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Incidence, growth and enterotoxin production of Staphylococcus aureus in insufficiently dried traditional beef ham “govedja pršuta” under different storage conditions
Food Control 27 (2012) 369e373
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Incidence, growth and enterotoxin production of Staphylococcus aureus in insufficiently dried traditional beef ham “govedja prsuta” under different storage conditions Andreja Rajkovic a, b, * a b
Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium Department of Food Safety and Food Quality Management, Faculty of Agriculture, Belgrade University, Nemanjina 6, 11080 Zemun, Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Received in revised form 21 March 2012 Accepted 27 March 2012
Market pressures for shorter product turnover and faster selling result in modified production practices of Serbian traditional dry, cured beef ham known as “govedja prsuta”. These are mainly characterized by incomplete maturation and drying of the hams, resulting in higher water activity levels of products that require correctly chosen intrinsic and extrinsic factors to control foodborne pathogens. An incidence of almost 90% has been found after the enrichment of Staphylococcus aureus in 20 tested samples produced by five different producers and marketed in different parts of Serbia. Although important these findings do not completely surprise due to known high prevalence of S. aureus. However, combined with market-driven shorter and incomplete maturation and drying of the products this high prevalence of low numbers resulted in growth and staphylococcal enterotoxin (SE) production in 35% of samples tested (7/20). Results of the analysis showed that enumerable S. aureus counts were found only in four out of twenty samples, but with counts ranging from 2.8 to 3.7 log CFU/g suggesting that actual outgrowth and toxin production perhaps occurred earlier in the production stages. Nevertheless, inoculation of enterotoxigenic isolates in the same type of product, previously found negative on the presence of S. aureus and SE, showed that both growth and SE production can occur even in the final product at 22 C and 37 C, but not at 12 C, regardless of whether vacuum or aerobic packaging of the product was applied. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Enterotoxin Dried Cured Ham
1. Introduction Staphylococcus aureus intoxication remains one of the most common causes of foodborne diseases (Bennett, 2005), which is often found in many different food products (Bartolomeoli, Maifreni, Frigo, Urli, & Marino, 2009; Guven, Mutlu, Gulbandilar, & Cakir, 2010; Pereira et al., 2009; Seo, Jang, & Moon, 2010). Improper holding times and temperatures were the most important factors associated with outbreaks involving S. aureus and in fact caused by the consumption of food containing heat stable enterotoxin(s) (Hedberg, Palazzi-Churas, Radke, Selman, & Tauxe, 2008). Although many foods harbour small numbers of S. aureus and can be eaten safely, time and temperature abuse of a food product can result in sufficient growth of present S. aureus causing formation of enterotoxins. Quantities between 100 and 200 ng of consumed * Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium. Tel.: þ32 92649904; fax: þ32 92255510. E-mail addresses: [email protected], [email protected]. 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.03.028
enterotoxin can cause symptoms of staphylococcal intoxication. These toxin levels are presumably reached when S. aureus population exceeds 105e106 CFU per gram (Bennett, 2005). The presence of relatively large numbers of enterotoxigenic S. aureus is a good circumstantial evidence that the food contains toxins, but the absence of S. aureus cells is not an evidence of toxin absence, therefore both cells and toxins should be part of investigation (Rajkovic, 2006). The classical antigenic types of S. aureus enterotoxins (SE) incriminated in staphylococcal food poisoning are SEA, SEB, SEC, SED and SEE. New serological types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER and SEU) were identified in the last decade (Blaiotta et al., 2004; Lovseth, Loncarevic, & Berdal, 2004; Omoe et al., 2004). Detection techniques for some of the staphylococcal enterotoxins are commercially available and require from one and a half to 24 h to be completed. Reported detection limits range from 0.5 to 2 ng enterotoxin per gram of food (Di Pinto, Forte, Ciccarese, Conversano, & Tantillo, 2004; Mathieu, Isigidi, & Devriese, 1992; Park, Akhtar, & Rayman, 1994; Vernozy-Rozand, Mazuy-Cruchaudet, Bavai, & Richard, 2004; Wieneke, 1991).
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Its ubiquitous prevalence, and versatile pathogenic character make staphylococcal intoxications one of the (re)emerging foodborne diseases still today (Newell et al., 2010). Its role on foodborne outbreaks caused by raw cured hams has been well documented (Atanassova, Meindl, & Ring, 2001; Portocarrero, Newman, & Mikel, 2002). EFSA reported that in 2.3% of the verified outbreaks caused by staphylococcal toxins in EU, 2009 vector was bovine meat or products thereof and about 18% were meat products in general (EFSA, 2011). Although this risk is well recognized no easily available information exits on its prevalence and the prevalence of its toxins in typical and largely consumed Serbian meat products. Among the most valued products are smoked, dry beef hams (beef prshuta). These products are produced locally in many households and sold on local and regional markets, but they are not to be confused with “Uzice Beef Prshuta” a product with Protected Designation of Origin originating from the region of mountain Zlatibor (south-western part of Serbia), which is produced of the most valuable parts of beef carcass (round muscles, loin muscles and tenderloin) originating from well-fed, 3- to 5-year-old cattle. The details of the production process are elaborated elsewhere (Tomic, Tomasevic, Radovanovic, & Rajkovic, 2008 and therein Radovanovic, Cavoski, Velickovic, & Carapic, 1990; Radovanovic, Cavocki, Velickovic, & Obradovic, et al., 1990). However, there are imitations of the U zicka Prsuta, some produced in an artisan way in the surrounding regions, and sold under the geographical names “Zlatibor” or “Uzice”. Others artisan or industrial beef Prsuta are no imitations, but direct competitors. In addition to typical steps of dry-curing process, these products are in general characterized by (extensive) smoking. The specificity of each products is mainly built at the processor level. At the level of consumer, the U zicka Prsuta has no guarantee premium in comparison with other beef Prsuta, which allows other products to compete with lower quality and higher margins (Bernardoni, Estève, Paus, & Reymann, 2008). Based on their intrinsic properties many of these products can theoretically host S. aureus and even allow its proliferation and toxin production. While this chance diminishes with correctly chosen raw materials, good hygienic practices and good manufacturing practices, and especially with sufficient maturation, drying and smoking of the product (GMP utilizes salting of up to 20 days, maturation, drying and smoking of up to 35 days with 8 h/a day of cold smoking), an economic situation requires cheaper (not necessarily sold at lower price) products to be placed on the market. The later results in the occurrence of commercial practices that yield products with higher aw and pH values, with lower antimicrobial effect of smoke components and made of lower quality raw materials, increasing the risk related to the presence of microbial foodborne hazards. Therefore the goal of the research described in this manuscript was to determine the occurrence of S. aureus and its main four enterotoxins (SEAeSED) in beef ham. Moreover, the same products were inoculated with a cocktail of three different S. aureus strains to investigate survival, growth and toxin production potential.
2. Materials and methods 2.1. Samples The total number of 20 packages of sliced and vacuum packaged raw dried beef ham were obtained from five different producers at point of sale. Every sample was divided in five parts, one for the initial microbiological analysis, three for inoculation study and one for the determination of initial pH and aw values with pH electrode designed for monitoring the pH of meat (Mettler Toledo AG, Urdorf, Switzerland) and Durotherm aw-Wert-messer (Haiterbach, Germany), respectively.
2.2. Microbiological analysis The native presence of S. aureus was determined using a standard spread plate technique on Baird-Parker agar at 37 C during 24 h. Samples that tested negative on S. aureus were subjected to the enrichment following the protocol of ISO 6888-3:2003 in modified Giolitti and Cantoni broth (Oxoid, Basingstoke, U.K.) with potassium tellurite and under anaerobic conditions. Presumptive S. aureus colonies were isolated on Baird-Parker agar (Oxoid). Selected colonies were confirmed by the coagulase activity test using rabbit plasma (BioRad, Hercules, CA, USA). Identification was confirmed by profile determination using API Staph system (bioMérieux, Marcy l’Etoile, France). 2.3. Enterotoxin analysis The SE presence was serologically determined by reversed passive latex agglutination technique using Oxoid SET-RPLA Kit for detection of Staphylococcal enterotoxins A, B, C and D (Shingaki et al., 1981). In short, 10 g of sample was mixed with 10 ml of sodium chloride solution (0.85%) in a homogeniser. The homogenized sample was centrifuged at 900 g at 4 C for 30 min and the obtained supernatant was passed through the 0.2e0.45 mm low protein-binding membrane filter to be used for assay of toxin content. Extracts were immediately analysed, as prescribed by the manufacturer. Except for the extraction step, the analysis done in the culture to check the enterotoxin production by isolated S. aureus (isolates grown in Tryptone Soya Broth, TSB, Oxoid for 24 h at 37 C) were performed on the same way. 2.4. Inoculation study A cocktail of three enterotoxigenic isolates coming from the screening of analysed samples were mixed in 1:1:1 ratio to obtain concentration of approx. 3 log CFU/ml. Previously found as negative, on both enterotoxin and S. aureus presence, meat samples of 50 g were inoculated with 1 ml of the inoculum suspension to obtain approx. 2.5 log CFU/g. The samples were vacuum or air packaged and stored for three weeks at 12 and 22 C and for two weeks at 37 C. Each sample was foreseen as a separate package. The packages were analysed daily during the storage on the presence of S. aureus and enterotoxins, as described above. 3. Results and discussion The experience has shown that staphylococci may be expected to exist, at least in low numbers, in probably all food products that are of animal origin or in those that are handled directly by humans, unless heat-processing steps are applied to affect their destruction (Jay, 2000). Therefore the success of prevention and the control of S. aureus growth and enterotoxins production will be mainly dependent on the inherent product characteristics and strict application of GMP, GHP and HACCP principles. The pH and aw found in 20 samples of purchased beef ham ranged from 5,11 to 5,60, and from 0.901 to 0.940, respectively (Table 1). Of 20 tested samples, four samples contained enumerable S. aureus counts of 3.2, 2.8, 3.7 log CFU/g belonging to producer 1 and 3.1 log CFU/g belonging to producer 4. Other 14 samples were found positive for the presence of S. aureus following the enrichment. Analysis of enterotoxins with SET-RPLA [Oxoid, Basingstoke, United Kingdom] showed all but seven of the samples to be free of detectable SEA, SEB, SEC and SED (Table 1). Four samples were found positive on the presence of SEA (encompassing 3 producers: producer 1, 2 and 4), two samples on both SEA and SED (one producer: producer 2), and one on SEB (one producer: producer 5). Surprisingly all enumerable S. aureus were
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Table 1 Staphylococcus aureus and staphylococcal enterotoxins presence in analysed dried cured beef ham samples. Producer
Samples containing S. aureus
Samples containing SE
aw and pH in the ham samples SE positive samples
1
4/4
2/4 SEA
2
4/4
1/4 SEA, 2/4 SEAeSED
3
3/4
No SE containing samples
4
4/4
1/4 SEA
5
3/4
1/4 SEB
a
aw 0.925e0.940 pH 5.1e5.3 aw 0.921e0.937 pH 5.2e5.5
aw 0.923 pH 5.4 aw 0.933 pH 5.5
SE negative samples
SEAeSEE production by isolated S. aureus under optimal growth conditions in TSBa 2 SEA isolates
aw 0.920 pH 5.3 aw 0.901e0.915 pH 5.2e5.6 aw 0.918e0.923 pH 5.2e5.5 aw 0.921e0.931 pH 5.2e5.5
2 SEA isolates and 1 SEAeSED isolate 2 SEA isolate 2 SEA isolates, 1 SEB isolate and 1 SEC isolates 1 SEA isolates and 1 SEB isolate
One isolate per package was analysed.
SEA positive isolates. These findings confirm that enumeration of S. aureus may lead to the false evaluation of the product safety. While all five producers had positive samples on the presence of S. aureus, four also had samples positive on SE. Based on elsewhere published data the differences in pH and aw between the products of different producers are relatively small and presumably account only for some of the observed differences. Other authors reported that the majority of S. aureus strains tested produced detectable amounts of SE aerobically at a pH of 5.1 (Hennekinne, Buyser, & Dragacci, 2011). SEA and SED production occurs under nearly all aw conditions allowing growth of S. aureus as long as all other conditions are optimal. Although the production of SEB and SEC is thought to be very sensitive to reductions in aw and hardly any is produced at aw 0.93 despite extensive growth, the current study showed that SEB can be found in products with aw lower than 0.93, possibly due to formation in earlier production stages (Ewald & Notermans, 1988; Qi & Miller, 2000). Presence of other type of background flora, perhaps use of lactate, nitrite or other components in brining mixture might have also contributed to the observed differences. The isolated S. aureus from each of the 18 S. aureus e positive samples were grown in TSB and in-vitro toxin production was controlled by SET-RPLA as instructed by the manufacture (Oxoid). Nine isolates produced SEA only, one produced SEA and SED, 2 produced SEB and one produced SEC. The remaining isolates produced none of SEAeSED enterotoxins, regardless of the growth to more than 7 log CFU/ml. The obtained data are in agreement with generally established knowledge that the most common toxin implicated in staphylococcal food poisoning is SEA (Balaban & Rasooly, 2000). Further characterisation of the isolates regarding other SE or SE like toxins, as well as regarding their growth characteristics, might provide valuable information on other food safety risks involved. The importance of this is illustrated by the fact that to date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. Moreover, any food that provides a convenient medium for growth of S. aureus (or other enterotoxigenic Staphylococcus species) in any of the stages of production may be involved in a staphylococcal food poisoning outbreak, SFP (Hennekinne et al., 2011).
Contamination, growth and enterotoxin production remain a prerequisite for SFP. To investigate the growth and toxin production in the same meat products a cocktail made of one SEAeSED, one SEB and one SEC producing isolates (equal ratios to make a final inoculum of approx. 2.5 log CFU/g) was used to inoculate six samples that were previously found negative on both S. aureus and four classical SE (Table 2). At 12 C in both aerobic and vacuum conditions no growth was observed during three weeks of storage, but enrichment showed presence of viable S. aureus at all times during three weeks with sampling done every three days. At 22 C results in food showed a very restricted growth resulting in an increase of S. aureus numbers of approx. 2.5 log CFU/g in 15 days in air, reaching maximal cell density of approx. 6.5 log CFU/g after three weeks of storage. At the same temperature the growth onset in vacuum packaging was delayed for another day. However, the variation around the mean counts (enumerations of each sample were done in duplicate) was somewhat higher than under aerobic conditions, indicating that vacuum provided certain stress on the S. aureus population probably favouring some fractions of present cells and stressing other ones (Rajkovic, Smigic, & Devlieghere, 2010; Rajkovic et al., 2009). At growth temperature of 37 C and all other conditions the same, the growth onset was for both aerobic and vacuum conditions about one day, reaching 7.2 log CFU/g after 4 days of storage. It is one of the key findings that in none of the conditions at 12 C was SE production observed. At 22 C and 37 C all samples contained detectable amounts of SEA and SED, but not of SEC and SEB toxins, after 15 days and 3 days, respectively. This detection SET-RPLA detection limit was reached only at S. aureus counts higher than 5 log CFU/g, but this does not preclude earlier onset of SE production. To verify if the lack of SEB and SEC production was due to insufficient growth the two respective isolates were inoculated in the additional slices of beef ham and incubated at 22 C and 37 C. Results showed that none of the two isolates was able to produce SEB and SEC after two weeks of storage, measured at the end of the storage period, regardless of final S. aureus count of 7 log CFU/g. This, together with the fact that used SET-RPLA method does not detect other SE but SEAeSED, and that some of the natively present S. aureus might have been injured or stressed to be recovered on the
Table 2 Staphylococcus aureus and staphylococcal enterotoxins in inoculated beef ham samples. Parameter
12 C Aerobic
Time to 5 log CFU/g Maximal cell densitya Enterotoxin production a
22 C Vacuum
No growth No SEA, SEB and SEC production
Aerobic
37 C Vacuum
15 Days 16 Days 6.5 0.3 log CFU/g (aerobic) to 6.7 0.7 log CFU/g (vacuum) in 21 days SEA and SED after 15 days
Mean values of two enumerations done on the same sample.
Aerobic
Vacuum
2 Days 7.2 0.6 log CFU/g in 4 days SEA and SED after 3 days
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selective medium during enumeration, could explain differences in the percentage of inoculated samples containing SE (100%) and native samples containing SE (35%). The range of environmental parameters over which S. aureus will produce enterotoxins is accepted to be narrower than the range over which it will grow and are influenced also by the composition of food (Jay, 2000). In general, enterotoxin production is substantially reduced at 20e25 C and it is generally accepted that enterotoxin production is unlikely to occur at temperatures below 10 C. Optimum enterotoxin production occurs at pH 6e7 and is influenced by atmospheric conditions, carbon and nitrogen source and salt level. Although reduced levels of aw may also inhibit toxin synthesis more than growth the toxin production has been reported at as low as aw ¼ 0.86 (under otherwise optimal conditions). Similar findings were reported by Portocarrero et al. (2002) who found that initially inoculated S. aureus populations of 8.57 and 8.12 log CFU/cm2 for salt and salt þ NO2 hams decreased to below the levels of detection after the fourth month of ageing. S. aureus was detected following enrichment for 75% of the inoculated and 62% of the control (non-inoculated) hams at the end of the ageing period. Staphylococcal enterotoxin was detected in 40% of the inoculated and 50% of the control hams following the ageing period. The NaCl content of these hams were 4.45/3.37% and aw values 0.94/0.91. Country-cured ham products obtained from retail stores in that study were all negative for S. aureus enterotoxin. These results indicated that higher salt content and lower aw values on commercially produced country-cured hams played an important role in controlling the growth and toxin production of S. aureus. In the case of our study it is important to note that while drying and curing of hams may or may not destroy S. aureus, the high salt content on the exterior will often inhibit the superficial growth of these bacteria. However, when the ham is sliced the moister interior can permit staphylococcal multiplication and thus sliced dried and cured hams should be refrigerated (FSIS-USDA, 2011). The vacuum package used in this study was not sufficient to prevent this. Not to different results were found by Wallin-Carlquist, Marta, Borch, and Radstrom (2010) reporting no growth of inoculated 6 log CFU of S. aureus on the small surface of Serrano hams stored for seven days at 23 C, but reporting SEA in the same samples. Obtained results in the current study confirm the ubiquitous prevalence of S. aureus in meat products and suggest that insufficiently dried and matured hams as studied here provide niche for growth of S. aureus and enterotoxin formation. Moreover, it has been seen that one sample can contain multiple enterotoxins leading to more complex doseeresponse scenarios. These hams should not be stored at temperatures higher than 12 C and preferably should be refrigerated. Moreover, the high prevalence of S. aureus and enterotoxins indicates that improved control of raw materials and all production stages is required. It has been recently reported that detectable amounts of SE are produced at S. aureus counts of as low as 3 log CFU/g under permissible conditions (Rajkovic et al., 2006). This suggests that possibly even stricter measures are to be implemented to control this microbial hazard, and that certainly no loose setting deviations from elaborated good practices and HACCP plans are to be made. Further investigations should show whether this niche can offer growth habitat to other potential microbial hazards leading possibly to muti-hazard exposure. The safety of the products needs to remain superior request to that of the commercial needs that may require faster turnover through short maturation time of cured meat products. Acknowledgement This work was supported by the FWO grant of Dr. Andreja Rajkovic
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Incidence, growth and enterotoxin production of Staphylococcus aureus in insufficiently dried traditional beef ham “govedja prsuta” under different storage conditions Andreja Rajkovic a, b, * a b
Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium Department of Food Safety and Food Quality Management, Faculty of Agriculture, Belgrade University, Nemanjina 6, 11080 Zemun, Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2012 Received in revised form 21 March 2012 Accepted 27 March 2012
Market pressures for shorter product turnover and faster selling result in modified production practices of Serbian traditional dry, cured beef ham known as “govedja prsuta”. These are mainly characterized by incomplete maturation and drying of the hams, resulting in higher water activity levels of products that require correctly chosen intrinsic and extrinsic factors to control foodborne pathogens. An incidence of almost 90% has been found after the enrichment of Staphylococcus aureus in 20 tested samples produced by five different producers and marketed in different parts of Serbia. Although important these findings do not completely surprise due to known high prevalence of S. aureus. However, combined with market-driven shorter and incomplete maturation and drying of the products this high prevalence of low numbers resulted in growth and staphylococcal enterotoxin (SE) production in 35% of samples tested (7/20). Results of the analysis showed that enumerable S. aureus counts were found only in four out of twenty samples, but with counts ranging from 2.8 to 3.7 log CFU/g suggesting that actual outgrowth and toxin production perhaps occurred earlier in the production stages. Nevertheless, inoculation of enterotoxigenic isolates in the same type of product, previously found negative on the presence of S. aureus and SE, showed that both growth and SE production can occur even in the final product at 22 C and 37 C, but not at 12 C, regardless of whether vacuum or aerobic packaging of the product was applied. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Enterotoxin Dried Cured Ham
1. Introduction Staphylococcus aureus intoxication remains one of the most common causes of foodborne diseases (Bennett, 2005), which is often found in many different food products (Bartolomeoli, Maifreni, Frigo, Urli, & Marino, 2009; Guven, Mutlu, Gulbandilar, & Cakir, 2010; Pereira et al., 2009; Seo, Jang, & Moon, 2010). Improper holding times and temperatures were the most important factors associated with outbreaks involving S. aureus and in fact caused by the consumption of food containing heat stable enterotoxin(s) (Hedberg, Palazzi-Churas, Radke, Selman, & Tauxe, 2008). Although many foods harbour small numbers of S. aureus and can be eaten safely, time and temperature abuse of a food product can result in sufficient growth of present S. aureus causing formation of enterotoxins. Quantities between 100 and 200 ng of consumed * Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium. Tel.: þ32 92649904; fax: þ32 92255510. E-mail addresses: [email protected], [email protected]. 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.03.028
enterotoxin can cause symptoms of staphylococcal intoxication. These toxin levels are presumably reached when S. aureus population exceeds 105e106 CFU per gram (Bennett, 2005). The presence of relatively large numbers of enterotoxigenic S. aureus is a good circumstantial evidence that the food contains toxins, but the absence of S. aureus cells is not an evidence of toxin absence, therefore both cells and toxins should be part of investigation (Rajkovic, 2006). The classical antigenic types of S. aureus enterotoxins (SE) incriminated in staphylococcal food poisoning are SEA, SEB, SEC, SED and SEE. New serological types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER and SEU) were identified in the last decade (Blaiotta et al., 2004; Lovseth, Loncarevic, & Berdal, 2004; Omoe et al., 2004). Detection techniques for some of the staphylococcal enterotoxins are commercially available and require from one and a half to 24 h to be completed. Reported detection limits range from 0.5 to 2 ng enterotoxin per gram of food (Di Pinto, Forte, Ciccarese, Conversano, & Tantillo, 2004; Mathieu, Isigidi, & Devriese, 1992; Park, Akhtar, & Rayman, 1994; Vernozy-Rozand, Mazuy-Cruchaudet, Bavai, & Richard, 2004; Wieneke, 1991).
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Its ubiquitous prevalence, and versatile pathogenic character make staphylococcal intoxications one of the (re)emerging foodborne diseases still today (Newell et al., 2010). Its role on foodborne outbreaks caused by raw cured hams has been well documented (Atanassova, Meindl, & Ring, 2001; Portocarrero, Newman, & Mikel, 2002). EFSA reported that in 2.3% of the verified outbreaks caused by staphylococcal toxins in EU, 2009 vector was bovine meat or products thereof and about 18% were meat products in general (EFSA, 2011). Although this risk is well recognized no easily available information exits on its prevalence and the prevalence of its toxins in typical and largely consumed Serbian meat products. Among the most valued products are smoked, dry beef hams (beef prshuta). These products are produced locally in many households and sold on local and regional markets, but they are not to be confused with “Uzice Beef Prshuta” a product with Protected Designation of Origin originating from the region of mountain Zlatibor (south-western part of Serbia), which is produced of the most valuable parts of beef carcass (round muscles, loin muscles and tenderloin) originating from well-fed, 3- to 5-year-old cattle. The details of the production process are elaborated elsewhere (Tomic, Tomasevic, Radovanovic, & Rajkovic, 2008 and therein Radovanovic, Cavoski, Velickovic, & Carapic, 1990; Radovanovic, Cavocki, Velickovic, & Obradovic, et al., 1990). However, there are imitations of the U zicka Prsuta, some produced in an artisan way in the surrounding regions, and sold under the geographical names “Zlatibor” or “Uzice”. Others artisan or industrial beef Prsuta are no imitations, but direct competitors. In addition to typical steps of dry-curing process, these products are in general characterized by (extensive) smoking. The specificity of each products is mainly built at the processor level. At the level of consumer, the U zicka Prsuta has no guarantee premium in comparison with other beef Prsuta, which allows other products to compete with lower quality and higher margins (Bernardoni, Estève, Paus, & Reymann, 2008). Based on their intrinsic properties many of these products can theoretically host S. aureus and even allow its proliferation and toxin production. While this chance diminishes with correctly chosen raw materials, good hygienic practices and good manufacturing practices, and especially with sufficient maturation, drying and smoking of the product (GMP utilizes salting of up to 20 days, maturation, drying and smoking of up to 35 days with 8 h/a day of cold smoking), an economic situation requires cheaper (not necessarily sold at lower price) products to be placed on the market. The later results in the occurrence of commercial practices that yield products with higher aw and pH values, with lower antimicrobial effect of smoke components and made of lower quality raw materials, increasing the risk related to the presence of microbial foodborne hazards. Therefore the goal of the research described in this manuscript was to determine the occurrence of S. aureus and its main four enterotoxins (SEAeSED) in beef ham. Moreover, the same products were inoculated with a cocktail of three different S. aureus strains to investigate survival, growth and toxin production potential.
2. Materials and methods 2.1. Samples The total number of 20 packages of sliced and vacuum packaged raw dried beef ham were obtained from five different producers at point of sale. Every sample was divided in five parts, one for the initial microbiological analysis, three for inoculation study and one for the determination of initial pH and aw values with pH electrode designed for monitoring the pH of meat (Mettler Toledo AG, Urdorf, Switzerland) and Durotherm aw-Wert-messer (Haiterbach, Germany), respectively.
2.2. Microbiological analysis The native presence of S. aureus was determined using a standard spread plate technique on Baird-Parker agar at 37 C during 24 h. Samples that tested negative on S. aureus were subjected to the enrichment following the protocol of ISO 6888-3:2003 in modified Giolitti and Cantoni broth (Oxoid, Basingstoke, U.K.) with potassium tellurite and under anaerobic conditions. Presumptive S. aureus colonies were isolated on Baird-Parker agar (Oxoid). Selected colonies were confirmed by the coagulase activity test using rabbit plasma (BioRad, Hercules, CA, USA). Identification was confirmed by profile determination using API Staph system (bioMérieux, Marcy l’Etoile, France). 2.3. Enterotoxin analysis The SE presence was serologically determined by reversed passive latex agglutination technique using Oxoid SET-RPLA Kit for detection of Staphylococcal enterotoxins A, B, C and D (Shingaki et al., 1981). In short, 10 g of sample was mixed with 10 ml of sodium chloride solution (0.85%) in a homogeniser. The homogenized sample was centrifuged at 900 g at 4 C for 30 min and the obtained supernatant was passed through the 0.2e0.45 mm low protein-binding membrane filter to be used for assay of toxin content. Extracts were immediately analysed, as prescribed by the manufacturer. Except for the extraction step, the analysis done in the culture to check the enterotoxin production by isolated S. aureus (isolates grown in Tryptone Soya Broth, TSB, Oxoid for 24 h at 37 C) were performed on the same way. 2.4. Inoculation study A cocktail of three enterotoxigenic isolates coming from the screening of analysed samples were mixed in 1:1:1 ratio to obtain concentration of approx. 3 log CFU/ml. Previously found as negative, on both enterotoxin and S. aureus presence, meat samples of 50 g were inoculated with 1 ml of the inoculum suspension to obtain approx. 2.5 log CFU/g. The samples were vacuum or air packaged and stored for three weeks at 12 and 22 C and for two weeks at 37 C. Each sample was foreseen as a separate package. The packages were analysed daily during the storage on the presence of S. aureus and enterotoxins, as described above. 3. Results and discussion The experience has shown that staphylococci may be expected to exist, at least in low numbers, in probably all food products that are of animal origin or in those that are handled directly by humans, unless heat-processing steps are applied to affect their destruction (Jay, 2000). Therefore the success of prevention and the control of S. aureus growth and enterotoxins production will be mainly dependent on the inherent product characteristics and strict application of GMP, GHP and HACCP principles. The pH and aw found in 20 samples of purchased beef ham ranged from 5,11 to 5,60, and from 0.901 to 0.940, respectively (Table 1). Of 20 tested samples, four samples contained enumerable S. aureus counts of 3.2, 2.8, 3.7 log CFU/g belonging to producer 1 and 3.1 log CFU/g belonging to producer 4. Other 14 samples were found positive for the presence of S. aureus following the enrichment. Analysis of enterotoxins with SET-RPLA [Oxoid, Basingstoke, United Kingdom] showed all but seven of the samples to be free of detectable SEA, SEB, SEC and SED (Table 1). Four samples were found positive on the presence of SEA (encompassing 3 producers: producer 1, 2 and 4), two samples on both SEA and SED (one producer: producer 2), and one on SEB (one producer: producer 5). Surprisingly all enumerable S. aureus were
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Table 1 Staphylococcus aureus and staphylococcal enterotoxins presence in analysed dried cured beef ham samples. Producer
Samples containing S. aureus
Samples containing SE
aw and pH in the ham samples SE positive samples
1
4/4
2/4 SEA
2
4/4
1/4 SEA, 2/4 SEAeSED
3
3/4
No SE containing samples
4
4/4
1/4 SEA
5
3/4
1/4 SEB
a
aw 0.925e0.940 pH 5.1e5.3 aw 0.921e0.937 pH 5.2e5.5
aw 0.923 pH 5.4 aw 0.933 pH 5.5
SE negative samples
SEAeSEE production by isolated S. aureus under optimal growth conditions in TSBa 2 SEA isolates
aw 0.920 pH 5.3 aw 0.901e0.915 pH 5.2e5.6 aw 0.918e0.923 pH 5.2e5.5 aw 0.921e0.931 pH 5.2e5.5
2 SEA isolates and 1 SEAeSED isolate 2 SEA isolate 2 SEA isolates, 1 SEB isolate and 1 SEC isolates 1 SEA isolates and 1 SEB isolate
One isolate per package was analysed.
SEA positive isolates. These findings confirm that enumeration of S. aureus may lead to the false evaluation of the product safety. While all five producers had positive samples on the presence of S. aureus, four also had samples positive on SE. Based on elsewhere published data the differences in pH and aw between the products of different producers are relatively small and presumably account only for some of the observed differences. Other authors reported that the majority of S. aureus strains tested produced detectable amounts of SE aerobically at a pH of 5.1 (Hennekinne, Buyser, & Dragacci, 2011). SEA and SED production occurs under nearly all aw conditions allowing growth of S. aureus as long as all other conditions are optimal. Although the production of SEB and SEC is thought to be very sensitive to reductions in aw and hardly any is produced at aw 0.93 despite extensive growth, the current study showed that SEB can be found in products with aw lower than 0.93, possibly due to formation in earlier production stages (Ewald & Notermans, 1988; Qi & Miller, 2000). Presence of other type of background flora, perhaps use of lactate, nitrite or other components in brining mixture might have also contributed to the observed differences. The isolated S. aureus from each of the 18 S. aureus e positive samples were grown in TSB and in-vitro toxin production was controlled by SET-RPLA as instructed by the manufacture (Oxoid). Nine isolates produced SEA only, one produced SEA and SED, 2 produced SEB and one produced SEC. The remaining isolates produced none of SEAeSED enterotoxins, regardless of the growth to more than 7 log CFU/ml. The obtained data are in agreement with generally established knowledge that the most common toxin implicated in staphylococcal food poisoning is SEA (Balaban & Rasooly, 2000). Further characterisation of the isolates regarding other SE or SE like toxins, as well as regarding their growth characteristics, might provide valuable information on other food safety risks involved. The importance of this is illustrated by the fact that to date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. Moreover, any food that provides a convenient medium for growth of S. aureus (or other enterotoxigenic Staphylococcus species) in any of the stages of production may be involved in a staphylococcal food poisoning outbreak, SFP (Hennekinne et al., 2011).
Contamination, growth and enterotoxin production remain a prerequisite for SFP. To investigate the growth and toxin production in the same meat products a cocktail made of one SEAeSED, one SEB and one SEC producing isolates (equal ratios to make a final inoculum of approx. 2.5 log CFU/g) was used to inoculate six samples that were previously found negative on both S. aureus and four classical SE (Table 2). At 12 C in both aerobic and vacuum conditions no growth was observed during three weeks of storage, but enrichment showed presence of viable S. aureus at all times during three weeks with sampling done every three days. At 22 C results in food showed a very restricted growth resulting in an increase of S. aureus numbers of approx. 2.5 log CFU/g in 15 days in air, reaching maximal cell density of approx. 6.5 log CFU/g after three weeks of storage. At the same temperature the growth onset in vacuum packaging was delayed for another day. However, the variation around the mean counts (enumerations of each sample were done in duplicate) was somewhat higher than under aerobic conditions, indicating that vacuum provided certain stress on the S. aureus population probably favouring some fractions of present cells and stressing other ones (Rajkovic, Smigic, & Devlieghere, 2010; Rajkovic et al., 2009). At growth temperature of 37 C and all other conditions the same, the growth onset was for both aerobic and vacuum conditions about one day, reaching 7.2 log CFU/g after 4 days of storage. It is one of the key findings that in none of the conditions at 12 C was SE production observed. At 22 C and 37 C all samples contained detectable amounts of SEA and SED, but not of SEC and SEB toxins, after 15 days and 3 days, respectively. This detection SET-RPLA detection limit was reached only at S. aureus counts higher than 5 log CFU/g, but this does not preclude earlier onset of SE production. To verify if the lack of SEB and SEC production was due to insufficient growth the two respective isolates were inoculated in the additional slices of beef ham and incubated at 22 C and 37 C. Results showed that none of the two isolates was able to produce SEB and SEC after two weeks of storage, measured at the end of the storage period, regardless of final S. aureus count of 7 log CFU/g. This, together with the fact that used SET-RPLA method does not detect other SE but SEAeSED, and that some of the natively present S. aureus might have been injured or stressed to be recovered on the
Table 2 Staphylococcus aureus and staphylococcal enterotoxins in inoculated beef ham samples. Parameter
12 C Aerobic
Time to 5 log CFU/g Maximal cell densitya Enterotoxin production a
22 C Vacuum
No growth No SEA, SEB and SEC production
Aerobic
37 C Vacuum
15 Days 16 Days 6.5 0.3 log CFU/g (aerobic) to 6.7 0.7 log CFU/g (vacuum) in 21 days SEA and SED after 15 days
Mean values of two enumerations done on the same sample.
Aerobic
Vacuum
2 Days 7.2 0.6 log CFU/g in 4 days SEA and SED after 3 days
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selective medium during enumeration, could explain differences in the percentage of inoculated samples containing SE (100%) and native samples containing SE (35%). The range of environmental parameters over which S. aureus will produce enterotoxins is accepted to be narrower than the range over which it will grow and are influenced also by the composition of food (Jay, 2000). In general, enterotoxin production is substantially reduced at 20e25 C and it is generally accepted that enterotoxin production is unlikely to occur at temperatures below 10 C. Optimum enterotoxin production occurs at pH 6e7 and is influenced by atmospheric conditions, carbon and nitrogen source and salt level. Although reduced levels of aw may also inhibit toxin synthesis more than growth the toxin production has been reported at as low as aw ¼ 0.86 (under otherwise optimal conditions). Similar findings were reported by Portocarrero et al. (2002) who found that initially inoculated S. aureus populations of 8.57 and 8.12 log CFU/cm2 for salt and salt þ NO2 hams decreased to below the levels of detection after the fourth month of ageing. S. aureus was detected following enrichment for 75% of the inoculated and 62% of the control (non-inoculated) hams at the end of the ageing period. Staphylococcal enterotoxin was detected in 40% of the inoculated and 50% of the control hams following the ageing period. The NaCl content of these hams were 4.45/3.37% and aw values 0.94/0.91. Country-cured ham products obtained from retail stores in that study were all negative for S. aureus enterotoxin. These results indicated that higher salt content and lower aw values on commercially produced country-cured hams played an important role in controlling the growth and toxin production of S. aureus. In the case of our study it is important to note that while drying and curing of hams may or may not destroy S. aureus, the high salt content on the exterior will often inhibit the superficial growth of these bacteria. However, when the ham is sliced the moister interior can permit staphylococcal multiplication and thus sliced dried and cured hams should be refrigerated (FSIS-USDA, 2011). The vacuum package used in this study was not sufficient to prevent this. Not to different results were found by Wallin-Carlquist, Marta, Borch, and Radstrom (2010) reporting no growth of inoculated 6 log CFU of S. aureus on the small surface of Serrano hams stored for seven days at 23 C, but reporting SEA in the same samples. Obtained results in the current study confirm the ubiquitous prevalence of S. aureus in meat products and suggest that insufficiently dried and matured hams as studied here provide niche for growth of S. aureus and enterotoxin formation. Moreover, it has been seen that one sample can contain multiple enterotoxins leading to more complex doseeresponse scenarios. These hams should not be stored at temperatures higher than 12 C and preferably should be refrigerated. Moreover, the high prevalence of S. aureus and enterotoxins indicates that improved control of raw materials and all production stages is required. It has been recently reported that detectable amounts of SE are produced at S. aureus counts of as low as 3 log CFU/g under permissible conditions (Rajkovic et al., 2006). This suggests that possibly even stricter measures are to be implemented to control this microbial hazard, and that certainly no loose setting deviations from elaborated good practices and HACCP plans are to be made. Further investigations should show whether this niche can offer growth habitat to other potential microbial hazards leading possibly to muti-hazard exposure. The safety of the products needs to remain superior request to that of the commercial needs that may require faster turnover through short maturation time of cured meat products. Acknowledgement This work was supported by the FWO grant of Dr. Andreja Rajkovic
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