Staphylococcus aureus is an important pathogen bacteria causing food poisoning and various infection in animals and humans. Haemolysin is one of the virulence factors of Staphylococcus aureus. The aims of the research were to characterize haemolysins of Staphylococcus aureus isolated from various food of animal origin, phenotypic- and genotypically. In the present study, eleven Staphylococcus aureus isolated from various food of animal origins from traditional markets and supermarkets in Yogyakarta, Sidoarjo, Jakarta, and Bandung were characterized for haemolysin, pheno- and genotypically. Characterization of haemolysin phenotypically based on haemolysis pattern of Staphylococcus aureus on sheep blood agar plate. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. The results of the studies showed that Staphylococcus aureus on sheep blood agar plates revealed an alpha haemolysis pattern (18,18%), beta haemolysis (27,27%) and gamma haemolysis (54,55%). Based on amplification of the gene encoding haemolysin of Staphylococcus aureus with specific primers showed hla genes (81,81%), and hla combined with hlb genes (18,18%). The amplification of gene hla and hlb had a single amplicon with a size of approximately 534 bp and 833 bp, respectively. The haemolysin characteristics of Staphylococcus aureus from various food of animal origin could be used as important information to control staphylococcal food poisoning.Keywords : Staphylococcus aureus, haemolysin, PCR, food of animal origin

Ariyanti, Dwi

Salasia, Siti Isrina Oktavia

Tato, Syarifudin

English

Staphylococcus aureus is an important pathogen bacteria causing food poisoning and various infection in animals and humans. Haemolysin is one of the virulence factors of Staphylococcus aureus. The aims of the research were to characterize haemolysins of Staphylococcus aureus isolated from various food of animal origin, phenotypic- and genotypically. In the present study, eleven Staphylococcus aureus isolated from various food of animal origins from traditional markets and supermarkets in Yogyakarta, Sidoarjo, Jakarta, and Bandung were characterized for haemolysin, pheno- and genotypically. Characterization of haemolysin phenotypically based on haemolysis pattern of Staphylococcus aureus on sheep blood agar plate. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. The results of the studies showed that Staphylococcus aureus on sheep blood agar plates revealed an alpha haemolysis pattern (18,18%), beta haemolysis (27,27%) and gamma haemolysis (54,55%). Based on amplification of the gene encoding haemolysin of Staphylococcus aureus with specific primers showed hla genes (81,81%), and hla combined with hlb genes (18,18%). The amplification of gene hla and hlb had a single amplicon with a size of approximately 534 bp and 833 bp, respectively. The haemolysin characteristics of Staphylococcus aureus from various food of animal origin could be used as important information to control staphylococcal food poisoning.
Keywords : Staphylococcus aureus, haemolysin, PCR, food of animal origin

Dwi Ariyanti

Siti Isrina Oktavia Salasia

Syarifudin Tato

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Indonesian Journal of Biotechnology

Characterization of Haemolysin of Staphylococcus aureus Isolated from Food of Animal Origin

32Ariyanti et al. I.J. Biotech.Characterization of Haemolysin of Staphylococcus aureus Isolated from Food of Animal OriginDwi Ariyanti1, Siti Isrina Oktavia Salasia2*, and Syarifudin Tato31Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta, Indonesia2Department of Clinical Pathology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia 3Department of Pharmacology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, IndonesiaAbstractStaphylococcus aureus is an important pathogen bacteria causing food poisoning and various infection in animals and humans. Haemolysin is one of the virulence factors of Staphylococcus aureus. The aims of the research were to characterize haemolysins of Staphylococcus aureus isolated from various food of animal origin, phenotypic- and genotypically. In the present study, eleven Staphylococcus aureus isolated  from various food of animal origins from traditional mark ets and supermarkets in Yogyakarta, Sidoarjo, Jakarta, and Bandung were characterized for haemolysin, pheno- and genotypically. Characterization of haemolysin phenotypically based on haemolysis pattern of Staphylococcus aureus on sheep blood agar plate. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. The results of the studies showed that Staphylococcus aureus on sheep blood agar plates revealed an alpha haemolysis pattern (18,18%), beta haemolysis (27,27%) and gamma haemolysis (54,55%). Based on amplification of the gene encoding haemolysin of Staphylococcus aureus with specific primers showed hla genes (81,81%), and hla combined with hlb genes (18,18%). The amplification of gene hla and hlb had a single amplicon with a size of approximately 534 bp and 833 bp, respectively. The haemolysin characteristics of  Staphylococcus aureus from various food of animal origin could be used as important information to control staphylococcal food poisoning.Keywords : Staphylococcus aureus, haemolysin, PCR, food of animal origins*)Corresponding author:Siti Isrina Oktavia SalasiaDepartment of Clinical Pathology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia E-mail: isrinasalasia@ugm.ac.idIntroductionStaphylococcus aureus is well known as bacterial pathogen of both human and animal. In humans, this bacteria causes food poisoning, toxic shock and variety of pyogenic infections (Le Loir et al., 2003). Staphylococcus aureus also could cause mastitis in cows, sheeps and goats, leading to severe economic losses worldwide (Roberson et al., 1994; Salasia et al., 2004).The primary habitat of S.aureus is in the nasal passage on the skin and hair of human and warm-blooded animals. The transmission of the organisms may occur through skin lesions, contaminated food, including milk and other animal products (Le Loir et al., 2003; Boerema, et al., 2006).Staphylococcus aureus is an important food-borne pathogen because of its ability to produce a wide range of extracellular protein toxins and virulence factors that contribute to pathogenecity of the organism (Dinges et al., 2000). The pathogenecity of S. aureus, is related to the production of a wide variety of exoproteins, including alpha and beta haemolysins which contributes to its Indonesian Journal of Biotechnology, June, 2011 Vol. 16, No. 1, pp.32-37I.J. Biotech.33Ariyanti et al.ability to cause diseases in many mammalian species (Da Silva, 2005).Staphylococcal haemolysin are identified as an important virulence factor that contribute for bacterial invasion and to escape from the host immune response. Alpha-haemolysin or alpha toxin considered to be a main pathogenicity factor because of its haemolitic, dermonecrotic and neurotoxic effects. Additionally, beta-haemolysin contains  sphingomyelinase that more active against sheep and bovine erythrocytes (Da Silva, 2005).Several studies indicated that haemolysins of S. aureus correlated well with mastitis. However, there are only few informations about haemolysins of S. aureus isolated from food of animal origin. This study was designed to determine the haemolysin of S. aureus isolated from various foods of animal origin based on their pheno- and genotypic characters. Materials and MethodsBacterial isolatesA total number of 11 S. aureus isolated from foods of animal origin were used in the present study (Table 1). The samples were collected from foods of animal origin from various cities. All isolates were identified as S. aureus based on Gram staining, coagulase and catalase test, and fermentation of mannitol salt agar (MSA). Molecular identification was done according to the amplification of 23S rRNA genes using PCR method. The program and primer design were performed as previously described (Straub et al., 1999) (Table 2).Haemolysins characterizationType of haemolysins were characterized based on the lysis zone of each S. aureus isolates on the blood agar plate after 24 h incubation at 37oC. The genes encoding for haemolysins of S. aureus were performed by amplification of hla genes for alpha haemolysin and hlb genes for beta haemolysins using PCR method. The PCR programs and the sequences of the primers are listed in Table 2.Isolation of DNAThe bacterial DNA was prepared with the QIAamp tissue kit as described by the manufacturer (Qiagen, Hilden, Germany). After cultivation of the isolates for 24 h at 37°C on blood agar plates, 5-10 colonies of the bacteria were suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8) containing 5 µl lysostaphin (1.8 U/µl; Sigma, St. Louis, Table 2. Oligonucleotide primers for amplification of 23S rRNA and genes encoding haemolysinGene Primer sequence Program(*) Reference23S rRNA5’AGC GAG TTA CAA AGG AGG AC 3’3’AGC TCA GCC TTA ACG AGT AC 5’1 Straub et al., 1999hla 5’GGT TTA GCC TGG CCT TC 3’3’CAT CAC GAA CTC GTT CG 5’2 Booth et al., 2001hlb 5’GCC AAA GCC GAA TCT AAG 3’3’GCG ATA TAC ATC CCA TGG C 5’2 Booth et al., 2001(*) 1 : 30 times 940C-120 s, 550C-120 s, 720C-60 s; 2 : 30 times 940C-30 s, 500C-30 s, 720C-60 s.Table 1. The origin of samples No Code Source City1 K1 Cheese Sidoarjo2 SR1 Packaged Milk Yogyakarta3 YK1 Fermented MilkYogyakarta4 RO Rollade Yogyakarta5 SS2 Sosis Yogyakarta6 RY6 Cake Yogyakarta7 B3 Meat ball Yogyakarta8 BL2 Cheese Bolen Bandung9 SY1 Packaged Milk Yogyakarta10 SI1 Packaged Milk Yogyakarta11 BW4 Brownies Jakarta 34Ariyanti et al. I.J. Biotech.Missouri, USA). After 1 h incubation at 37°C, 25 µl of proteinase K (14,8 mg/ml; Sigma) and 200 µl  of buffer AL (containing reagents AL1 and AL2) were added. The suspension was incubated for 30 min at 56°C and for 10 min at 95°C, and after a spin for a few seconds an amount of 420 µl ethanol was added to each sample and placed in a spin column. After centrifugation for 1 min, the QIAamp spin columns were placed in a clean collection tube and the samples were washed twice with 500 µl of buffer AW. After a second wash and a centrifugation for 3 min, the QIAamp spin columns were placed in a clean 2 ml microfuge tube and the DNA was twice eluted with 200 µl and 100 µl of buffer AE, respectively. The DNA could be stored at -20°C. Amplification reactions with PCR were perfomed by program and specific primer according to the references for 23S rRNA and hla and hlb genes.  PCR products were then separated by gel electrophoresis in a 1.5% w/v agarose (Seakem) gel run in 0.5 × TBE buffer. The 1 kb Plus DNA ladder (Invitrogen, Netherlands) was used as a size marker. Resulting bands were visualized with cyber safe by UV transillumination.Results and DiscussionAccording to Gram staining, biochemical properties and to the analysis of the PCR products of 23S rRNA specific region, all isolates in the present study could be identified as S. aureus. All cultures investigated were Gram positive, positive for coagulase and catalase, and fermented of MSA (Table 3). A comparable PCR-based system for identification of S. aureus isolated from various origins had already been used by numerous authors (Straub et al., 1999; Annemüller et al., 1999;  Akineden et al., 2001; Salasia et al., 2004).The types of haemolysins of S. aureus on the sheep blood agar plate, revealed alpha hemolysis for 2 isolates (18,18%), beta haemolysis for 3 isolates (27,27%) (Figure 1) and gamma hemolysis for 6 isolates (54,55%) (Table 4). Table 3. Identification of S. aureus Code Food Sample Source of IsolateGram StainingFermen-tation of MSA*Cata-lase Coa-gulase 23S rRNA GeneK1 Cheese Sidoarjo + + + + +SR1 Packaged Milk Yogyakarta + + + + +YK1 Fermented Milk Yogyakarta + + + + +RO Rollade Yogyakarta + + + + +SS2 Sosis Yogyakarta + + + + +RY6 Cake Yogyakarta + + + + +B3 Meat ball Yogyakarta + + + + +BL2 Cheese Bolen Bandung + + + + +SY1 Packaged Milk Yogyakarta + + + + +SI1 Packaged Milk Yogyakarta + + + + +BW4 Brownies Jakarta + + + + +* MSA = mannitol salt agarFigure 1. Alpha-haemolysis (A) and beta-hemolysis (B) of Staphylococcus aureus isolates K1 and YK1 on the sheep blood agar plate.                   A                                         BI.J. Biotech.35Ariyanti et al.Figure 2. PCR Amplification of hla gene of S. aureus isolated from food, with the amplicon size of approximately 534 bp, in 2% of agarose gel electrophoresis. M= marker DNA ladder, K+/- = positive/negative control.Figure 3. PCR amplification of hlb  gene of S. aureus isolated from food, with the amplicon size of approximately 833 bp, in 2% of agarose gel electrophoresis. M= marker DNA ladder, K+/- = positive/negative control.By PCR amplification of the gene encoding haemolysin of S. aureus with specific primers could be observed hla gene for 9 isolates (81,81%), and 2 isolates with two genes in combination hla and hlb (18,18%). The size of amplicon for hla gene was approximately 534 bp and 833 bp for hlb gene (Figure 2 and 3).The S. aureus isolates with hla genes expressed alpha-, beta- and gamma-haemolysis on the sheep blood agar. The S. aureus with two genes in combination of hla and hlb expressed phenotypically as alpha- and beta- haemolysis. The genotypes of haemolysin of S. aureus in the recent study seemed to be not having correlation with the expression of their phenotypes. It might be influenced by many factors on the level of genetic or phenotypic. Blood collected from various animals on agar plates could show variable haemolysis pattern of S. aureus. The sheep blood shows an alpha, beta or gamma haemolysis of S. aureus on agar plates. However, the horse blood specifically showing a delta haemolysis of S. aureus on agar plates (Da Silva, 2005). Most of S. aureus isolated from human have usually an alpha haemolytic character, because the human platelets and monocytes are more sensitive to the alpha toxin. S. aureus isolated from animal are mostly characterized beta toxin because of the sensitivity of animal erythrocytes to this toxin (Todar, 2005). S. aureus isolated from foods Table 4. Characterization of haemolysins of S. aureus Code Food Sample Source of IsolateHaemolysis Type  on Sheep Blood Agar Platehla gene hlb geneK1 Cheese Sidoarjo α + -SR1 Packaged Milk Yogyakarta β + -YK1 Fermented Milk Yogyakarta β + -RO Rollade Yogyakarta Γ + -SS2 Sausage Yogyakarta γ + -RY6 Cake Yogyakarta γ + -B3 Meat ball Yogyakarta γ + -BL2 Cheese Bolen Bandung γ + -SY1 Packaged Milk Yogyakarta γ + -SI1 Packaged Milk Yogyakarta α + +BW4 Brownies Jakarta β + +1000 bp534 bp300 bp1000 bp833 bp500 bp36Ariyanti et al. I.J. Biotech.used in this study might have more complex haemolysin characters. Cross contamination between humans and food might occur via humans with skin lesions or via nasal discharge (Sandel and Mc. Killip, 2002). Food were probably affected by food contamination during handling or food processing. The temperature plays also an important role to the haemolysis character and in the mechanism of  hot cold lysis for beta toxin (Todar, 2005). The sheep blood agar used in this study could show alpha, beta and gamma haemolytics, nor delta hemolytic. The age of the sheep was also reported influence to the type of haemolytic of S. aureus. In this study was used blood from a sheep with age of about 1 year old, that might contain of haemoglobin with the concentration higher than the younger (Mitruka and Rawnsley, 1981). Agar plate that contain old sheep blood may cause a dark back ground of plates that can confusing on the haemolysis examination. S. aureus with hla gene should express an alpha haemolytic. However, in this research some isolates revealed gamma haemolytic on blood sheep agar plate. It is quite possible the haemolytic type were influenced by high concentration of haemoglobin on sheep blood.The existence of hla and hlb genes in S. aureus isolates are important for this strains, related to staphylococcal infection cases that caused food poisoning. This study demonstrated that the hla and hlb genes widely distributed among S. aureus isolated from food of animal origins. Indeed, although their true incidence in staphylococcal infection cases that caused food poisoning has still need to be clarified, it is now thought likely that their role has been underestimated.In conclusions, the variable pattern of haemolysins of S. aureus established by the characteristic properties of samples which were highly determined by contamination both of animal product (milk, meat, and egg) as the basic material, or  human as a food processor. Environment was also plays an important role for the hygiene and sanitation of the food process and storage. The haemolysin characteristics of  S. aureus from various food of animal origin could be used as important information to control staphylococcal food poisoning.AcknowledgementsThis research was a part of the research results funded by Ministry of Education Republic of Indonesia through Hibah Kompetensi Program 2008-2010. The authors would acknowledge drh. Sugiyono, M.Sc. for help in the research in Clinical Pathology Laboratory and drh. Khusnan, MP., Academy of Farming Brahmaputra for help in collecting rabbit and sheep blood. ReferencesAkineden, Ö., Annemüller, C., Hassan, A. A., Lämmler, C., Wolter, W., and Zschöck, M. 2001. Toxin genes and other charac-teristics of Staphylococcus aureus isolates from milk of cows with mastitis. Clin. Diagn. Lab. Immunol. 8, 959-964.Annemüller, C., Lämmler, C., and Zschöck, M. 1999. Genotyping of Staphylococcus aureus isolated from bovine mastitis. Vet. Microbiol. 69, 217-224.Boerema, J. A., R. Clemens and G. Brightwell, 2006. Evaluation of Molecular Methods to Determine Enterotoxigenic Status and Molecular Genotype of Bovine, Ovine, Human and Food Isolats of Staphylococcus aureus. Int. J. Food. Miro-biol. 107 (2), 192-201.Booth, M. C., Pence, L. M., Mahasresthi, P., Callegan, M. C., Gilmore, M. S. 2001. Clonal Association among Staphylococ-cus aureus Isolates from Various Sites of Infection. Infect Immun., 69, 345-352Da Silva, R. E., Boechat, J. U. D., Martins, J. C. D., Ferreira, W. P. B. , Sequera, A. P. S., and Da silva, N. 2005. Hemolysin Production by Staphylococcus aureus Species Isolated from Mastitic Goat Milk in Brazilian Dairy Herd. Science. Small rum. res., 56, 271-275.Dinges, M. M., Orwin, P. M., Schlievert, P. M., 2000. Enterotoxin of Staphylococcus aureus. Clin. Microbiol. Rev., 13, 16-34. I.J. Biotech.37Ariyanti et al.Le Loir, Y., Baron, F. and Gautier, M. 2003. Staphylococcus aureus and Food Poison-ing. Genet. Mol. Res., 2(1), 63-76. Rennes Cedex.Mitruka, B. M. and Rawnsley, H. M. 1981. Clinical, Biochemical and Hematologi-cal Reference Values in Normal Experi-mental Animals and Normal Human. 2nd. Year Book Medical Publishers Inc.Roberson, J.R., L.K. Fox, D.D. Hancock, J.M. Gay, and T.E. Besser. 1994. Ecology of Staphylococcus aureus isolated from various sites on dairy farms. J. Dairy Sci., 77, 3354.Salasia, S.I.O., Khusnan, Z. Lämmler, C. and Zschöck, M. 2004. Comparative Studies on Pheno- and Genotypic Properties of Staphylococcus aureus, Isolated from Bo-vine Subclinical Mastitis in Central Java in Indonesia and Hesse In Germany. J.Vet. Sci., 5 (2), 103-109. Sandel, M. K. and Mc. Killip, J. L. 2002. Viru-lence and Recovery of Staphylococcus aureus Relevant to the Food Industry Using Improvements on Traditional Approaches. Food Control. 15, 5-10.Straub, J. A., Hertel, C and Hammes, W. P. 1999. A 23S rRNA-Targeted Polymerase Chain Reaction-Based System for De-tection of Staphylococcus aureus in Meat Starter Cultures and Dairy Product. J. Food. Prot., 62, 1150-1156.Todar, K. 2005. Todar Online Textbook of Bacteriology. Staphylococcus. University of Wincosin-Madison Department of Bacteriology. www.text bookofbacte-riology.net/staph.html. Download 1 November 2007.

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Characterization of Haemolysin of Staphylococcus aureus Isolated from Food of Animal Origin

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