Enumeration, Antimicrobial Resistance and Typing of Salmonella enterica: Profile of Strains Carried in the Intestinal Contents of Pigs at Slaughter in Southern Brazil
Background: Despite a strong association between Salmonella isolation and slaughter hygiene, as measured by the Enterobacteriaceae levels on pre-chill carcass surfaces, a high variation in this association was observed between sampling days within the same slaughterhouse. It was hypothesised that in a scenario of high exposure on the farm, batches with a high prevalence of carrier pigs shedding a high number of Salmonella may enhance the risk of contamination on some slaughter days. Thus, the aim of this study was to assess the profile of Salmonella carried in the intestinal contents of slaughter pigs.
Materials, Methods & Results: Ten pig batches slaughtered in a slaughterhouse were investigated for the presence of Salmonella. From each pig, the following samples were taken: i. blood collected at bleeding; ii. sponges rubbed on the carcass surface after bleeding and before chilling; iii. fragment of the ileocecal region of the intestine. Serum samples were subjected to a ELISA-Typhimurium test. Sponges were investigated for the presence of Salmonella and total aerobic mesophilic (TAM) and Enterobacteriaceae (EC) bacterial counts. Salmonella was enumerated in the intestinal contents. Selected Salmonella strains were subjected to an antimicrobial resistance disk diffusion test, macro-restriction with Xba-I (PFGE) and whole genome sequencing (WGS). From the 50 sampled pigs, 96% were positive in the ELISA-Typhimurium test and 64% were Salmonella-positive in the intestinal contents. The amount of Salmonella in the intestinal content samples was highly variable, and the mean log of fitted distributions of Salmonella in the batch ranged from -2.97 to 2.25 cfu.g-1. The slaughter process achieved a logarithmic reduction, ranging from 0.64 to 2.35 log cfu.cm-2 for TAM and from 0.55 to 2.57 log cfu.cm-2 for EC. Salmonella was isolated from 16% of the carcasses after bleeding; this frequency decreased to 8% at the pre-chill step. All positive pre-chill carcasses originated from pigs carrying Salmonella in the intestinal content and from batches with a high number of carrier pigs. Salmonella Typhimurium and its monophasic variant were the most frequent in the intestinal contents and carcasses. Resistance was detected against ampicillin (42.5%), tetracycline (42.5%), sulfonamide (40%), gentamicin (25%) and ciprofloxacin (12.5%). Regarding colistin, 85% of the tested strains were classified as non-susceptible. The monophasic variant S. Typhimurium strains subjected to PFGE and WGS presented different profiles; several antimicrobial resistance genes were identified and all belonged to ST-19.
Discussion: In this study, almost all sampled pigs entering the slaughter line had been exposed to Salmonella on the farm and a high number were carrying Salmonella in their guts. While the three batches with Salmonella-positive carcasses at the pre-chill step presented TAM media that was not significantly different from the other batches, there was a higher number of positive pigs carrying Salmonella in their intestinal contents. Moreover, the batch with the highest number of positive carcasses also presented the highest Salmonella mean count in their intestinal contents. The profile of Salmonella carried in the intestinal content of slaughter pigs proved to be highly variable in terms of the frequency, number of bacteria, serovars, antimicrobial resistance, and genotypes. Results indicate that the day-to-day variability in the prevalence and number of Salmonella in the intestinal contents of slaughter batches is likely to influence the frequency of contaminated pre-chill carcasses. Salmonella Typhimurium isolated from the intestinal contents of slaughter pigs may belong to genotypes involved in human disease and may carry several antimicrobial resistance genes. These aspects should be taken into account when planning Salmonella control in swine.
Alban L., Baptista F.M., Mogelmose V., Sorensen L.L., Christensen H., Aabo S. & Dahl J. 2012. Salmonella surveillance and control for finisher pigs and pork in Denmark- A case study. Food Research International. 45: 656-665.
Almeida F., Seribelli A.A., Medeiros M.I.C., Rodrigues D.P., Varani A.M., Luo Y., Allard M.W. & Falcão J.P. 2018. Phylogenetic and antimicrobial resistance gene analysis of Salmonella Typhimurium strains isolated in Brazil by whole genome sequencing. PLOS One. 13(8): e0201882.
Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., Pyshkin A.V., Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A. & Pevzner P.A. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computing Biology. 19(5): 455-477.
Baptista F.M., Dahl J. & Nielsen L.R. 2010. Factors influencing Salmonella carcass prevalence in Danish pig abattoirs. Preventive Veterinary Medicine. 95(3-4): 231-238.
Bolger A.M., Lohse M. & Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30(15): 2114-2120.
Bone A., Noel H., Le Hello S., Pihier N., Danan C., Raguenaud M.E., Salah S., Bellali H., Vaillant V., Weill F.X. & Jourdan-da Silva N. 2010. Nationwide outbreak of Salmonella enterica serotype 4,12:i:- infections in France, linked to dried pork sausage. Euro Surveillance. 15(24): pii = 19592.
Borch E., Nesbakken T. & Christensen H. 1996. Hazard identification in swine slaughter with respect to foodborne bacteria. International Journal of Food Microbiology. 30: 9-25.
Brasil. Ministério da Agricultura Pecuária e Abastecimento. 2018. Instrução Normativa Nº 58 de 17 de dezembro de 2018. Available at: <http://www.in.gov.br/materia/-/asset_publisher/Kujrw)TZC2Mb/content/id/55882955>. [Accessed online in December 2018].
Brasileiro A.C.M., Santos M.A.S., Sá C.V.G.C., Rodrigues C.S. & Haddad J.P.A. 2017. National prevalence of Salmonella spp. in pork slaughterhouses under Federal Inspection in Brazil, 2014/2015. In: 12th International Symposium on the Epidemiology and Control of Biological, Chemical and Physical Hazards in Pigs and Pork (Foz do Iguaçu, Brazil). pp.55-58.
Buncic S. & Sofos J. 2012. Interventions to control Salmonella contamination during poultry, cattle and pig slaughter. Food Research International. 45: 641-655.
Busschaert P., Geeraerd A.H. & Van Impe J.F. 2010. Estimating distributions out of qualitative and (semi) quantitative microbiological contamination data for use in risk assessment. International Journal of Food Microbiology. 138: 260-269.
Carriço J.A., Pinto F.R., Simas C., Nunes S., Sousa N.G., Frazão N., de Lencastre H. & Almeida J.S. 2005. Assessment of band-based similarity coefficients for automatic type and subtype classification of microbial isolates analyzed by pulsed-field gel electrophoresis. Journal of Clinical Microbiology. 43(11): 5483-5490.
Clinical and Laboratory Standards Institute (CLSI). 2018. Performance standards for antimicrobial disk and dilution susceptibility test for bacteria isolated from animals; Approved standard. CLSI supplement VET08. 4th edn. Wayne: Clinical and Laboratory Standards Institute, 99p.
Clinical and Laboratory Standards Institute (CLSI). 2018. Performance standards for antimicrobial susceptibility testing. CLSI document M100-S28. 28th edn. Wayne: Clinical and Laboratory Standards Institute, 282p.
Corbellini L.G., Bianco Júnior A., Costa E.F., Duarte A.S.R., Albuquerque E.R., Kich J.D., Cardoso M. & Nauta M. 2016. Effect of slaughterhouse and day of sample on the probability of a pig carcass being Salmonella-positive according to the Enterobacteriaceae count in the largest Brazilian pork production region. International Journal of Food Microbiology. 228: 58-66.
Davies P. 2017. Salmonella control in pigs in the USA - focus on slaughter hygiene. Available at: <https://www.pig333.com/articles/salmonella-control-in-pigs-in-the-usa-%E2%80%93-focus-on-slaughter-hygiene_13103/>. [Accessed online in July 2018].
De Busser E.V., De Zutter L., Dewulf J., Houf K. & Maes D. 2013. Salmonella control in live pigs and at slaughter. The Veterinary Journal. 196(1): 20-27.
Delignette-Muller M.L. & Dutang C. 2015. fitdistrplus: an R Package for Fitting Distributions. Journal of Statistical Software. 64(4): 1-34.
Di Ciccio P., Ossiprandi M.C., Zanardi E., Ghidini S., Belluzzi G., Vergara A. & Ianeri A. 2016. Microbiological contamination in three large-scale pig slaughterhouses in Northern Italy. Italian Journal of Food Safety. 5: 219-223.
European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). 2017. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA Journal. 15(12): 5077-5305.
European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2018. Breakpoint tables for interpretation of MICs and zone diameters. Version 9.0, 2018. Available at: < http://www.eucast.org/clinical_breakpoints/>. [Accessed online in December 2018].
Garneau-Tsodikova S. & Labby K.J. 2016. Mechanisms of resistance to aminoglycoside antibiotics: overview and perspectives. MedChemComm. 7(1): 11-27.
Gymoese P., Sorensen G., Litrup E., Olsen J.E., Nielsen E.M. & Torpdahl M. 2017. Investigations of outbreaks of Salmonella enterica serovar Typhimurium and its monophasic varians using whole-genome sequencing, Denmark. Emerging Infectious Diseases. 3(10): 1631-1639.
International Standard Organization (ISO). 2002. ISO6579:2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Geneva: ISO, 27p.
Kich J.D., Coldebella A., Mores N., Nogueira M.G., Cardoso M., Fratamico P.M., Call J.E., Fedorka-Cray P. & Luchansky J.B. 2011. Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil. International Journal of Food Microbiology. 151: 307-313.
Kich J.D., Costa E.F., Triques N., Nogueira M.G, Dalla Costa O., Coldebella A., Kummer A. & Cardoso M. 2016. Assessment of different cut-off values of the ELISA-Typhimurium for the discrimination of swine herds with Salmonella isolation. Semina: Ciências Agrárias. 37: 3107-3113.
Kich J.D., Schwarz P., Silva L.E., Coldebella A., Piffer I.A., Vizzoto R. & Cardoso M. 2007. Development and application of an enzyme-linked immunosorbent assay to detect antibodies against prevalent Salmonella serovars in swine in southern Brazil. Journal of Veterinary Diagnostic Investigation. 19(5): 510-517.
Larsen M.V., Cosentino S., Rasmussen S., Friis C., Hasman H., Marvig R.L., Jelsbak L., Sicheritz-Pontén T., Ussery D.W., Aarestrup F.M. & Lund O. 2012. Multilocus sequence typing of total genome sequenced bacteria. Journal of Clinical Microbiology. 50(4): 1355-1361.
Looft T., Johson T.A., Allen H.K., Bayles D.O., Alt D.P., Stedtfeld R.D., Sul W.J., Stedfeld T., Chai B., Cole J.R., Hashsham S.A., Tiedje J.M. & Stanton T.B. 2012. In-feed antibiotic effects on the swine intestinal microbiome. Procedures of the National Academy of Science USA. 109: 1691-1696.
Lopes G.V., Pissetti C., Pellegrini D.C.P., Silva L.E. & Cardoso M. 2015. Resistance phenotypes and genotypes of Salmonella enterica subsp. enterica isolates from feed, pigs, and carcasses in Brazil. Journal of Food Protection. 78(2): 407-413.
Liu J.Y., Liao T.L., Huang W.C., Liu Y.M., Wu K.M., Lauderdale T.L., Tsai S.F., Kuo S.C. & Kuo H.C. 2018. Increased mcr-1 in pathogenic Escherichia coli from diseased swine, Taiwan. Journal of Microbiology, Immunology and Infection. https://doi.org/10.1016/j.jmii.2018.10.011
Liu Y.Y., Wang Y., Walsh T.R., Yi L.X., Zhang R., Spencer J., Doi Y., Tian G., Dong B., Huang X., Yu L.F, Gu D., Ren H., Chen X., L. L., He D., Zhou H., Liang Z., Liu J.H. & Shen J. 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet. Infectious Diseases. 16(2): 161-168.
Meneguzzi M., Kich J.D., Rebelatto R., Pissetti C., Kuchiishi S.S., Reis A.T., Guedes R.M.C., Leão J.A. & Reichen C. 2017. Salmonella clinical isolates from Brazilian pig herds: genetic relationship and antibiotic resistance profiling. In: 12th International Symposium on the Epidemiology and Control of Biological, Chemical and Physical Hazards in Pigs and Pork (Foz do Iguaçu, Brazil). pp.170-174.
Paim D. 2016. Perfil de excreção de Salmonella em suínos ao abate e presença de carcaças positivas no pré-resfriamento. 52f. Porto Alegre, RS. Dissertação (Mestrado em Ciências Veterinárias) - Programa de Pós-graduação em Ciências Veterinárias, Universidade Federal do Rio Grande do Sul.
Panzenhagen P.H.N., Paul N.C., Conte Junior C.A., Costa R.G., Rodrigues D.P. & Shah D.H. 2018. Genetically distinct lineages of Salmonella Typhimurium ST313 and ST19 are present in Brazil. International Journal of Medical Microbiology. 308: 306-316.
Pearce R.A., Bolton D.J., Sheridan J.J., McDowell D.A, Blair I.S. & Harrington D. 2004. Studies to determine the critical control points in pork slaughter hazard analysis and critical control point systems. International Journal of Food Microbiology. 90: 331-339.
Pissetti C., Werlang G.O., Kich J.D. & Cardoso M. 2017. Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses. Pesquisa Veterinária Brasileira. 37(11): 1253-1260.
Pribul B.R., Festivo M.L., Souza M.M.S. & Rodrigues D.P. 2016. Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil. Brazilian Journal of Microbiology. 47: 196-201.
R Core Team. 2017. A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available at: . [Accessed online in May 2018].
San Roman B., Garrido V., Sánchez S., Martínez-Ballesteros I., Garaizar J., Mainar-Jaime R.C., Migura-Garcia L. & Grilló M.J. 2018. Relationship between Salmonella infection, shedding and serology in fattening pigs in low-moderate prevalence areas. Zoonoses and Public Health. 65: 481-489.
Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14): 2068-2069.
Silva L.E., Dias V., Ferronatto A., Guerra P., Berno L., Triches N., Kich J.D., Corbellini L.G. & Cardoso M. 2012. Longitudinal dissemination of Salmonella enterica clonal groups through the slaughter process of Salmonella-positive pig batches. Journal of Food Protection. 75(9): 1580-1588.
Silva N., Junqueira V.C.A., Silveira N.F.A., Taniwaki M.H., Santos R.F.S. & Gomes R.A.R. 2010. Manual de Métodos de Análises Microbiológicas de Alimentos e Água. 4.ed. São Paulo: Varela, 624p.
Tavares M.F.P. 2013. Avaliação da influência da etapa do abate Shechita na população de Salmonella sp. e de micro-organismos indicadores em carcaças de frango. 60f. São Paulo, SP. Dissertação (Mestrado em Ciências dos Alimentos) - Programa de Pós-graduação em Ciências dos Alimentos, Universidade de São Paulo.
van Hoek H.A.M., Jonge R.A., van Overbeek M.W., Bouw E., Pielaat A., Smid J.H., Malorny B., Junker E., Löfström C., Pedersen K., Henk A.J.M. & Heres L. 2012. A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line. International Journal of Food Microbiology. 153(1-2): 45-52.
World Health Organization (WHO) 2017. WHO list of Critically Important Antimicrobials for human medicine. Available at: . [Accessed online in June 2018].
Zankari E., Hasman H., Cosentino S., Vestergaard M., Rasmussen S., Lund O., Aarestrup F.M. & Larsen M.V. 2012. Identification of acquired antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy. 67(11): 2640-2644.
Zhang J., Chen L., Wang J., Yassin A.K., Butaye P., Kelly P., Gong J., Guo W., Li J., Li M., Yang F., Feng Z., Jiang P., Song C., Wang Y., You J., Yang Y., Price S., Qi1 K., Kang Y. & Wang C. 2018. Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry. Nature. 8: 3705-3714.
Zhang S., Yin Y., Jones M.B., Zhang Z., Deatherage Kaiser B.L., Dinsmore B.A., Fitzgerald C., Fields P.I. & Deng X. 2015. Salmonella serotype determination utilizing high-throughput genome sequencing data. Journal of Clinical Microbiology. 53(5): 1685-1692.
Zweifel C., Baltzer D. & Stephan R. 2005. Microbiological contamination of cattle and pig carcasses at five abattoirs determined by swab sampling in accordance with EU Decision 2001/471/EC. Meat Science. 69(3): 559-566.
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