Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary

https://doi.org/10.1016/j.ijfoodmicro.2007.07.010Get rights and content

Abstract

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene (tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0 × 10CFU/ml. Farm size had no significant effect (P > 0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes (seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.

Introduction

Staphylococcus aureus is the most prevalent and economically significant pathogen causing intramammary infections in dairy ruminants (Akineden et al., 2001, Cabral et al., 2004, Katsuda et al., 2005). The organism is responsible for approximately 30% to 40% of all mastitis cases (Asperger and Zangerl, 2003). S. aureus can gain access to milk either by direct excretion from udders with clinical or subclinical staphylococcal mastitis or by contamination from the environment during handling and processing of raw milk (Scherrer et al., 2004, Jørgensen et al., 2005c). When the udder is infected, S. aureus is excreted in the milk with large fluctuations in counts ranging from zero to 108 CFU/ml (Asperger and Zangerl, 2003).

In the European Union, criteria have been established for the S. aureus content in raw bovine milk intended for processing without prior heat treatment. The m value, which separates acceptable from marginally acceptable quality, is 5.0 × 102 CFU/ml or 2.70 log10 CFU/ml; and the M value separating marginally acceptable from defective quality is 2.0 × 103 CFU/ml or 3.30 log10 CFU/ml (Council of the European Communities, 1992).

Foodborne diseases have a major public health impact. It is estimated that in the United States alone foodborne illnesses affect 6 million to 80 million people each year, causing up to 9000 deaths, and cost about 5 billion US dollars (Balaban and Rasooly, 2000). S. aureus is considered the third most important cause of disease in the world among the reported foodborne illnesses (Asperger and Zangerl, 2003, Normanno et al., 2005, Boerema et al., 2006). The growth of S. aureus in foods presents a potential public health hazard because many strains of S. aureus produce enterotoxins (SEs) that cause food poisoning if ingested (Akineden et al., 2001, Cenci-Goga et al., 2003, Boerema et al., 2006). Milk and dairy foods have frequently been implicated in staphylococcal food poisoning, and contaminated raw milk is often involved (De Buyser et al., 2001). SEs are a family of exoproteins forming a single chain with a molecular weight ranging from 26,000 to 29,600 Da (Balaban and Rasooly, 2000, Asperger and Zangerl, 2003, Normanno et al., 2005). Unlike the producer organism, SEs are remarkably heat resistant, showing D-values of 3 min to 8 min at 121 °C (Asperger and Zangerl, 2003). As a result, they may be present in foods even when viable S. aureus are absent (Jørgensen et al., 2005c). Traditionally, five classical antigenic SE types (SEA, SEB, SEC, SED, and SEE) were recognized. However, in recent years, the existence of new types of SEs, including enterotoxin-like (SEl) toxins (SEG, SEH, SEI, SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElR, SElU, SElU2, and SElV) has been reported and their genes described (Letertre et al., 2003, Lina et al., 2004, Omoe et al., 2004, Jørgensen et al., 2005a, Bania et al., 2006, Boerema et al., 2006, Hata et al., 2006, Thomas et al., 2006). A distantly related protein, toxic shock syndrome toxin 1 (TSST-1), also produced by S. aureus, was the first toxin shown to be involved in the toxic shock syndrome of humans and animals (Akineden et al., 2001).

Over the last decade, various typing methods have been developed for the characterization of S. aureus isolates (Hata et al., 2006). Phenotyping methods have gradually been supplemented or replaced with genotyping methods. Numerous techniques have been described for S. aureus genotyping, of which pulsed-field gel electrophoresis (PFGE) is considered to be the “gold standard” because of its discriminatory power and reproducibility (Weller, 2000). The polymerase chain reaction (PCR) has been introduced as a simple technique for the detection of enterotoxigenic strains (Asperger and Zangerl, 2003, Kwon et al., 2004). Although the PCR-based approach is specific, highly sensitive, and rapid, it can only demonstrate the presence of enterotoxin genes in S. aureus isolates rather than the production of the SE protein (Boerema et al., 2006).

The main objectives of this study were to enumerate S. aureus in milk from dairy farms of different sizes and to characterize both pheno- and genotypically the S. aureus strains isolated from the mammary quarter milk of mastitic cows and from bulk tank milk. Genotyping was performed by PFGE analysis, and the prevalence of genes encoding various enterotoxins was determined by multiplex PCR.

Section snippets

Farms

Twenty farms (7 large, 4 medium-size, and 9 small farms designated as LF1 to LF7, MF8 to MF11, and SF12 to SF20, respectively) were enrolled in the study carried out from June 2005 through August 2006. The farms were located in the eastern part of Hungary, at a distance of 15 km to 100 km from one another. Herd size varied from 4 cows to 520 cows of Holstein Friesian and/or Hungarian Red Pied breeds. The S. aureus counts in bulk tank milk were determined four times on each farm throughout the

Isolation and enumeration of S. aureus

The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0 × 103 CFU/ml, which was in accordance with a previous report by Stephan, Buehler & Lutz (2002), who determined that the S. aureus counts ranged from 1.0 × 101 CFU/ml to 3.0 × 103 CFU/ml in bulk tank milk samples in Switzerland. No staphylococci were recovered from bulk milks collected from farms LF2, LF7, MF10, MF11, SF19, and SF20. The mean S. aureus counts were high (> 3.30 log10 CFU/ml or 2.0 × 103

Conclusions

S. aureus, the most prevalent pathogen causing mastitis in dairy cows, is regularly found in bulk tank milk because the principal source of microbial contamination of raw milk is the infected udder. For this reason, S. aureus counts in bulk milk are related to the mastitis situation of the herd and may range from less than 10 to several thousands CFU/ml. S. aureus was recovered from the majority (55%) of bulk milk samples examined in this research. The spread of antibiotic resistant bacteria

Acknowledgments

The authors gratefully acknowledge the collaboration and cooperation of the 20 farms involved in this study. This research was supported in part by grants from the Austro-Hungarian Action Fund (460-7/2005) and the Institute of Milk Hygiene, Milk Technology, and Food Science of the University of Veterinary Medicine at Vienna.

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