Multiplex PCR and RPLA Identification of Staphylococcus aureus Enterotoxigenic Strains From Bulk Tank Milk

Abstract

Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains.

Occurrence of methicillin-resistant strains of Staphylococcus aureus at a goat breeding farm

“…Similar data were reported by Zouharova and Rysanek (2008) in bulk tank cow's milk samples (15.9%). MRSA was found only in goat's milk samples and the irregular detection of MRSA in bulk tank milk samples was probably due to substantial dilution of contaminated milk and thus to substantially reduced likelihood of MRSA detection.…”

Occurrence and antibiotic resistance of enterotoxigenic Staphylococcus aureus in raw ovine and caprine milk in Greece

“…Similarly, several studies reported that none of the strains isolated from bovine and CM and dairy products harbored the seb gene (Akineden et al 2001;Lyra et al 2013). In other studies, however, the seb gene was detected in S. aureus isolates from milk and dairy products at various frequencies (Jørgensen et al 2005;Zouharova and Rysanek 2008). The fact that seb was the most commonly detected SE genotype in isolates from OM in our study is in agreement with the findings of previous studies (Arcuri et al 2010;Peles et al 2007).…”

“…None of the strains in our study harbored the see gene, in agreement with other investigations on S. aureus isolates from milk and dairy products (Arcuri et al 2010;Carfora et al 2015;Cremonesi et al 2007;Jørgensen et al 2005;Poli et al 2007;Zouharova and Rysanek 2008).…”

“…Some authors reported a predominant and systematic co-detection of seg and sei genes (Rosec and Gigaud 2002) whereas others have reported their presence individually or in different combinations with other se genes (Arcuri et al 2010;Cremonesi et al 2007;Zouharova and Rysanek 2008). The association between seg and sei has been attributed to their location within the same gene cluster (egc) in the genomic island mSAb (also known as SaPIn3/m3) (Malachowa and DeLeo 2010).…”

“…Since, to date, no commercial tests for the "new" types of SEs exist, S. aureus isolates containing the corresponding genes should be considered as potential SE producers. Α review of the literature seems to indicate an increase in the number of potentially enterotoxigenic S. aureus isolates from milk and dairy products carrying "new" SE genes in addition to the classical ones (Arcuri et al 2010;Rosec and Gigaud 2002;Zouharova and Rysanek 2008). It should be noted, however, that in our study the prevalence of se genes may have been underestimated because only the isolates testing positive for SE production via immunoassays were tested with PCR, as well as due to the fact that the isolates were not tested for the presence of the recently described se genes (sek-ser and seu).…”