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Behavior and Enterotoxin Productionby Coagulase Negative Staphylococcusin Cooked Ham, Reconstituted SkimmedMilk, and Confectionery CreamAna Maria Oliveira, Norma Teruko Nago Miya, Anderson S. Sant’Ana, and Jose Luiz Pereira

Abstract: In this study, the behavior and enterotoxin production by 10 different coagulase negative Staphylococcus (CNS)strains inoculated in cooked ham, reconstituted skimmed milk, and confectionery cream in the presence or absence ofbackground microbiota have been investigated. After inoculation (103 CFU/g), foods were incubated at 25, 30, and37 ◦C and aerobic mesophilic and CNS counts were carried out at 12, 24, 48, and 72 h. Staphylococcal enterotoxins(SE) detection was performed by SET-RPLA (Oxoid, Basingstoke, U.K.) and mini-Vidas R© (bioMerieux, La Balme lesGrottes, France). CNS counts increased during incubation and approached 106 to 107 CFU/g after 12 h at 37 ◦C in the3 foods studied. At 25 ◦C, counts reached 106 to 107 CFU/g only after 24 to 48 h. The interference of backgroundmicrobiota on CNS behavior was only observed when they grew in sliced cooked ham, which presented a high initialtotal count (105 CFU/g). Significantly higher counts of CNS isolated from raw cow’s milk in comparison with foodhandlers isolates were found in reconstituted milk and confectionery cream. Although CNS strains were able to produceSEA, SEB, and SED in culture media, in foods, in the presence or absence of background microbiota S. chromogenesLE0598 was the only strain able to produce SEs. Despite the scarcity of reports on CNS involvement with foodbornedisease outbreaks, the results found here support the CNS growth and SE production in foods even in the presence ofbackground microbiota and may affect food safety.

Keywords: coagulase-negative staphylococci, confectionery cream, enterotoxigenic staphylococci, enterotoxin, foodsafety, ham, skimmed milk powder

IntroductionEnterotoxigenic Staphylococcus is the second most relevant

pathogen associated with foodborne diseases outbreaks in Brazil.The microorganism has been implicated in more than 572 out-breaks between 1999 and 2007 (Anvisa 2008). The disease is causedby the consumption of foods containing preformed SE (Le Loirand others 2003).

In food microbiology laboratories, the ability of coagulase andthermonuclease (TNase) enzymes are commonly linked to thepotential virulence of Staphylococcus strains. In recent years, thisassociation has led regulatory agencies and industries to focus onand establish microbiological methods and specifications based onthe presence of either coagulase or TNase enzymes. Coagulasepositive staphylococci (CPS), which are relevant for food safety,belong to S. aureus, S. hyicus, and S. intermedius species (Holt 1994).However, the main drawback of linking coagulase production tothe enterotoxigenic potential is that it may lead to false negative

MS 20100248 Submitted 3/8/2010, Accepted 6/7/2010. Authors Oliveira, Miya,and Pereira are with Dept. of Food Science, Faculty of Food Engineering, Univ. ofCampinas (UNICAMP), Campinas, SP, Brazil. Author Sant’Ana is with Dept.of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, Univ. ofSao Paulo (USP), Sao Paulo, SP, Brazil. Direct inquiries to author Pereira (E-mail:pereira@fea.unicamp.br).

results in the reports. This may clearly affect food safety, sincealthough some of the staphylococci strains may not express co-agulase production when growing in culture media, they may beable to produce SEs (Veras and others 2008). Furthermore, somestaphylococci strains may also not possess coagulase genes (calledCNS) and will not be able to produce this enzyme in culture me-dia; however, they will be able to produce SE in culture media orfoods.

Within the CNS group, S. epidermidis, S. saprophyticus, S. simu-lans, S. conhii, S. chromogenes, S. xylosus, S. equorum, S. capitis, andS. lentus are the main species isolated from foods with no abilityto produce SEs (Bautista and others 1988; Harvey and Gilmour1990; Vernozy-Rozand and others 1996; Veras and others 2008;Zell and others 2008; Coton and others 2010). CNS are knownfor their relevance in the processing of fermented foods (Irlinger2008; Zell and others 2008; Coton and others 2010). However,CNS’ ability to produce SE (Vernozy-Rozand and others 1996;Udo and others 1999; Zell and others 2008) and linking withfoodborne disease outbreaks (do Carmo and others 2002; Verasand others 2008) may raise concerns about their presence in foods.

Although several studies on behavior and SE production by S.aureus or CPS have been found in the literature (Meyrand andothers 1998; Vernozy-Rozand and others 1998; Delbes and oth-ers 2006; Fujikawa and Morozumi 2006; Kim and others 2009;Le Marc and others 2009), data on CNS behavior and entero-toxin production in foods are very scarce. However, these data

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may be very relevant in order to provide information to evaluatemethodological and surveillance approaches on enterotoxigenic-ity of Staphylococcus spp. Thus, in this study, we have investigatedthe behavior and production of enterotoxin by CNS strains iso-lated from foods and food handlers in cooked ham, confectionerycream, and reconstituted skimmed milk at abuse temperatures.Microbial antagonism of background flora on CNS growth andenterotoxin production was also evaluated.

Material and Methods

CNS strains and their enterotoxigenic potentialEnterotoxigenic CNS strains from Microbial Toxins Lab cul-

ture collection at the Univ. of Campinas (Brazil) were used in thisstudy. These strains presented characteristics of Staphylococcus spp.according to Gram staining, catalase, coagulase and TNase tests,and when grown in Baird–Parker agar (BPA) (Oxoid, Basingstoke,U.K.), as described by Lancette and Bennett (2001). First, entero-toxigenic potential of Staphylococcus spp. used was confirmed usingmembrane-over-agar method as described by Robbins and oth-ers (1974) and Pereira and others (1996). Enterotoxins detection(A, B, C, and D) was performed using Reverse Passive LatexAgglutination method (SET-RPLA, Oxoid, Basingstoke, U.K.)as described by Fujikawa and Igarashi (1988). This method pre-sented a sensitivity of 0.25 ng/mL. Based on these experiments,10 enterotoxigenic CNS strains isolated from food handlers (n =5) (hands and nasal cavity) and from raw cow’s milk (n = 5) wereused. These strains were able to produce at least one type of SE(A, C, or D) but none of the strains produced SEB (Table 1).Therefore, CNS strains showing enterotoxigenic potential wereidentified to the species level using API Staph (bioMerieux, LaBalme les Grottes, France) (Table 1).

FoodsFoods selected for studying behavior and SE production by CNS

were reconstituted skimmed milk, sliced cooked ham, and confec-tionery cream. These foods simulate different scenarios in whichStaphylococcus spp. may contaminate and grow in foods: growth af-ter rehydration (milk powder), recontamination of a cooked prod-uct by handling or equipment when portioning (cooked ham), andhome-made preparation of confectionery cream with limited shelflife. These types of foods have also been associated with staphylo-coccal poisoning (Jett and others 2001).

Skimmed milk powder and cooked ham were purchased fromretail stores in the city of Campinas, Brazil. Skimmed milk powderwas rehydrated with sterile distilled water as established by theproducer (15 to 20 g in 200 mL of distilled water). Confectionery

Table 1– Staphylococcus spp. isolated from raw cow’s milk andfood handlers used in this study and SE produced.1

Staphylococcus spp. Type of SE produced

CNS isolated from S. xylosus (LE0198) SEA, SEC, and SEDraw cow’s milk S. warneri (LE0298) SEA

S. hyicus (LE0398) SEA, SEC, and SEDS. xylosus (LE0498) SEAS. chromogenes (LE0598) SEC and SED

CNS isolated from S. epidermidis (LE0698) SEDfood handlers S. warneri (LE0798) SEA, SEC, and SED

S. epidermidis (LE0898) SECS. epidermidis (LE0998) SEDS. hominis (LE1098) SED

1Strains were identified using API-Staph (bioMerieux). CNS = coagulase negative Staphy-lococcus; SE = Staphylococcal enterotoxins.

cream was prepared according to bakery recipes (100 g of sugar,100 g of corn starch, 8 mL of a 1% tintanil yellow colorant solution,1 egg yolk, and 2 L whole milk). Ingredients were thoroughlymixed, then heated while being stirred until consistency for bakerywas obtained. The foods (100 to 200 g) were then distributedinto sterile closed flasks. The behavior of Staphylococcus spp. wasassessed in the presence and absence of background microbiota.In order to study the CNS behavior in the absence of backgroundmicrobiota, foods were heated in an autoclave (121 ◦C/5 min),while nonheated foods were used to evaluate the CNS behaviorin the presence of background microbiota.

Preparation of CNS cell suspensionsand inoculation of foods

CNS strains were individually cultivated in Brain Heart Infusion(BHI) broth (Difco, Sparks, Md., U.S.A.) at 37 ◦C at 24 h. Theywere centrifuged twice (3000 × g for 10 min at 4 ◦C) and the su-pernatants discarded. Cell sediments were washed and resuspendedin sterile 0.1% peptone water to give a final concentration of106 CFU/mL using McFarland standard scale. Counts were con-firmed by serial decimal dilution and inoculation in BPA plates.

Each food was aseptically inoculated to have a final popula-tion of 103 CFU/mL of each of the 10 CNS strains studied.This resulted in 30 trials that were performed in duplicate at 3different temperatures (25, 30, and 37 ◦C) (n = 180). The in-oculum level used was based on recommendations of the Instituteof Food Technologists (2003) microbial challenge tests. The CNSbehavior and enterotoxin production were evaluated at 25, 30, and37 ◦C, to simulate temperature abuse. Microbial counts and en-terotoxin detection were performed after 0, 12, 24, 48, and 72 h.Noninoculated samples were used as control.

Microbial analysisEnumeration of aerobic mesophylic microorganisms and CNS

was performed with Plate Count Agar (PCA) and BPA (Oxoid,Basingstoke, U.K.) as described by Morton (2001) and Lancetteand Bennett (2001), respectively. Five to ten colonies grown inBPA from each sample and sampling time were taken for con-firmation (Lancette and Bennett 2001). The pH was 6.6, 6.0,and 6.5 for reconstituted skimmed milk, sliced cooked ham, andconfectionery cream, respectively.

Enterotoxin detection in inoculated foodsEnterotoxins detection in foods inoculated with CNS was per-

formed using mini-Vidas R© (bioMerieux) and SET-RPLA (Oxoid,Basingstoke, U.K.). First, the mini-Vidas R© (bioMerieux) test wasperformed to assess the presence of SEs. This method presents asensitivity of 1 ng/g and allows the detection of 7 SEs: A, B, C1,

C2, C3, D, and E (Vernozy-Rozand and others 1998). However, asit uses antienterotoxins polyclonal antibodies, it does not allow thediscrimination of the type of SE produced. However, SET-RPLAuses antienterotoxins monoclonal antibodies and may detect SEsA to D with a sensitivity of 0.25 ng/mL (Fujikawa and Igarashi1988). Therefore, this test was used to determine the type of SEproduced. The results of both methods were expressed as presenceor absence of SE in foods studied.

Enterotoxin detection by mini-Vidas. Sliced cooked hamand confectionary cream extracts were obtained after homoge-nizing samples and buffer (2.5 mol/L TRIS, 10 g/L Tween, and10 g/L sodium azide) at 1:1 proportion. After that, filtration tubescontaining 2 mL of sample suspension were centrifuged (494 × gfor 15min, 4 ◦C). The pH of filtrate was maintained between 6 and

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8, and 0.5 mL of food extracts were placed into the sample well ofmini-Vidas R© (bioMerieux) reagent strip. Reconstituted skimmedmilk samples were inoculated directly into the Vidas R© strip afterbeing heated at 80 ◦C/3 min in order to inactivate endogenous al-kaline phosphatase (Vernozy-Rozand and others 1998). Outputsgave relative fluorescence values (RFV), and results were con-sidered positive and negative when RFV were >0.13 or <0.13,respectively.

Enterotoxin identification by SET-RPLA. Samples werediluted and homogenized with 0.85% saline solution at 1:1 pro-portion. Then, they were centrifuged (900 × g for 30 min, 4 ◦C).Twenty-five microliters of samples supernatants were placed atmicrotitulation plate wells. After that, 25 μL of latex-sensitizedparticles solution was added and plates were kept at room temper-ature for 24 h without agitation. Agglutination of latex particlesobserved against a black bottom was considered a positive result.

Statistical analysisCounts of CNS were transformed to log10 and box plot graphs

were prepared. Multivariate variance analysis was performed forthe 3 different foods studied (sliced cooked ham, reconstitutedskimmed milk, and confectionery cream), considering differenttimes of sampling (12, 24, 48, and 72h) (Johnson and Wich-ern 1982; Dunn and Clark 1987). Based on multivariate varianceanalysis results, a 3-way variance analysis (Dunn and Clark 1987)for each of the sampling times was performed. Variables analyzedwere temperature of incubation (25, 30, and 37 ◦C), source ofCNS strain (food or human origin), and type of sample (with orwithout background microbiota). These variables were describedwith the use of box plot graphs after stratification for the vari-ables that were significant. Statistical analysis was performed usingSPSS for Windows (version 7.5; SPSS, Inc., Sao Paulo, SP, Brazil).Significant differences were considered when P ≤ 0.05.

Results

CNS strain identification and SEproduction in culture media

Table 1 shows the 10 Staphylococcus spp. identified by API-Staph(bioMerieux) and used in this study. All these strains were co-agulase and TNase negative, excepting S. xylosus (LE0198 andLE0498), S. hyicus (LE0398), and S. chromogenes (LE0598) isolatedfrom raw cow’s milk, and S. epidermidis (LE0898 and LE0998) iso-lated from food handlers that were positive for TNase production.All the strains studied have shown ability to produce at least 1 typeof SE. In particular, 4 CNS (S. xylosus LE0198, S. hyicus LE0398,S. chromogenes LE0598, S. warneri LE0798) produced more than 1type of SE. The enterotoxin of type D was the most frequentlyproduced by staphylococcal strains (n = 7), followed by SEA,which was produced by 5 different strains. No CNS producedSEB.

CNS growth in foodsCounts in PCA and BPA for all control samples at time zero

were <101 CFU/g, excepting aerobic mesophilic microorganismsin sliced cooked ham (5.94 log10CFU/g). Overall CNS countsincreased during incubation and approached 106 to107 CFU/gafter 12 h at 37 ◦C in the 3 foods studied. At 25 ◦C, counts reached106 to 107 CFU/g only after 24 to 48 h. CNS counts reached aplateau after 24 h and then were stabilized (data not shown).Although in confectionery cream and reconstituted skimmed milkCNS growth was always observed, in sliced cooked ham, several

CNS strains were inhibited depending on time and temperatureof incubation (P < 0.05) (data not shown).

In reconstituted skimmed milk inoculated with CNS strains,growth was significantly different after 12 h at 37 ◦C (P < 0.05),but no difference was found after this time (P > 0.05) (Figure 1A).In Figure 2, it can be seen that raw cow’s milk CNS strains onlyreached significantly higher counts (P < 0.05) when compared toCNS strains isolated from food handlers after 12 h of incubation.Statistical analysis have shown that only for reconstituted skimmedmilk, time × temperature, source of strains × time, and presenceof background microbiota × time were significant (P < 0.05).It has been found that temperature was significant after 12 and24 h of incubation, while the interaction temperature × sourcewas significant only after 12 h. After 72 h, a significant differ-ence (P < 0.05) was found for the reconstituted skimmed milk inwhich background microbiota was present or not. Staphylococcushyicus (LE0398) and S. warneri (LE0798) were most sensitive tomicrobial competition with background microbiota, being inhib-ited at 25, 30, and 37 ◦C after 72 h (P < 0.05). CNS strains S.warneri (LE0298), S. epidermidis (LE0698), and S. hominis (LE1098)were only inhibited at 25 ◦C, while S. xylosus (LE0498) and S.epidermidis (LE0898) were only inhibited at 37 ◦C (P < 0.05).

In sliced cooked ham (Figure 3) it can be seen that CNS grewbetter in the absence of background microbiota (P < 0.05). Tem-perature significantly affected (P < 0.05) CNS growth after 12and 24 h (Figure 1B). However, the interaction source × pres-ence of background microbiota was only significant (P < 0.05)after 12 h of incubation. For all incubation times, the presence ofbackground microbiota affected (P < 0.05) CNS growth.

In confectionery cream, CNS grew better at 37 ◦C/12 h; how-ever, after this time no significant difference was found regardingtime × temperature (Figure 1C). Considering the source of theCNS strains growing in this food, results indicated that CNS strainsisolated from raw cow’s milk reached higher counts (P < 0.05)after 12 h of incubation. The interaction temperature × sourcesignificantly affected CNS growth after this period of incubation(Figure 4).

SE production in foods by CNSStaphylococcus chromogenes LE0598 isolated from raw cow’s milk

was the only CNS strain able to produce SEs in inoculated foods.This strain produced SE in all incubation times (12, 24, 48, and72 h) and temperatures (25, 30, and 37 ◦C) studied in cookedham and confectionery cream in the presence or absence of back-ground microbiota. However, in reconstituted skimmed milk, inthe absence of background microbiota, S. chromogenes LE0598 wasonly able to produce SE after 48 h at 25 ◦C. Growth of S. chro-mogenes LE0598 in foods resulted in production of SEC and SED.Although it was found that S. warneri LE0298 was able to produceSE in reconstituted skimmed milk in the presence of backgroundmicrobiota after 24 h incubation at 25 and 30 ◦C, SE was onlydetected by ELFA method (mini-Vidas, bioMerieux). Accordingto RPLA, it was found that S. warneri LE0298 produced SEC andSED, but with doubtful results.

DiscussionAlthough most of foodborne disease outbreaks in which Staphy-

lococcus was the agent were caused by S. aureus (Miwa and others2001; do Carmo and others 2002; do Carmo and others 2003;Do Carmo and others 2004; Colombari and others 2007; Lopezand others 2008), incidence and enterotoxigenic potential of CNSstrains in foods have been overlooked. Despite this, studies have

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shown the enterotoxigenic potential of some CNS strains by test-ing SE production in culture media or through detection of SEgenes (Udo and others 1999; Srinivasan and others 2006; Zell andothers 2008). However, limitations of these approaches are thecapability of Staphylococcus to overcome hurdles imposed by thepresence of background microbiota and to find optimum condi-tions to grow and produce SE in foods. However, the knowledge

of CNS behavior in foods is of major relevance to provide infor-mation about their potential to cause foodborne disease outbreaks.Therefore, in this study, the inoculation of 10 different CNS strainsin different foods kept at abuse temperatures was carried out toobserve their ability to grow, producing SEs that impair food safety.

As sliced cooked ham and confectionery cream are highly ma-nipulated, they were chosen for the evaluation of the behavior of

Figure 1–Box plot of coagulase negativeStaphylococcus (CNS) counts (log10 CFU/g) inthe presence and absence of backgroundmicrobiota at 25, 30, and 37 ◦C according tothe time and temperature of incubationreconstituted skimmed milk (A), sliced cookedham (B), and confectionery cream (C).

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CNS at abuse temperatures. In addition, rehydrated skimmed milkhas been chosen because of common association of milk and dairyproducts with staphylococcal intoxication (Oliver and others 2005;Oliver and others 2009). Overall CNS growth in all foods and tem-peratures studied reached higher counts than 105 CFU/g after 12 hof incubation (data not shown). The interference of backgroundmicrobiota on CNS behavior was observed when their growth inconfectionery cream and reconstituted skimmed milk was com-pared with growth in sliced cooked ham. As confectionery creamand reconstituted skimmed milk presented low microbial load attime zero (<101 CFU/g), no significant difference (P < 0.05)on CNS growth in samples with and without background micro-biota. However, as sliced cooked ham presented counts at timezero as high as 105 CFU/g, CNS growth was clearly affected. Inthe presence of background microbiota, although CNS grew after12 and 24 h of incubation, a clear decrease in their population was

found after 48 and 72 h (P < 0.05), showing weak competitivenessof CNS strains. Although CNS counts have declined in the pres-ence of background microbiota, aerobic mesophylic counts werebetween 108 and 109 CFU/g. This is reinforced by the fact thatin sliced cooked ham samples with no background microbiota,CNS counts as high as 107 to 108 CFU/g were found after 72 hof incubation. These data indicate that if CNS contamination oc-curs in foods when background microbiota count is low, thesemicroorganism will be prone to grow, produce SE, and cause afoodborne disease outbreak. However, if this contamination takesplace when background microbiota is already established, CNSsurvival and growth may not occur. The presence of a previousestablished background microbiota was so outstanding that thesource of CNS strains inoculated (raw cow’s milk or food han-dler isolates) have not significantly affected the behavior of thesemicroorganisms in sliced cooked ham (P > 0.05). On the other

Figure 2–Box plot of coagulase negative Staphylococcus (CNS) counts (log10 CFU/g) in the presence and absence of background microbiota at 25, 30,and 37 ◦C according to the time and source of strains (reconstituted skimmed milk).

Figure 3–Box plot of coagulase negative Staphylococcus (CNS) counts (log10 CFU/g) at 25, 30, and 37 ◦C according to the time and presence or not ofbackground microbiota (sliced cooked ham).

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Figure 4–Box plot of coagulase negative Staphylococcus (CNS) counts (log10 CFU/g) at 25, 30, and 37 ◦C in the presence or not of backgroundmicrobiota according to the time and source of strains (reconstituted skimmed milk).

hand, significant difference (P < 0.05) after 12 h of incubationwas found on CNS growth when raw cow’s milk or food handlerisolates were inoculated in reconstituted skimmed milk and con-fectionery cream. In addition, the significantly higher counts ofCNS isolated from raw cow’s milk (P < 0.05) in comparison withfood handlers isolates may indicate the better adaptation of the firstto the nutrients found in milk, present as whole in reconstitutedskimmed milk, or as part in confectionery cream.

Currently, more than 20 types of SE are described (Pereira andothers 1991; Munson and others 1998; Balaban and Rasooly 2000;Orwin and others 2001; Omoe and others 2005; Thomas andothers 2006; Ono and others 2008) and enterotoxigenic Staphylo-coccus counts higher than 106 CFU/g or mL in foods are reportedas critical for enterotoxins being produced in amounts to causediseases (Balaban and Rasooly 2000; Le Loir and others 2003).Our results have shown that, although CNS strains were able toproduce SEA, SEB, and SED in culture media when growingin foods, in the presence or absence of background microbiota,only S. chromogenes LE0598 was able to produce SEs. Althoughsome data indicate that foodborne pathogens are weak competi-tors against background microbiota (McKellar 2007; Alomar andothers 2008), some Staphylococcus species may adapt better andgrow and produce SEs in foods. Despite the fact that S. warneriLE0298 produced SEA in culture media, as detected by RPLAmethod, SEC and SED detection in sliced cooked ham could bedue to unspecific reactions. Cross-reaction between food compo-nents and antienterotoxin latex-sensitized particles leading to falsepositive results have been reported by Pereira and others (1996).However, analyses using mini-Vidas (Enzyme linked immunoflu-orescent assay method–-ELFA) have shown positive results for SEproduction by S. warneri LE0298 confirming that cross-reactiondid not occur.

Although there are scarce reports on CNS involvement withfoodborne diseases outbreaks (do Carmo and others 2002; Verasand others 2008), the results found here support the CNS growthand SE production in foods even in the presence of backgroundmicrobiota. Thus, due to the enterotoxigenic potential of someCNS strains, stakeholders should also consider these microorgan-isms when establishing regulations at governmental and industrial

levels. To our understanding, this modification would certainlyreduce the uncertainty of methods for Staphylococcus enumerationand reinforce the surveillance system focused on enterotoxigenicStaphylococcus. In turn, this would certainly result in improvementsin the public health and in the safety of foods, in which entero-toxigenic Staphylococcus spp. play an important role as agents offoodborne diseases.

AcknowledgmentsThe authors acknowledge FAPESP (Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo), CNPq (Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico), Capes (Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior), and FAEP(Fundo de Apoio ao Ensino e Pesquisa) for the financial supportfor this study.

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