staphylococcal enterotoxins detected were sea (6) and seb (3). All isolates were sensitive to essential oils tested, indicating that these can be an alternative to food preservation.
Occurrence of Salmonella, Listeria monocytogenes, and enterotoxigenic Staphylococcus in goat milk from small and medium-sized farms located in Minas Gerais State, Brazil
2015, Journal of Dairy Science
Consumption of goat milk has been increasing due to its nutritional characteristics and health benefits. Therefore, assessment of the presence of foodborne pathogens in this product is critical to ensure its safety to consumers. The present study aimed to identify common foodborne pathogens in raw goat milk. Fifty-three samples of raw goat milk from 11 farms were collected and cultured for the presence of Salmonella spp. and Listeria monocytogenes, as well as for enumeration and isolation of coagulase-positive and coagulase-negative Staphylococcus (CPS and CNS, respectively). All samples tested negative for Salmonella spp. and L. monocytogenes. The CPS counts in raw goat milk samples were predominantly less than 2 log cfu/mL (n = 39), and CNS counts were predominantly higher than 3 log cfu/mL (n = 42). Based on Staphylococcus counts, 51 isolates were selected (CPS = 26; CNS = 25) and tested by PCR for the presence of classic enterotoxin-encoding genes (sea, seb, sec, sed, and see). Only 3 isolates (CPS = 2, CNS = 1) were negative for all enterotoxin-encoding genes tested, and the genotype sec and see was the most frequent (n = 16), followed by sea, sec, and see (n = 13) and sec (n = 13); sed was not detected in any isolate. The frequencies of enterotoxin-encoding genes for CPS and CNS were similar, demonstrating the equivalence of both groups in harboring these virulent markers. These results suggest that Salmonella and L. monocytogenes are not frequent contaminants of raw goat milk, but that Staphylococcus spp. that are capable of producing enterotoxins are prevalent; therefore, consumers of raw goat milk and products made from raw milk are at risk.
Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis
2015, Microbial Pathogenesis
The aim of this study was to investigate genes involved in adhesion expression, biofilm formation, and enterotoxin production in isolates of Staphylococcus spp. from goats with subclinical mastitis and associate these results with the staphylococcal species. One hundred and twenty-four isolates were identified and polymerase chain reaction (PCR) was performed to detect the following genes: cna, ebpS, eno, fib, fnbA, fnbB, bap, sea, seb, sec, sed and see. The most commonly Staphylococcus species included S. epidermidis, S. lugdunensis, S. chromogenes, S. capitis ss capitis and S. intermedius. With the exception of fnbB, the genes were detected in different frequencies of occurrence in 86.3% of the Staphylococcus spp. isolates. Eno (73.2%) and bap (94.8%) were more frequently detected in coagulase-negative staphylococci (CNS); ebpS (76%), fib (90.9%) and fnbA (87%) were the most frequent genes in coagulase-positive staphylococci (CPS). Regarding enterotoxins, genes sed (28.2%) and see (24.2%) had a higher frequency of occurrence; sec gene was more frequently detected in CPS (58.8%). There was no association between the presence of the genes and the Staphylococcus species. Different virulence factors genes can be detected in caprine subclinical mastitis caused by CNS and CPS. The knowledge of the occurrence of these virulence factors is important for the development of effective control and prevention measures of subclinical mastitis caused by CNS and CPS in goats.

View all citing articles on Scopus

¹: Tel.: +55 31 3499 2106.

²: Tel.: +55 31 3371 9466.

View full text

Article preview

Veterinary Microbiology

Abstract

Introduction

Section snippets

Sources and strains of S. aureus

Production and detection of SEA, SEB, and SEC

Specificity and reproducibility testing

Discussion

References (15)

Development and use of PCR primers for the investigation of C1, C2 and C3 enterotoxin types of Staphylococcus aureus strains isolated from food-borne outbreaks

Int. J. Food Microbiol.

Hemolysin production by Staphylococcus species isolated from mastitic goat milk

Small Rumin. Res.

Differences between Danish bovine and human Staphylococcus aureus isolates in possession of superantigens

Vet. Microbiol.

Superantigens: microbial agents that corrupt immunity

Lancet Infect. Dis.

Staphylococcal enterotoxin genes of classical and new types detected by PCR in France

Int. J. Food Microbiol.

Characterization of leukocytotoxic and superantin-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis

Vet. Microbiol.

Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland

Vet. Microbiol.

Cited by (57)

Relationship between virulence factors and antimicrobial resistance in Staphylococcus aureus from bovine mastitis

Staphylococcus aureus isolated from handmade sweets: Biofilm formation, enterotoxigenicity and antimicrobial resistance

Detection of biofilm related genes, classical enterotoxin genes and agr typing among Staphylococcus aureus isolated from bovine with subclinical mastitis in southwest of Iran

Antimicrobial activity of essential oils of Origanum vulgare L. and Origanum majorana L. against Staphylococcus aureus isolated from poultry meat

Occurrence of Salmonella, Listeria monocytogenes, and enterotoxigenic Staphylococcus in goat milk from small and medium-sized farms located in Minas Gerais State, Brazil

Virulence factors genes of Staphylococcus spp. isolated from caprine subclinical mastitis