Phenotypic and genotypic characterization of Staphylococcus aureus isolated from pork sausage
DOI:
https://doi.org/10.33448/rsd-v10i2.12041Keywords:
Biofilm; Enterotoxins; Methicillin; Public health; Sublethal stress.Abstract
The objective of this study was to characterize the genotype and phenotype of S. aureus isolates from pork sausages. Fifty samples of pork sausages were collected, counts of coagulase-positive Staphylococcus were made and isolates were obtained to identify S. aureus species. In the isolates, the presence of genes sea, seb, sec, and sed was surveyed, the methicillin-resistance was assessed and the production of biofilm in Congo red agar, stainless steel, polyethylene, glass, and pork casing was tested. The capacity of biofilm formation was assessed after the exposure to sublethal stress. Of the samples tested, 12% had counts superior to what is permitted by the legislation. S. aureus was isolated in 44% of the samples. Of these, 54% had only the gene sed and 32% had genes sec and sed, 73% were classified as methicillin-resistant S. aureus (MRSA). Of the MRSA isolates, 62% had only gene sed and 35% had both genes found in this study. Regarding the biofilm formation in Congo red agar, 68% of S. aureus isolates were considered as biofilm formers. After undergoing the sublethal stress, most of the biofilm former isolates proceeded to form biofilm and the non-biofilm former isolates responded in a distinct manner. The condition in which the sublethal stress greatly induced the biofilm formation was the cold. Biofilm production was observed only in the stainless steel and pork casing in 71% and 57% of the isolates tested, respectively. Thus, we stress the importance of implementing good manufacturing practices within the industry to control microbial contamination and biofilm formation.
References
Andretta, M. (2019). Serro artisanal cheese produced in Brazil has a microbial safety status for consumers. https://www.locus.ufv.br/bitstream/handle/123456789/25766/texto%20completo.pdf?sequence=1&isAllowed=y
Bauer, A. W., Kirby, W. M., Sherris, J. C. & Turch, M. (1966), Antibiotic susceptibility testing by a standardized single 457 disk method. American Journal of Clinical Pathology, 45 (4), 493-496.
Botelho, C. V. (2017). Staphylococcus coagulase positiva e Staphylococcus aureus resistentes a antibióticos em cadeia produtiva de carne suína. https://www.locus.ufv.br/bitstream/handle/123456789/18691/texto%20completo.pdf?sequence=1
Brasil - Ministério da Agricultura e do Abastecimento. (2017). Decreto nº 9.013, de 29 de março de 2017. Regulamento de inspeção industrial e sanitária de produtos de origem animal. https://www.gov.br/agricultura/pt-br/assuntos/aquicultura-e-pesca/legislacao/legislacao-geral-da-pesca/decreto-no-9-013-de-29-03-2017.pdf/view#:~:text=Regulamenta%20a%20Lei%20n%C2%BA%201.283,de%20produtos%20de%20origem%20animal
Brasil - Ministério da Agricultura e do Abastecimento. (2001). Resolução nº 12, de 02 de janeiro de 2001. Regulamento técnico sobre padrões microbiológicos para alimentos. http://portal.anvisa.gov.br/documents/33880/2568070/RDC_12_2001.pdf/15ffddf6-3767-4527-bfac-740a0400829b#:~:text=1%C2% BA%20Aprovar%20o%20REGULAMENTO%20T%C3%89CNICO,1977%2C%20e%20demais%20disposi%C3%A7%C3%B5es%20aplic%C3%A1veis
Carfora, V., Caprioli, A., Marri, N., Sagrafoli, D., Boselli, C., Giacinti, G., Giangolini, G., Sorbara, L., Dottarelli, S., Battisti, A. & Amastite, S. (2015). Enterotoxin genes, enterotoxin production, and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy. International Dairy Journal, 42, 12-15.
Carmo, L. S., Dias, R. S., Linardi, V. R., Sena, M. J., Santos, D. A., Faria, M. E., Pena, E. C., Jett, M. & Heneine, L. G. (2002). Food poisoning due to enterotoxigenic strains of Staphylococcus present in Minas cheese and raw milk in Brazil. Food Microbiology, 19 (1), 9-14.
Chang, C. M., Chiang, M. L. & Chou, C.C. (2004). Responses of heat-shocked Vibrio parahaemolyticus to subsequent physical and chemical stresses. Journal of Food Protect, 67 (10), 2183-2188.
Chen, Q., Xie, S., Lou, X., Cheng, S., Liu, X., Zheng, W., Zheng, Z. & Wang, H. (2019). Biofilm formation and prevalence of adhesion genes among Staphylococcus aureus isolates from different food sources. Microbiology Open, 9 (1), 1-11.
CLSI - Clinical and Laboratory Standards Institute. (2019). Performance Standards for Antimicrobial Susceptibility Testing - Twenty Fourth Informational Supplement. https://clsi.org/media/2663/m100ed29_sample.pdf
Cunha, M. R. L. S., Calsolari, R. A. O. & Júnior, J. P. A. (2007). Detection of Enterotoxin and Toxic Shock Syndrome Toxin 1 Genes in Staphylococcus, with Emphasis on Coagulase Negative Staphylococci. Microbiology and Immunology, 51 (4), 381-390.
De Buyser, M. L., Dufour, B., Maire, M. & Lafarge, V. (2001). Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. International Journal of Food Microbiology, 67 (1-2), 1-17.
Denayer, S., Delbrassinne, L., Nia, Y. & Botteldoorn, N. (2017). Food-borne outbreak investigation and molecular typing: high diversity of Staphylococcus aureus strains and importance of toxin detection. Toxins, 9 (12), 407.
Di Ciccio, P., Vergara, A., Festino, A. R., Paludi, D., Zanardi, E., Ghidini, S. & Lanieri, A. (2015). Biofilm formation by Staphylococcus aureus on food contact surfaces: Relationship with temperature and cell surface hydrophobicity. Food Control, 50, 930-936.
Donlan, R. M. & Costerton, J. M. (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews, 15 (2), 167-193.
El-Maghraby, M. S., Hassan, M. A., Hassanin, F. S. & Shawky, N. A. (2018). Detection of Enterotoxigenic Staphylococcus aureus in Meat Product Sandwiches Using Multiplex PCR. Benha Veterinary Medical Journal, 35 (1), 190-196.
Franco, B. D. G. M. & Landgraf, M. (2008), Microbiologia dos Alimentos. Atheneu.
Freeman, D. J., Falkiner, F. R. & Keane, C. T. (1989). New method for detecting slime production by coagulase-negative staphylococci. Journal Clinical Pathology, 42 (8), 872–874.
Friedriczewski, A. B., Gandra, E. Á., Da Conceição, R. C., Cereser, N. D., Moreira, L. M. & Timm, C.D. (2018). Biofilm Formation by Coagulase-Positive Staphylococcus aureus Isolated from Mozzarella Cheese Elaborated with Buffalo Milk and its Effect on Sensitivity to Sanitizers. Acta Scientiae Veterinariae, 46 (1), 6.
Galvão, N. N., Chiarini, E., Destro, M. T., Ferreira, M. A. & Nero, L. A. (2012). PFGE characterisation and adhesion ability of Listeria monocytogenes isolates obtained from bovine carcasses and beef processing facilities. Meat science, 92 (4), 635–643.
Germano, P. M. L., & Germano, M. I. S. (2015). Higiene e vigilância sanitária de alimentos. Manole.
Giaouris, E., Heir, E., Hébraud, M., Chorianopoulos, N., Langsrud, S., Moretro, T., Habimana, O., Desvaux, M., Renier, S. & Nychas, G. J. (2014). Attachment and biofilm formation by foodborne bacteria in meat processing environments: causes, implications, role of bacterial interactions and control by alternative novel methods. Meat Science, 97 (3), 298-309.
Goulart, A. R., Lacerda, I. C. A. & Dias, R. S. (2016). Potencial risco de intoxicação alimentar por Staphylococcus spp. enterotoxigênicos isolados de bolos com cobertura e recheio. NBC Periódico Científico do Núcleo de Biociências, 6 (11), 11-17.
Haaber, J., Cohn, M. T., Frees, D., Andersen, T. J. & Ingmer, H. (2012). Planktonic aggregates of Staphylococcus aureus protect against common antibiotics. Plos One, 7 (7).
Iñiguez-Moreno, M., Gutiérrez-Lomelí, M., Guerrero-Medina, P. J. & Avila-Novoa, M. G. (2018). Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. Brazilian Journal Microbiology, 49 (2), 310-319.
Jefferson, K. K. (2004). What drives bacteria to produce a biofilm? FEMS Microbiology Letters, 236 (2), 163-173.
Khan, F., Shukla, I., Rizvi, M., Mansoor, T. & Scharma, S. C. (2011). Detection of biofilm formation in Staphylococcus aureus. Does it have a role in treatment of MRSA infections. Trends in Medical Research, 6 (2), 116-123.
Kluytmans, J. A. J. W. (2010). Methicillin-resistant Staphylococcus aureus in food products: cause for concern or case for complacency?. Clinical Microbiology and infection, 16 (1), 11-15.
Lee, A., De Lencastre, H., Garau, J., Kluytmans, J., Malhotra-Kumar, S., Peschel, A. & Harbarth, S. (2018). Methicillin-resistant Staphylococcus aureus. Nature Reviews Disease Primers, 4 (1), 1-23.
Lee, J. S., Bae, Y. M., Lee, S. & Lee, S. Y. (2015). Biofilm formation of Staphylococcus aureus on various surfaces and their resistance to chlorine sanitizer”, Journal of food science, 80 (10), 2279- 2286.
Lianou, A. & Koutsoumanis, K. P. (2012). Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions. International Journal of food microbiology, 160 (2), 171–178.
Linhares, L. L., Yang, M., Sreevatsan, S., Munoz-Zanzi, C. A., Torremorell, M. & Davies, P. R. (2015). The effect of anatomic site and age on detection of Staphylococcus aureus in pigs. Journal of Veterinary Diagnostic Investigation, 27 (1), 55-60.
Lowy, F. D. (1998). Staphylococcus aureus infections. The New England Journal of Medicine, 339 (8), 520-532.
Marques, L. M. P. (2017). Caracterização fenotípica e genotípica de Staphylococcus aureus isolados de queijo minas frescal” https://app.uff.br/riuff/bitstream/1/3305/1/Marques%2c%20Leila%20M%c3%a1rcia%20Peres%20%5bDisserta%c3%a7%c3%a3o%2c%202014%5d.pdf
Milan, C., Agostinetto, A., Conceição, R. C. S., Gonzalez, H. L. & Timm, C.D. (2015). Sanitizer resistance of biofilm-forming Salmonella isolated from meat products. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 67 (2), 642-646.
Oppliger, A., Moreillon, P., Charrière, N., Giddey, M., Morisset, D. & Sakwinska, O. (2012). Antimicrobial resistance of Staphylococcus aureus strains acquired by pig farmers from pigs. Applied and Environmental Microbiology, 78 (22), 8010-8014.
Ordóñez, J. A. (2005). Tecnologia de alimentos: Alimentos de Origem Animal. ArtMed.
Reiter, K. C., Da Silva, T. G., De Oliveira, C. F. & D’Azevedo, P. A. (2011). High biofilm production by invasive multiresistant staphylococci. Apmis, 119 (11), 776-781.
Robert, S. & Chambers, S. (2005). Diagnosis and management of Staphylococcus aureus infections of the skin and soft tissue. Internal Medicine Journal, 35, 97-105.
Rode, M. T., Langsrud, S., Holck, A. & Moretro, T. (2007). Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions. International Journal of Food Microbiology, 116 (3), 372-383.
Rosa, J. V., Kaefer, K., Da Conceição, N. V. D., Da Conceição, R. C. S. & Timm, C. D. (2017). Formação de biofilme por Vibrio parahaemolyticus isolados de pescados. Pesquisa Veterinária Brasileira, 37 (4), 339-345.
Sambrook, J. & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Sankomkai, W., Boonyanugomol, W., Kraisriwattana, K., Nutchanon, J., Boonsam, K., Kaewbutra, S. & Wongboot, W. (2020). Characterisation of classical enterotoxins, virulence activity, and antibiotic susceptibility of Staphylococcus aureus isolated from Thai fermented pork sausages, clinical samples, and healthy carriers in northeastern Thailand. Journal of Veterinary Research, 64 (2), 289-297.
Santa, O. R. D., Alvarez, D. C., Santa, H. S. D., Zanette, C. M., De Freitas, R. J. S., De Macedo, R. E. F. & Terra, N. N. (2012). Microbiota of sausages obtained by spontaneous fermentation produced in the South of Brazil. Ciência e Tecnologia de Alimentos, 32 (4), 653-660.
Sasaki, T., Tsubakishita, S., Tanaka, Y., Sakusabe, A., Ohtsuka, M., Hirotaki, S., Kawakami, T., Fukata, T. & Hiramatsu, K. (2010). Multiplex‐PCR method for species identification of coagulase‐positive staphylococci. Journal of Clinical Microbiology, 48 (3), 765-769.
Savariraj, W. R., Ravindran, N. B., Kannan, P., Paramasivam, R., Senthilkumar, T. M. A., Kumarasamy, P. & Rao, V. A. (2019). Prevalence, antimicrobial susceptibility and virulence genes of Staphylococcus aureus isolated from pork meat in retail outlets in India. Journal of food safety, 39 (1).
Schmid, D., Fretz, R., Winter, P., Mann, M., Höger, G., Stöger, A., Ruppitsch, W., Ladstätter, J., Mayer, N., De Martin, A. & Allerberger, F. (2009). Outbreak of staphylococcal food intoxication after consumption of pasteurized milk products, June 2007, Austria. Wiener Klinische Wochenschrift, 121 (3-4), 125-131.
Shylaja, M., Goud, S. S. S., Samatha, K. & Pradeep, C. H. (2018). Studies on the incidence of Staphylococcus aureus and its enterotoxins in different meat and meat products. The Pharma Journal, 7 (4), 669-673.
Silva, J.R. (2019). Caracterização fenotípica de Staphylococcus aureus isolados de alimentos de origem animal e de outras fontes relacionadas. https://wp.ufpel.edu.br/ppgveterinaria/files/2019/09/Julia-Rosin-da-Silva.pdf
Smith, K., Perez, A., Ramage, G., Lapin, D., Gemmell, C. G. & Lang, S. (2008). Biofilm formation by Scottish clinical isolates of Staphylococcus aureus. Journal of Medical Microbiology, 57 (8), 1018-1023.
Song, J. W., Yang, S. J., Shin, S., Seo, K. S., Park, Y. H. & Park, K. T. (2016). Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitic milk in Korea. Journal of food protection, 79 (10), 1725-1732.
Strube, M. L., Hansen, J. E., Rasmussen, S. & Pedersen, K. (2018). A detailed investigation of the porcine skin and nose microbiome using universal and Staphylococcus specific primers. Scientific Reports, 8 (1), 1-9.
Tallent, S., Hait, J., Bennett, R. W. & Lancette, G. A. (2016). Staphylococcus aureus. In: U.S. Food and Drug Administration, Bacteriological analytical manual, chap. 12. https://www.fda.gov/food/laboratory-methods-food/bam-chapter-12-staphylococcus-aureus
Thapaliya, D., Forshey, B. M., Kadariya, J., Quick, M. G., Farina, S., O’Brien, A., Nair, R., Nworie, A., Hanson, B., Kates, A., Wardyn, S. & Smith, T. C. (2017). Prevalence and molecular characterization of Staphylococcus aureus in commercially available meat over a one-year period in Iowa, USA. Food microbiology, 65, 122-129.
Tutenel, A. V., Pierad, D., Hoff, J. V., Cornelis, M. & Zutter, L. D. (2003). Isolation and molecular characterization of Escherichia coli O157 isolated from cattle pigs and chickens at slaughter. International Journal of Food Microbiology, 84 (1), 63–69.
USDA - United States Department of Agriculture. (2019). Livestock and Poultry: World Markets and Trade. https://downloads.usda.library.cornell.edu/usda-esmis/files/73666448x/g445ct12h/ff365k146/Livestock_poultry.pdf
Valiatti, T. B., Barcelos, I. B., Calegari, G. M., Silva, W. M. C., Almeida, F. K. V., Dos Prazeres, P. F. L., Sobral, F. O. S., Romão, N. F. & Gasparotto, P. H. G. (2016). Avaliação microbiológica de linguiças tipo frescal comercializadas em supermercados do município de Ji-Paraná, Rondônia. Revista da Universidade do Vale do Rio Verde, 14 (2), 678-686.
Wong, H. C., Peng, P. Y., Han, J. M., Chang, C. Y. & Lan, S. L. (1998). Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in Vibrio parahaemolyticus. Infection and Immunity, 66 (7), 3066-3071.
Zhang, Y., Xu, D., Shi, L., Cai, R., Li, C. & Yan, C. (2018). Association between agr type, virulence factors, biofilm formation and antibiotic resistance of Staphylococcus aureus isolates from pork production. Frontiers in microbiology, 9, 1876.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2021 Kauana Kaefer; Débora Rodrigues Silveira; Juliana Fernandes Rosa; Thaís Gonçalves Gonçalves; Thamíris Pereira de Moraes; Cláudio Dias Timm
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
1) Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
2) Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
3) Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.
