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  3. Investigating contamination and genotyping of Staphylococcus aureus coagulase gene in traditional cheeses of villages around Malekan and Mia Ndoab cities.

Investigating contamination and genotyping of Staphylococcus aureus coagulase gene in traditional cheeses of villages around Malekan and Mia Ndoab cities.

Number of pages: 145 File Format: word File Code: 32087
Year: 2011 University Degree: Master's degree Category: Biology - Environment
Tags/Keywords: Coagulase - Coagulase gene genotyping - Dairy products - Local cheese - PCR - pollution - Staphylococcal poisoning - Staphylococcus aureus - Traditional cheeses
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  • Summary of Investigating contamination and genotyping of Staphylococcus aureus coagulase gene in traditional cheeses of villages around Malekan and Mia Ndoab cities.

    Dissertation for receiving a master's degree

    Inclination: Microbiology

    Abstract

    Staphylococcus aureus is the third most important cause of foodborne diseases. Milk is the primary material for the growth of Staphylococcus aureus and milk products are a known source of staphylococcal poisoning. This study was conducted to determine the prevalence of Staphylococcus aureus in local cheeses. A total of 103 samples were randomly collected from the villages around Malekan and Miandoab and tested with culture and PCR methods. According to the results of this study, 7 cases (11.6%) of the samples were infected with Staphylococcus aureus and another 7 cases (11.6%) of the samples were infected with Staphylococcus intermedius and according to the results obtained in this Study 2.23% of samples were infected with coagulase-positive staphylococci and 76.8% of samples were infected with coagulase-negative staphylococci. The results of this study showed that compliance with sanitary conditions during production is necessary and the use of pasteurized milk is important in the preparation of cheese. style="direction: rtl;">Keywords: Local cheese - Staphylococcus aureus - Cultivation - PCR - Malekan and Miandoab.

    Introduction:

    Cheese is one of the most important human phenomena, which has a great contribution to human nutrition, and while providing the necessary ingredients for growth, it is effective in making the daily food pleasant. In rural and nomadic areas, due to the lack of cold storage to store milk and the pleasant taste of traditional cheeses, the production and consumption of this product is common.

    Since cheese is one of the most important milk products consumed in the country and the consumption of traditional cheese is also popular in many parts of the country, its microbial contamination should be taken into consideration.

    Locally produced cheeses are divided into several categories. which is based on animal milk such as buffalo, cow, sheep, goat and mixed cheese (cow milk + sheep milk) from which cheese is prepared.

    Staphylococcus genus is one of the important bacteria in food hygiene and human and domestic diseases. Among the different species of this genus, some cause food poisoning, some cause skin ulcers, boils, and some cause very dangerous hospital infections, and sometimes they are resistant to treatment. Staphylococcus is one of the genera with opportunistic bacteria on the body surfaces of warm-blooded organisms. The most important problem in terms of public health, especially in terms of food hygiene, is food poisoning caused by enterotoxin produced by St. aureus.

    The characteristics of poisoning caused by Staphylococcus aureus are: The incubation period is short, 30 minutes to 8 hours, vomiting, abdominal pain and contractions, weak pulse, sweating, and low body temperature. In this poisoning, death is very rare and recovery occurs within 24-48 hours. This poisoning is one of the most common food poisonings in the world and is among the top 3 causes of food poisoning in most countries (6,10).

    The culture methods that are used to isolate coagulase-positive staphylococcus aureus are time-consuming and apparently false positive responses are observed in their results. In recent studies, new methods using polymerase chain reaction (PCR) [1] have shown good efficiency.

    Therefore, due to the importance of the subject, the present study was conducted to determine the prevalence of coagulase-positive Staphylococcus aureus in local cheeses based on culture and PCR methods.

    rtl;">Research Objectives

    Investigation of the prevalence of Staphylococcus coagulase positive in local cheeses in the villages around Malekan and Miandoab using the culture method.

    Investigation of the prevalence of Staphylococcus aureus in local cheeses in the villages around Melkan and Miandoab based on the PCR method.

    Comparison of the results of investigation by culture and PCR method

    Staphylococcus genus[1]

    Staphylococcus are found everywhere. These bacteria are among the most common microorganisms. Despite the identification of antimicrobial substances and the improvement of public health, which played an effective role in reducing the prevalence and mortality of staphylococcal diseases in the 20th century, staphylococci are still considered one of the most important pathogens. These bacteria are responsible for more than 80% of purulent diseases with clinical symptoms. The primary natural habitat of staphylococci is the body surface of mammals, where microorganisms are found in large numbers. If the defense barrier of the host body is damaged due to injury or surgery and these microorganisms penetrate deep tissues, their aggressive power will be revealed. If this bacterium enters the general circulation of the body, it can cause acute endocarditis and widespread metastatic conditions. The main factor that caused these microorganisms to survive despite the production of a wide range of antibiotics effective on staphylococci (in the last 40 years) is the strains resistant to penicillin and its derivatives and the presence of these strains in severe hospital infections. Grape clusters grow. The name staphylococcus is derived from the Greek root staphyl (meaning a bunch of grapes) and cocos (meaning a seed or grain). For the first time, this word was used to describe the microorganisms that were present in the purulent secretions of surgical wounds. Since young strains produce yellow pigment in staphylococcal infections, this microorganism is called Staphylococcus aureus and can be distinguished from strains with less pathogenic power (staphylococci producing white colonies). Pigment production is a variable feature in staphylococci and its relation to pathogenicity is not certain. Today, the ability to produce coagulase [2] has replaced the pigment type of colonies. The ability to produce coagulase is the most useful diagnostic criterion for Staphylococcus aureus (7).  

    1-1-1- Classification of staphylococci

    Staphylococci are spherical and gram-positive bacteria. In general, the classification of Staphylococcus aureus is as follows: Prokaryote ? Kingdom Order Firmibacterium ? Class Class Micrococcaceae ? Family Family

    Staphylococcus ? Genus

    Aureus ? Species

    The Micrococcaceae family has 4 genera, which are: Staphylococcus, Micrococcus, Stomatococcus and Plano coconut Among the mentioned genera, only staphylococci are capable of causing disease, and other genera are usually harmless and live on plants and mucus of different animals (7).

  • Contents & References of Investigating contamination and genotyping of Staphylococcus aureus coagulase gene in traditional cheeses of villages around Malekan and Mia Ndoab cities.

    List:

     

    Abstract. .. .1

    The first chapter: An overview of the research background

    Introduction. .. . 2. Research objectives. 3. Staphylococcus genus. 5

    Staphylococcus aureus.. 7

    Morphology.. 7

    Structure features. .. .7

    Biological and biochemical characteristics. 8 Pettidoglycan 9

    Polysaccharide.. 10

    Acideticoic.. 10

    Protein. A.. 11

    Physiology.. 11

    Cultural characteristics .. 11

    Metabolism.. 12

    Biochemical identification.. . 13

    Classification methods.. . 13

    Determining the type with bacteriophage.. 13

    Determining the types.. 14

    Genetics.. 14

    Resistance.. 15

    Effective factors in pathogenicity.. 15

    Protein receptors.. 16

    Extracellular enzymes.. 16

    Guaculase.. 17

    Clamping factor.. 17

    The cause of coagulase polymorphism. 18

    lipases.. 18

    hyaluronidase.. 18

    staphylokinase (fibrinolysin). 19

    Heat-resistant nuclease.. 19

    Cytolytic toxins. 19. Alpha toxin (hemolysin-alpha). .19

    Beta toxin (sphingomyelinase). 19. Delta toxin.. 19. Gamma toxin.. 20. Lecocidin. .22

    Production of enterotoxin.. . 23

    Exfoliative toxin... 23

    Extraintestinal disease... 23

    Staphylococcus food poisoning. 20

    Symptoms of food poisoning... 25

    The second chapter: Research method

    Materials and work methods. 27

    Selective environment.. 28

    Confirmation tests. .. .30

    Catalase test.. .30

    Glucose test.. .30

    Mannitol test.. .30

    Coagulase test.. .31

    Microscopic observation.. .32

    How to take samples.. .32

    Ready Making a sample for testing. . 32. DNA extraction.. 33. DNA quality determination using electrophoresis on agarose gel. . 34

    Determining the quantity and quality of DNA using spectrophotometry. 34

    Polymerization chain reaction. 35

    Electrophoresis of PCR products. 36

    Separation of duplicate parts. . . 37

    Loading duplicated products. 37.

    Chapter Three: Research Results

    The results of identifying Staphylococcus aureus by culture. 32. The results of DNA quality determination using electrophoresis on agarose gel. 41

    Results of polymerase chain reaction based on Coa gene. . 42

    Sequencing results.. 43

    Chapter four: discussion and conclusion

    Conclusion and suggestion.. . 51

    List of sources.. 95

    English abstract.. 98

    Source:

    List of Persian sources

    1- Barvan P., 1386, An introduction to gene cloning, DNA analysis translated by Tabatabai, M., fifth edition House of Biology, pp. 225 to 240

    2- Brown, B., 1386, Genome 3, translated by Fard Esfahani, P. Valeh Yaari., first edition, Khosravi Publications, pp. 76 to 79.

    3- Taj Bakhsh, H. 1375, Genetics of bacteria, fourth edition, Tehran University Press, pp. 466-495.

    4- Jaklik, P.  1382, Zinser microbiology translated by Rahimi, M., pages 25 to 48.

    5- Jamshidian, M., 1372, Bacteriology and mycology diagnosis guide for veterinary clinic, Shahid Chamran University, pages 152 to 148.

    6- Razweiler, V., 1387, Pathogenic microbes in food and myology of poisoning Foods, Tehran University Publications, pages 127-135. 7- Shimi, A., 1376, Veterinary Bacteriology and Bacterial Diseases, Jihad Publishing Institute affiliated with Jihad University, pages 85-94. 8- Standard and Industrial Research Institute of Iran, 1364, method of identification and counting of staphylococcus aureus quagmosome registered in food, print Fourth, Standard 1194

    9- Institute of Standard and Industrial Research of Iran., 1370, Panirsafid, third edition, Standard 2344

    10- Institute of Standard and Industrial Research of Iran., 1374, Code of Application and General Methods of Microbial Food Testing, seventh edition, standard number 2344

    11- Institute of Standard1374, Code of practice and general method of microbial testing of food, 7th edition, standard number 2344. 11- Institute of Standard and Industrial Research of Iran, 1375, preparation of food samples and counting of different microorganisms, first revision of the 10th edition, standard number 356. Mashhad and Qochan cities to Listeria monocytogenes, Staphylococcus aureus and Escherichia coli pathogens. The 12th National Environmental Health Conference of Iran, Shahid Beheshti University of Medical Sciences, Faculty of Health.

    13- Marhamatizadeh, M.H., Karim, G., Nik Afroz, R., Picer, J. (2018). Investigation of the contamination of traditional cheese with Staphylococcus aureus quagmire registered in Kazeroon city. Food and nutrition science. Year 4. Number 2. Pages 33-44.

    14- Karim, G.  1387, Microbial food tests, Tehran University Press, pages 128-138

    15- Karim, G., 1380, its milk products, Sepehr Press, pages 191-185.

    List of non-Persian sources

    16- Abbar, F.M. (2001). Tahir Mohammed M. Identification of som enterotoxigenic str ains of Staphylococci form locally processed cheese. Food Microbiology, 3.pp:33-36.

    17-Almeido Filho E., Nader Faiho.A.(2001). Occurrence Staphylococcus aureus frescal type of cheese. Rev Saud publica, 34.pp:578-580.

    18- Atanassova, V., Meindl, A. and Ring, C. (2001). Prevalence of Staphylococcus aureus and Staphylococcal enterotoxins in raw pork and uncooked smoked ham a comparison of classical coltoring detection and RFLP-PCR, International Journal of food microbiology, Vol68, PP - 105-113

    19-Claudia, M., Ander, D.P.B., Hidalgo Campos, M.R., Borges, L. J., Kipnis, A. (2005). Comparison of Staphylococcus aureus isolates form food handlers, raw bovine, food Microbiology 76pp:567-571

    20-Dastmalchi, H., Ahmadi, M., Mardani, K. and Batavani, R.A. (2009). Molecular typing of Staphylococcus aureus isdated form bovine mastitis based on polymorphism of the coagulase gene in the north west of Iran, Veterinary microbiology., vol 137, pp 202-206.

    21- Donnelly, C.B., Blak, L.A., Lewis, K.H. (2005). Occurrence of coagulase-positive staphlococci in Chaddar chesse, Applied Microbiology vol.12,No.4.pp:311-315

    22-Hasaliu,R.,Beli,E.,Terpollari,j.(2009).The influence of storage temperature of cheese on the incidence, Agricultural science,41 (2)

    23-Ikeda.T.,Morimoto.Y.,Makino.S.,Keui.(2006).Surveillance of Staphylococcus in some markets in Albania, Research Journal of Agricultural Science, 41(21)

    24- Johnv.H. Judithf. R, and Barryd.C. (1998). Molecluar Typing of Staphylococcus aureus based on PCR restriction fragment length Polymorphism and DNA sequence analysis of the coaglase gene., Journal of clinical microbiology, Apr. 1998, P. 1083-1089

    25- Kumar.S, Alin K., Ramavtar. S and Govid.S.(2008). Epidemiological typing of Staphylococcus aureus by DNA restriction fragment length polymorphism of coa gene. veterinarski arhiv 78(1), 31-38

    26-Lamperll, H., Villard, L., Cha Beuvier, E., Borgers, E., Maurin, F.F. Mazer Olles, G., NoY. Kodjo, A. (2006). Idification and biotyping of coagulase positive staphylococci in ripe French raw milk cheese and their in vitro ability to produce entrotaxin. Revue Med Vet, 155.pp:92-96

    27- Little, C.L., Rhoades, J.R., Sagoo, S.K., Harris, j., Greenwood, M., Mithani, V.rana, K. (2008). Mclauchlin, J. Microbiological quality of retail chess made form raw thermizd or pasteurizd milk in the UK,.food Microbiology 25.pp:304-312.

    28-Masatcioglu.T.M.Avsar.YK.(2005). Effect of flavorings, storage condition and storage time on survival of Staphylococcus aureus in Surk cheese, J. food prot., 68. pp: 1487-91.

    29- Mcdevitt, D. Vaudaux, p. , and , T.j. Foster, (2001)., Genetic evidence that bound coagulase of Staphylocococcus aureus is not clumping factor., Infection and immunity, Apr. 1992, P.1514 - 1523

    30-Obrien, M., Hunt, K., Mcsweenety, S., Jordan, K. (2009). Occurrence of food borne pathogens in Irish farhouse cheese,.food Microbiology 26.pp:910-914.

    31-Omer, A., Abdulwahed, A., Schneider, E. and Usleber, E. (2008).

Investigating contamination and genotyping of Staphylococcus aureus coagulase gene in traditional cheeses of villages around Malekan and Mia Ndoab cities.
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