Occurrence and Characterization of Enterotoxigenic Potential of S taphylococcus Isolated from Dairy Products

OCCURRENCE AND CHARACTERIZATION OFENTEROTOXIGENIC POTENTIAL OF STAPHYLOCOCCUSISOLATED FROM DAIRY PRODUCTSISABELA MATEUS MARTINS1, DIRCE YORIKA KABUKI, NORMA TERUKO NAGO MIYA and JOSÉ LUIZ PEREIRA

Department of Food Science, School of Food Engineering; University of Campinas (UNICAMP), Cidade Universitária “Zeferino Vaz”, s/n, CEP13083-862, CP 6121, São Paulo, Brazil

1Corresponding author.TEL: +55-(19)-991693376;+55-(19)-35212175;FAX: +55-(19)-35212153;EMAIL: [email protected]

Received for Publication November 18, 2013Accepted for Publication March 24, 2014

doi: 10.1111/jfs.12112

ABSTRACT

Staphylococcus deserves our close attention because of its ability to produce a largeamount of enterotoxins, which, if ingested, cause staphylococcal food poisoning.Cross-contamination after milk pasteurization, caused especially by improperhandling, is considered the most recurrent source of contamination of dairy prod-ucts. Therefore, this research aimed to quantify and analyze the presence ofcoagulase-positive and coagulase-negative staphylococci from 104 dairy productssamples to identify phenotypically the species and to evaluate the in vitro produc-tion of classical enterotoxins (SEA, SEB, SEC, SED and SEE) and the presence ofpossible coding genes of these enterotoxins. The average staphylococcal countvalues in different food groups ranged from 3.7 to 6.15 log colony-forming units(cfu)/g. Among the staphylococcal strains, 74.3% (110/148) were coagulase-negative Staphylococcus and 25.7% (38/148) were coagulase-positive Staphylococ-cus, and 13 species were identified. From the 111 selected isolates, one (0.9%)strain produced enterotoxin, which was identified as S. aureus, coagulase positive,isolated from “Minas” half-cured cheese, whose count was 6.28 log cfu/g. Contraryto expectations, no coding genes were present in the isolate analyzed. For thisresearch, the analyzed samples showed high staphylococcal concentrations that donot exclude a possible risk of producing the already described enterotoxins. Theyalso demonstrated poor hygiene during the production and commercialization ofdairy products.

PRACTICAL APPLICATIONS

Staphylococcus aureus is one of the most common agents in bacterial food poison-ing outbreaks. As milk is a major nutrient for infants, children, convalescents andold people, the presence of classical enterotoxins in milk and dairy products is amatter of concern. The information present in this research is important to alertabout the inadequate quality of Brazilian milk products, and efforts from the gov-ernment and the entire productive chain are required to attain consumer safety.

INTRODUCTION

Staphylococcus are microorganisms widespread in nature,and humans and animals are their main sources(Bannerman 2003). They are opportunistic bacteria andsome species are etiologic agents of various infections andpoisonings in humans and animals, causing acute infec-tions, septicemia, toxemias, toxic shock syndrome and

staphylococcal food poisoning (Baird-Parker 1990; Le Loiret al. 2003).

Some staphylococcal strains have the ability to produceseveral virulence factors that contribute to their pathogenic-ity. Among them, there are the enterotoxins, proteins thatcause staphylococcal food poisoning, classified as one of themost common gastroenteritis worldwide, which is causedby the ingestion of preformed staphylococcal enterotoxins

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Journal of Food Safety ISSN 1745-4565

185Journal of Food Safety 34 (2014) 185–192 © 2014 Wiley Periodicals, Inc.

in foods (Balaban and Rasooly 2000). Staphylococcal gas-troenteritis is a short-term disease (24–48 h), whose symp-toms include nausea, vomiting, abdominal pain andpossible diarrhea (Jay et al. 2005). Staphylococcus aureus isone of the most common agents in food poisoning out-breaks. Its strains produce several types of enterotoxins,being the main causative pathogen of subclinical mastitis indairy cattle (De Buyser et al. 2001; Le Loir et al. 2003; Omoeet al. 2005).

Twenty different types of staphylococcal enterotoxinshave been described. They are thermostable and resistant togastrointestinal enzymes (Balaban and Rasooly 2000). Themost common types detected are the enterotoxins A, B, C, Dand E (SEA-SEE), named as classical enterotoxins, which areresponsible for 95% of staphylococcal gastroenteritis cases(Bergdoll 1989). However, detection of new enterotoxins(SEG-SEU) and their encoding genes are increasinglycommon (Omoe et al. 2005; Ono et al. 2008).

Many different foods, including meat and chicken prod-ucts, eggs and milk products, can be considered good sub-strates for Staphylococcus development. Some are commonlyimplicated in staphylococcal food poisoning outbreaks. Themain sources of contamination are humans (handlers thatcontaminate food by manual contact or by respiratorytract), and this is related to the fact that 30% to 50% of thepopulation are carriers of Staphylococcus on skin andmucosa (De Buyser et al. 2001; Le Loir et al. 2003).

In Brazil, the microbiological standards established byRDC resolution no. 12/2001 of the National Agency forSanitary Vigilance (ANVISA) set limits only for coagulase-positive Staphylococcus (CPS) counting in foods (Brasil2001). This is correlated to the fact that only enterotoxigenicStaphylococcus should produce coagulase (Lancette andBennett 2001). However, several studies have shown theability of coagulase-negative Staphylococcus (CNS) toproduce enterotoxins in laboratory culture medium (Veraset al. 2008; Zell et al. 2008; Al-Tarazi et al. 2009; Oliveiraet al. 2011), besides, there are several reports of poisoningoutbreaks associated with the CNS species (Omori and Kato1959; Breckinridge and Bergdoll 1971; Simeão do Carmoet al. 2002; Veras et al. 2008). The risks for the presence ofCNS in food should be reconsidered because they are com-monly found in raw foods, such as milk, and some strainsare traditionally used as starter cultures for the productionof fermented foods (Zell et al. 2008).

This study aimed to evaluate the profile of contamina-tion of coagulase-positive and coagulase-negative staphylo-cocci in dairy products to identify the species isolatedusing biochemical tests and to evaluate the enterotoxigenicpotential of isolates by detection of classic enterotoxinsSEA, SEB, SEC, SED and SEE (in vitro) and their respectivecoding genes, comparing genotypic and phenotypicprofiles.

MATERIALS AND METHODS

Food Samples

A total of 104 samples of dairy products were analyzed,where 24 were homemade ice cream samples, 20 pasteurizeddairy creams, 20 cheese pâtés, 20 samples of “Minas” half-cured cheese and 20 samples of “Minas” fresh cheese. Thesamples were purchased in Campinas, SP, Brazil, trans-ported to the laboratory in ice boxes and submitted tomicrobiological analyzes described below.

Sample units of 25 g were removed from the packingunder aseptic conditions and were homogenized in a stom-acher (Seward, Worthing, United Kingdom) with 225 mL ofsodium citrate 2% (Labsynth, Diadema, Brazil) and serialdilutions were performed.

Counting, Isolation and Identificationof Staphylococcus

The count of Staphylococcus was performed using themethod recommended by American Public Health Associa-tion (Lancette and Bennett 2001). Dilutions of samples wereplated on Baird-Parker agar (Difco, Detroit, USA). Theplates were incubated at 35C for 48 h and we performed thepresumptive count of colony-forming units (cfu/g of food).

Not only the typical colonies were selected (circular,black, smooth, convex, with perfect edges, surrounded by anopaque zone and /or transparent halo), which are usuallycharacterized as CPS, but also atypical colonies (gray colorwithout transparent halo). Therefore, five colonies of eachsample were selected and transferred to brain heart infusion(BHI) agar (Difco), followed by incubation at 37C for 24 hfor morphological confirmation by Gram staining and bio-chemical tests of catalase, rabbit plasma coagulase andthermoresistant nuclease in toluidine blue DNA agar(Lancette and Bennett 2001), and also furazolidone suscep-tibility test (Von Rheinbaben and Hadlok 1981).

A total of 111 isolates (27 CPS and 84 CNS), randomlyselected, were submitted to phenotypic profile characteriza-tion according to the API-Staph system (bioMérieux, Marcyl’Etoile, France), following the manufacturer’s instructions fora taxonomic identification of species. Additional tests indi-cated by the manufacturer and according to the table of di-fferentiation between species of the genus Staphylococcus(Bannerman 2003) were necessary to identify the species ofsome isolates, whose results from the kit showed doubtfulprofiles. Therefore, the isolates were tested for antimicrobialsusceptibility to polymyxin B 300 U (Cefar, São Paulo, Brazil)and novobiocin 5 μg (Laborclin, Pinhais, Brazil) using thedisk diffusion method on Mueller-Hinton agar (Difco),according to the methodology established by the Clinical andLaboratory Standards Institute guide (CLSI 2003).

STAPHYLOCOCCUS IN DAIRY PRODUCTS I.M. MARTINS ET AL.

186 Journal of Food Safety 34 (2014) 185–192 © 2014 Wiley Periodicals, Inc.

Determination of Enterotoxin Production

For the evaluation of enterotoxin production in liquidculture medium, the 111 isolates were cultivated in 5 mL ofBHI broth at 35C for 24 h, from where an aliquot of 2 mLwas taken and added to 23 mL of BHI broth, incubated at35C for 48 h under stirring at 170 rpm (New BrunswickScientific, New Jersey, USA; Chou and Chen 1997; CunhaNeto et al. 2002). The cultures were centrifuged (Beckman-Coulter, Fullerton, USA) at 9,000 × g for 10 min at 4C andan aliquot of 500 μL of supernatant was applied on the kitVIDAS SET “Staph enterotoxin” (bioMérieux, France) forthe detection of enterotoxins SEA, SEB, SEC, SED and SEEusing the equipment Mini VIDAS (bioMérieux, France).The sensitivity of the equipment for the detection of entero-toxin is 0.05 – 0.1 ng/mL.

Detection of Classical Enterotoxin Genes

According to the methodology adapted from (Martín et al.2003; Zouharova and Rysanek 2008), the DNA was isolatedfrom 0.5 mL of BHI broth incubated for 18 h at 35C, underagitation (170 rpm). Cells were separated by centrifugationat 13,000 × g for 10 min and, after removal of supernatant,they were resuspended in 100 μL of Tris-EDTA buffer(10 mM Tris chloride, 1 mM EDTA [pH 8,0]) and incu-bated at 100C (boiled) for 20 min. After being centrifugedagain, supernatants were stored at −20C and subsequentlyused as template DNA for PCR reaction.

For the detection of five enterotoxin-encoding genes(SEA, SEB, SEC, SED and SEE), the PCR technique wasapplied. The method and primers were based on the workof Mehrotra et al. (2000), with some modifications andadaptations on the reaction final volume, annealing tem-perature and time of cycles. Five reference strains ofS. aureus were used as positive controls for enterotoxin pro-duction: ATCCs 13,565 (SEA producer), 14,458 (SEB),23,235 (SED), 27,664 (SEE) and FRI 1,230 (SEC).

The reaction mixtures were prepared to make a finalvolume of 25 μL containing PCR buffer 1x (100 mM Tris-HCl, [pH 8.3], 500 mM KCl) (LGC), 1.5 mM MgCl2,200 μM of dNTPs, 20 pmol of specific primers for eachgene, 0.5 U of Taq DNA polymerase and 1 μL of DNA. Thefinal volume was adjusted by adding sterile ultrapure water.

The DNA amplification was performed in a thermocy-cler Mastercycler epgradient S 534 (Eppendorf, Hamburg,Germany) using the following cycle: initial denaturation at94C for 5 min, 35 cycles of amplification (94C for 1 min,53C for 1 min, 72C for 1 min) and final extension at 72Cfor 7 min. The amplified PCR products were distingui-shed by standard gel electrophoresis in a 2% (w/v) agarosegel (Invitrogen, Carlsbad, USA) stained with Syber Safe(Invitrogen) and visualized using a transilluminator.

Markers (100 bp DNA ladder, Invitrogen) were included ineach gel electrophoresis.

RESULTS AND DISCUSSION

Counting, Isolation and PhenotypicIdentification of Staphylococcus

The dairy products analyzed, especially the “Minas” freshcheese, are considered as having very high humidity, consti-tuting a favorable media for the development of pathogenicbacteria, especially Staphylococcus. These bacteria, whenpresent in a large population (>5 log cfu/g) and underproper conditions (temperature, pH, water activity andoxidation-reduction potential), can be considered as poten-tial producers of staphylococcal enterotoxins (Jablonski andBohach 2001; Jay et al. 2005).

The isolates selected from preliminary counts on BairdParker agar (BP) and characterized by gram-positive coccigrouped like a “bunch of grapes” and catalase positive weretested for furazolidone susceptibility and production ofcoagulase and thermonuclease.

The furazolidone susceptibility test could separate iso-lates from two different genera (Micrococcus sp. and Staphy-lococcus sp.). Of all isolates, 16.4% (29/177) were classifiedas Micrococcus sp. and 83.6% (148/177) were classified asStaphylococcus sp. Micrococcus was detected in 10.6% (11/104) of the products investigated, where one was an icecream sample (4.2%), five were “Minas” half-cured cheese(25%) and five were “Minas” fresh cheese (25%). Vargunand Vatansever (2007), using the same test, found the fre-quency of 6.2% of isolates as Microccoccus sp. in milk anddairy cream samples. Esper (2006), however, found higherpercentage of Micrococcus sp. in ricotta cheese, a total of62.6% of isolates. Few studies perform this differentiationbetween Staphylococcus sp. and Micrococcus sp. This is indis-pensable because of the biochemical similarity of bothgenera, which can result in erroneous identification.

Among the isolates confirmed as Staphylococcus sp.,74.3% (110/148) were characterized as coagulase negativeand 25.7% (38/148) as coagulase positive. All CPS isolateswere obtained from cheese and the CNS isolates from theothers products (Table 1). The average values of CNS, CPSand Micrococcus sp. counts are presented in Table 2.

When evaluating each group of dairy products, the icecream samples showed elevated CNS population (reaching4 log cfu/g) and 66.7% (16/24) of samples were contami-nated. This amount is excessive and causes concerns aboutsafety when consuming these products because CNS hasbeen associated with food poisoning outbreaks. Vargun andVatansever (2007) detected seven isolates of Staphylococcusisolated from dairy products that produced classical entero-toxins, and four of them were coagulase negative.

I.M. MARTINS ET AL. STAPHYLOCOCCUS IN DAIRY PRODUCTS


The high prevalence of coagulase-negative staphylococci indairy products and their pathogenic potential have not beenclearly identified, but this is increasingly necessary to assessthe microbiological safety of these products (Irlinger 2008).

Although Staphylococcus has been detected in only onesample of pasteurized dairy cream, the counts reached 5 logcfu/g. This suggests flaws in thermal processing or post-pasteurization contamination, followed by storage tempera-ture abuse, stimulating the proliferation of microorganisms in“heat-treated” food. In addition to the high count value indairy cream, we reached counts of 6 log cfu/g in some samplesof cheese (amounts considered sufficient for the possible pro-duction of enterotoxins). The ubiquity of this pathogen innature and the non-adoption of Good Manufacturing Prac-tices (GMP) in milk production, from milking to its receptionin dairy plant, represent a risk factor to consumers and raisethe need for greater attention to hygiene and sanitary controlin the processing of these products.

The cheese were the products with the worst microbiologi-cal quality (related to the presence of Staphylococcus), with a

minimum count of 3 log cfu/g and the most part of thesamples (56% or 14/25) presented counts >5 log cfu/g, a valueconsidered a risk for the production of enterotoxins. In pâtéproducts, even with counting in only three samples, in two ofthem, the numbers were relevant (3 and 4 log cfu/g).

We believe that the differences in the frequency of coagu-lase positive and coagulase negative found in milk and milkproducts could be attributed to the different ways ofhygienic care when obtaining raw material and on theimplementation of manufacturing process. The perfor-mance of comparisons between results is difficult becausethe presence of Staphylococcus comes from subclinical mas-titis, handling of dairy cattle during milking and sanitaryprocessing conditions, which vary by location (Fagundeset al. 2010).

In Brazil, the microbiological standards for food estab-lished by RDC resolution no. 12/2001 of ANVISA set limitsonly for counting of CPS (Table 3; Brasil 2001). Therefore,the majority of works in Brazil related to staphylococci juststudy S. aureus and CPS, this being one of the main differ-entials of this research.

CPS was detected in 30% (12/40) of “Minas” cheesesamples (half-cured and fresh) and all samples (12/12) pre-sented counts above the allowed limit. The consumption ofthese products represents a health hazard and reveals thepoor hygienic quality of the cheese. Similar results are com-monly found in Brazil. In researches by André et al. (2008)and Komatsu et al. (2010), the samples of “Minas” freshcheese that presented counts higher than the allowed limitreached 54.2% (13/24) and 88% (44/50), respectively.Aragon-Alegro et al. (2007) found that 75% (12/16) of thecheese samples evaluated were in discordance with themicrobiological parameters. These studies show a large

TABLE 1. NUMBER AND PERCENTAGE OF POSITIVE SAMPLES FORCOAGULASE-NEGATIVE AND COAGULASE-POSITIVESTAPHYLOCOCCUS

Product

Number of samples (%)

n CNS CPS

Ice cream 24 16 (66.6) 0 (0)Dairy cream 20 1 (5) 0 (0)Pâté 20 3 (15) 0 (0)“Minas” cheese (half-cured) 20 8 (40) 3 (15)“Minas” cheese (fresh) 20 12 (60) 9 (45)Total 104 40 (38.5) 12 (11.5)

TABLE 2. AVERAGE COUNT OFCOAGULASE-NEGATIVE STAPHYLOCOCCUS(CNS), COAGULASE-POSITIVESTAPHYLOCOCCUS (CPS) ANDMICROCOCCUS SP. IN DAIRY PRODUCTS

Product

Average count (SD) (log cfu/g)

CNS CPS Micrococcus sp.

Ice cream 3.7 (3.95) – 1.7 (na)Dairy cream 5.46 (na) – –Pâté 3.8 (3.81) – –“Minas” cheese (half-cured) 5.6 (5.75) 5.6 (5.69) 5.58 (5.57)“Minas” cheese (fresh) 5.5 (5.54) 6.15 (6.25) 4.96 (4.91)

na, not applicable; SD, standard deviation.

TABLE 3. PERCENTAGE OF SAMPLES NOTCOMPLIANT WITH THE MICROBIOLOGICALCRITERIA ESTABLISHED IN RDC RESOLUTION12/2001* FOR COAGULASE-POSITIVESTAPHYLOCOCCUS

Product Limit* (log cfu/g) Noncompliant samples (%)

Ice cream 2.7 0Dairy cream 2 0Pâté 3 0“Minas” cheese (half-cured) 3 9/9 (100)“Minas” cheese (fresh) 3 3/3 (100)

* Brazilian Health Surveillance Agency – ANVISA (Brasil 2001).



amount of cheese that are currently produced and consid-ered as inappropriate for consumption, the negligence ofhygiene measures, possible failures in the procedures forcleaning and sanitizing production lines, in the control ofraw material and in the attention to the thermal processingrequired for the production of these foods, and possibleabuses on the temperature practiced both in the transportand storage of the product, which are enhancing agents ofcontamination levels. The sanitary measures, the implemen-tation of GMP and the application of Hazard Analysis andCritical Control Points during the production and commer-cialization of these products are very important, besides theneed for a more rigid surveillance.

A total of 111 isolates were identified using the API-Staphsystem, which proved to be very efficient for the identifica-tion of S. aureus, S. xylosus, S. auricularis and S. lentus,S. epidermidis and S. cohnii. However, it has a database witha restricted number of species because of the high diversityof Staphylococcus species already described, being necessary,in some cases, the use of additional biochemical tests forbetter and more reliable characterization of the species. Thislimitation is corroborated by other studies (Burriel 1997;Pereira 2006; Borges et al. 2008; Ruegg 2009).

With the complementation of antibiotic susceptibilitytests, we identified 13 different Staphylococcus species infood (Fig. 1). The staphylococci microbiota present in eachtype of food analyzed had a great diversity of species. Thegreatest number of species was detected in ice cream (ninespecies), followed by “Minas” fresh cheese (eight species),“Minas” half-cured cheese (six species), pâté (five species)and dairy cream (two species). Three species were pre-dominant in ice cream samples: S. aureus (24.4%),S. saprophyticus (22%) and S. cohnii ssp. cohnii (22%). Wecan observe that we detected S. aureus in ice cream samples,but none were coagulase positive. In pasteurized dairycream samples, only two species were identified, S.

auricularis (50%) and S. cohnii ssp. cohnii (50%). In pâtésamples, the prevalence was for S. xylosus (44.4%), followedby S. epidermidis (22.2%). In “Minas” half-cured samples,there was a high prevalence of S. aureus (53.1%), followedby S. saprophyticus (21.9%), and in “Minas” fresh cheese, themost frequent species was S. xylosus (25.9%), followed byS. aureus (22.2%).

There are few studies in this area that perform speciesidentification, mainly because the great majority just ana-lyzes S. aureus or CPS in food. Routine laboratories do notusually identify CNS at the species level. However, a precisebacteriological identification at the species or subspecieslevel is necessary to improve the quality of epidemiologicalinvestigations to evaluate their pathogenic significance andto devise specific management practices (Irlinger 2008).

At first, only the typical colonies were considered as pos-sibly being coagulase positive and thermonuclease-positiveStaphylococcus, but it is not what it is normally observed.Among the coagulase-negative isolates detected in thisstudy, 14.3% (12/84) were typical colonies on BP agar, andamong them, four were thermonuclease-positive; the otherswere all thermonuclease-negative (8 typical and 72 atypicalcolonies). Between the coagulase-positive isolates, 81.5%(22/27) were typical colonies and 18.5% (5/27) atypicalcolonies, being all of them thermonuclease-positive. Fromthe 34 typical colonies related, just 22 were characterized asS. aureus.

Similarly, Esper (2006) verified that four isolates ofcoagulase-positive staphylococci, found in ricotta cheese,had atypical colonies in BP agar. Nascimento et al. (2001)also observed that CPS, including S. aureus, could presentuncharacteristic colony growth, leading to failures in thequantification of these bacteria in specific culture medium,being of extreme importance to perform confirmatorybiochemical tests. Therefore, non-enterotoxigenic andenterotoxigenic Staphylococcus may present similar results

FIG. 1. FREQUENCY OF STAPHYLOCOCCUSSPECIES ISOLATED FROM DAIRY PRODUCTS



regarding the formation of typical and atypical colonies onBP and their production of coagulase and thermonuclease(Bennett 1996).

Enterotoxin Production Capacity andDetection of Classical Enterotoxin Genes

Regarding the enterotoxin production of the 111 isolatesanalyzed, one (0.9%) presented positive result using theVIDAS SET “Staph enterotoxin” (bioMérieux-SA). Thestrain was identified as S. aureus, coagulase-positive, iso-lated from “Minas” half-cured cheese, whose count was6.28 log cfu/g (6.18 log cfu/g of CPS and 5.58 log cfu/g ofCNS), considered as high enough for the possible produc-tion of enterotoxins in food. The relative fluorescence valuepresented by the isolate was 0.17, a value very close to theminimum considered positive (>0.13), being characterizedas a low-producing enterotoxin strain.

Because of the probability that there was no gene expres-sion to produce enterotoxins, the strains were also evaluatedfor the presence of specific coding genes for classical entero-toxins (SEA, SEB, SEC, SED, SEE), but the genes were notdetected by the PCR technique used in this study.

Currently, the detection of new coding genes of nonclas-sical enterotoxins (SEG-SEU) from Staphylococcus isolatedfrom food is more common (Aydin et al. 2011; Tang et al.2012). Besides, the discrepancy between the results from thedetection of enterotoxins by the immunological methodand the detection of genes has been related. In a study byPereira et al. (2009), some results on the immunologicalmethod were positive for SEA-SEE; however, the detectedgenes were related to nonclassical enterotoxins (SEG, SEH,SEI, SEJ). The comparison of phenotypic and genotypicresults indicated no correlation in 20% of samples, beingpositive for enterotoxin expression, but not for the detectionof genes (6%); positive for the presence of the genes, but noexpressed enterotoxin (4%); positive immunologicalmethod for SEA-SEE, but other genes were detected (10%).

McLauchlin et al. (2000), Rosec and Gigaud (2002) andJorgensen et al. (2005) observed discrepancies betweenresults, with the absence of coding gene detection.Zouharova and Rysanek (2008) had a higher level of genedetection than enterotoxin detection (McLauchlin et al.2000).

There are some hypotheses to explain the non-concordance between phenotypic and genotypic analysesfor the detection of staphylococcal enterotoxins, which alsomay explain the positive enterotoxin detection, but theabsence of coding genes on the isolated S. aureus in thisstudy. One would be that the immunoenzymatic interactionof Mini VIDAS has been more extensive, involving entero-toxins beyond the classical ones and presenting positiveresult, but which were not confirmed on the genetic analysis

(Pereira et al. 2009). Another suggestion is the occurrence ofvariations in the sequences of the genes analyzed, hinderingthe correct connection of the primers in the PCR reaction,besides the possible presence of an enterotoxin, which hasnot yet been reported with a immunological cross-reactionwith the classical toxins (McLauchlin et al. 2000; Jorgensenet al. 2005). Kérouanton et al. (2007) detected variant genesof SEA and SED in isolates with discrepant results, conclud-ing that often, the primers used could not have a broadrange when there were variations on the coding genes forS. aureus enterotoxins. DNA sequence analysis can be asolution to reveal additional differences in the genes thatmay be present in the isolates.

In this study, despite having only one strain that pro-duced classical enterotoxins, there was a very high numberof Staphylococcus in various products analyzed, which doesnot exclude the possible risk of producing other previouslydescribed enterotoxins and shows the lack of hygienic careand good practices during the production and marketing ofthese foods.

The Brazilian legislation (Brasil 2001), in addition toestablishing the count of CPS in food, should also requirethe count of CNS, but more important and correct wouldbe the determination of staphylococcal enterotoxin detec-tion (SCN and SCP) in food because this way, it would bepossible to control the quality of marketed products, toprevent the many outbreaks caused by these microorgan-isms and also to decrease the impacts on the economy fromleave of absence and expenses with public health services.For this end, many studies are still needed, besides thedevelopment of kits for the rapid detection of not only clas-sical enterotoxins, but also new enterotoxins, whose encod-ing genes are increasingly found in foods and may be amajor cause of many poisoning outbreaks.

CONCLUSIONS

The dairy products analyzed in this study had risk factorsassociated with their consumption because of the highpopulation of Staphylococcus detected, confirming them asvehicles for the transmission of potentially pathogenic bac-teria. Species of coagulase-positive staphylococci weredetected only in “Minas” fresh and half-cured cheese, butthe coagulase-negative ones were found in all dairy prod-ucts analyzed. The correlation between typical coloniesgrown on BP agar and coagulase-positive and enter-otoxigenic strains must not be followed as a rule; atypicalcolonies should be studied and not ignored.

In the dairy foods studied, one CPS isolate was detectedas potentially being able to produce classical enterotoxins invitro, being identified as S. aureus and isolated from asample of “Minas” half-cured cheese. There was a 99.1%



concordance between the genotypic and phenotypic testsused for the detection of enterotoxins.

The high counts and the presence of CPS and CNS onsamples may represent a potential risk of producing otheralready-known enterotoxins, not completely eliminating therisk related to the high counts of Staphylococcus found inthe milk products studied.

Acknowledgments

The authors are grateful to the National Council for Scien-tific and Technological Development (CNPq) for the schol-arship, the Oswaldo Cruz Institute (FIOCRUZ) forproviding enterotoxin-producing strains and Espaço daEscrita/General Coordination of UNICAMP for the Englishcorrections.

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Occurrence and Characterization of Enterotoxigenic Potential of S taphylococcus Isolated from Dairy Products