Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina

Detalhes bibliográficos
Autor(a) principal: Camargo, Anderson Carlos
Data de Publicação: 2013
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/5150
Resumo: Listeria monocytogenes is a foodborne pathogen often associated to beef cuts and meat products. Its presence in beef cuts is usually related to cross contamination during processing, once L. monocytogenes is naturally part of its environmental microbiota. The consumption of food contaminated with this pathogen, mainly ready-to-eat products, can determine listeriosis, a severe disease specially to risk groups such as pregnant women, children, elderly, and immune-compromised adults, leading to mortality rates of 20 to 30% in cases of systemic infection. Despite listeriosis being considered as an infrequent disease, its high severity and mortality and consequent economic losses determine a wide range of studies focusing on L. monocytogenes. The present study aimed the assessment of the occurrence of Listeria spp. and L. monocytogenes in a beef processing plant and beef cuts, and the characterization of the obtained isolates by phenotypic and molecular methodologies. A total of 272 environmental, utensils and beef cuts samples was collected between 2009 and 2012 in a slaughterhouse located in Minas Gerais state, Brazil. Samples were obtained by surface swabbing (400 cm²) and subjected to Listeria spp. and L. monocytogenes detection according protocols ISO 11290-1 and ISO 11290-2. Suspect isolates were subjected to biochemical identification, molecular serotyping, sequencing of atypical PCR products, pulsed field gel electrophoresis (PFGE), detection of virulence genes by PCR (inlA, inlB, inlC, inlJ, plcA, hlyA, actA e iap), and phenotypic tests of antimicrobials resistance. Listeria spp. was identified in 61 samples (22.4%), being L. monocytogenes in 23 (8.5%), L. innocua in 46 (16.9%), and L. seeligeri in 1 (0.37%). A total of 231 isolates was obtained and identified as L. monocytogenes (96), L. innocua (129), and L. seeligeri (6). Among L. monocytogenes isolates, 1/2c or 3c serotype was the most prevalent (74/96), being present in 21 of the 23 positive samples; 15 isolates were identified as serotype 4b atypical and 7 as serotype 1/2b, 3b or 7. PFGE demonstrated the genetic diversity of the isolates and indicated possible contamination routes in the beef processing environment. The isolates were clustered in 6 major groups and 20 pulsetypes after macro-restriction by ApaI and AscI. Isolates with distinct serotypes were grouped in same groups, but never in a same pulsetype. Isolates obtained from distinct samples presented identical genetic profile, as observed in isolates obtained from distinct sampling visits. All tested virulence genes were identified in all L. monocytogenes isolates. Based on PFGE, 20 isolates were selected and tested by phenotypic tests to identify their resistance profiles against 15 antimicrobials; the isolates presented sensitivity to all tested antimicrobials, but 19 isolates were resistant to sulfamethoxazole and 2 also to trimethoprim. Other 2 isolates presented multidrug resistance (gentamicin, tobramycin, clindamycin, trimethoprim, and sulfamethoxazole) and 8 isolates presented intermediary resistance to erythromycin. The obtained data indicate the persistence of L. monocytogenes strains in the beef environment processing of the studied slaughterhouse, possible contamination routes, and the occurrence of multi-drug resistant isolates, to antimicrobials usually considered in the listeriosis treatment.
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spelling Camargo, Anderson Carloshttp://lattes.cnpq.br/6707579945973222Pinto, Paulo Sérgio de Arrudahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793537Y3Nero, Luís Augustohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763792E2Pinto, José Paes de Almeida Nogueirahttp://lattes.cnpq.br/95810730119637662015-03-26T13:47:16Z2013-11-292015-03-26T13:47:16Z2013-07-18CAMARGO, Anderson Carlos. Phenotypic and molecular characterization of Listeria monocytogenes isolates obtained from a beef processing facility. 2013. 85 f. Dissertação (Mestrado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2013.http://locus.ufv.br/handle/123456789/5150Listeria monocytogenes is a foodborne pathogen often associated to beef cuts and meat products. Its presence in beef cuts is usually related to cross contamination during processing, once L. monocytogenes is naturally part of its environmental microbiota. The consumption of food contaminated with this pathogen, mainly ready-to-eat products, can determine listeriosis, a severe disease specially to risk groups such as pregnant women, children, elderly, and immune-compromised adults, leading to mortality rates of 20 to 30% in cases of systemic infection. Despite listeriosis being considered as an infrequent disease, its high severity and mortality and consequent economic losses determine a wide range of studies focusing on L. monocytogenes. The present study aimed the assessment of the occurrence of Listeria spp. and L. monocytogenes in a beef processing plant and beef cuts, and the characterization of the obtained isolates by phenotypic and molecular methodologies. A total of 272 environmental, utensils and beef cuts samples was collected between 2009 and 2012 in a slaughterhouse located in Minas Gerais state, Brazil. Samples were obtained by surface swabbing (400 cm²) and subjected to Listeria spp. and L. monocytogenes detection according protocols ISO 11290-1 and ISO 11290-2. Suspect isolates were subjected to biochemical identification, molecular serotyping, sequencing of atypical PCR products, pulsed field gel electrophoresis (PFGE), detection of virulence genes by PCR (inlA, inlB, inlC, inlJ, plcA, hlyA, actA e iap), and phenotypic tests of antimicrobials resistance. Listeria spp. was identified in 61 samples (22.4%), being L. monocytogenes in 23 (8.5%), L. innocua in 46 (16.9%), and L. seeligeri in 1 (0.37%). A total of 231 isolates was obtained and identified as L. monocytogenes (96), L. innocua (129), and L. seeligeri (6). Among L. monocytogenes isolates, 1/2c or 3c serotype was the most prevalent (74/96), being present in 21 of the 23 positive samples; 15 isolates were identified as serotype 4b atypical and 7 as serotype 1/2b, 3b or 7. PFGE demonstrated the genetic diversity of the isolates and indicated possible contamination routes in the beef processing environment. The isolates were clustered in 6 major groups and 20 pulsetypes after macro-restriction by ApaI and AscI. Isolates with distinct serotypes were grouped in same groups, but never in a same pulsetype. Isolates obtained from distinct samples presented identical genetic profile, as observed in isolates obtained from distinct sampling visits. All tested virulence genes were identified in all L. monocytogenes isolates. Based on PFGE, 20 isolates were selected and tested by phenotypic tests to identify their resistance profiles against 15 antimicrobials; the isolates presented sensitivity to all tested antimicrobials, but 19 isolates were resistant to sulfamethoxazole and 2 also to trimethoprim. Other 2 isolates presented multidrug resistance (gentamicin, tobramycin, clindamycin, trimethoprim, and sulfamethoxazole) and 8 isolates presented intermediary resistance to erythromycin. The obtained data indicate the persistence of L. monocytogenes strains in the beef environment processing of the studied slaughterhouse, possible contamination routes, and the occurrence of multi-drug resistant isolates, to antimicrobials usually considered in the listeriosis treatment.Listeria monocytogenes é um patógeno de origem alimentar frequentemente associado à carne bovina e produtos cárneos. A presença de L. monocytogenes em cortes cárneos normalmente está associada a contaminações cruzadas durante o processamento, já que esta bactéria está naturalmente presente no ambiente industrial. O consumo de alimentos contaminados por L. monocytogenes, principalmente alimentos prontos para consumo, pode causar a listeriose, considerada uma doença grave que acomete principalmente indivíduos de grupos de risco como gestantes, crianças, idosos e adultos imunocomprometidos, levando a uma alta taxa de mortalidade (20 a 30%) nos casos de infecção sistêmica. A listeriose é considerada uma doença rara, mas L. monocytogenes tem sido alvo de muitas pesquisas nas últimas décadas devido à severidade da doença, da alta taxa de mortalidade e aos prejuízos econômicos causados. Assim, o objetivo do presente estudo foi verificar a ocorrência de Listeria spp. e L. monocytogenes em uma planta de processamento de carne bovina e cortes cárneos e caracterizar por técnicas fenotípicas e moleculares os isolados obtidos. Um total de 272 amostras de ambiente, utensílios e cortes cárneos foi analisado entre os anos de 2009 e 2012, todas coletadas em um frigorífico localizado no Estado de Minas Gerais. Todas as amostras foram obtidas por swab superficial (400 cm²) e submetidas à detecção de Listeria spp. e L. monocytogenes pelos protocolos ISO 11290-1 e ISO 11.290-2 (2004) e isolados suspeitos foram submetidos a identificação bioquímica, sorotipagem molecular, sequenciamento dos produtos atípicos, eletroforese em gel em campo pulsado (PFGE), detecção de genes de virulência (inlA, inlB, inlC, inlJ, plcA, hlyA, actA e iap) e testes de resistência a antimicrobianos. Listeria spp foi identificada em 61 (22.4%) das amostras analisadas, L. monocytogenes em 23 (8.5%), L. innocua em 46 (16.9%), e L. seeligeri em 1 (0.37%). Um total de 231 isolados foi obtido e identificado como L. monocytogenes (n = 96), L. innocua (n = 129), L. seeligeri (n = 6). Entre os isolados de L. monoytogenes, sorotipo 1/2c ou 3c foi o mais prevalente (74/96), estando presente em 21/23 amostras, seguido pelo sorotipo 4b atípico (15/96) e sorotipo 1/2b, 3b ou 7 (7/96). PFGE mostrou a diversidade genética entre os isolados e indicou possíveis rotas de contaminação dentro do ambiente de processamento de carne bovina. Os 96 isolados foram agrupados em 6 grupos subdivididos em 20 pulsotipos através da combinação dos padrões de restrição das enzimas ApaI e AscI. Diferentes sorotipos compartilharam mesmos grupos genéticos, mas não o mesmo pulsotipo. Ainda foi verificado que isolados recuperados de diferentes origens apresentaram mesmo perfil genético, e alguns isolados obtidos em diferentes visitas também foram agrupados no mesmo pulsotipo. Todos os genes de virulência (inlA, inlB, inlC, inlJ, plcA, hlyA, actA e iap) investigados foram detectados em todos os isolados de L. monocytogenes. De maneira geral, os 20 isolados testados (um representante de cada pulsotipo) apresentaram sensibilidade à maioria dos antimicrobianos, entretanto 19 isolados foram resistentes a sulfametoxazole e dois também foram resistentes a trimetropim. Outros dois isolados apresentaram múltipla resistência (gentamicina, tobramicina, clindamicina, trimetoprim, e sulfametoxazole) e oito mostraram resistência intermediária a eritromicina. Os dados obtidos indicam a persistência de cepas de L. monocytogenes no ambiente de processamento de carne bovina, possíveis rotas de contaminação, e a ocorrência de isolados multi-resistentes a antimicrobianos usualmente utilizados no tratamento de listeriose.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Medicina VeterináriaUFVBRBiotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. deListeriaCarne bovinaPFGEListeriaBeefPFGECNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::INSPECAO DE PRODUTOS DE ORIGEM ANIMALCaracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovinaPhenotypic and molecular characterization of Listeria monocytogenes isolates obtained from a beef processing facilityinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1420227https://locus.ufv.br//bitstream/123456789/5150/1/texto%20completo.pdf6a4ef5b8564c78036999d045362d4595MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain147634https://locus.ufv.br//bitstream/123456789/5150/2/texto%20completo.pdf.txt91930ac4052d7cc56e8cc39730684672MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3567https://locus.ufv.br//bitstream/123456789/5150/3/texto%20completo.pdf.jpg7d2224481c5bd2933be2848bfdc1daf8MD53123456789/51502016-06-17 06:43:04.838oai:locus.ufv.br:123456789/5150Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-06-17T09:43:04LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
dc.title.en.fl_str_mv Phenotypic and molecular characterization of Listeria monocytogenes isolates obtained from a beef processing facility
title Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
spellingShingle Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
Camargo, Anderson Carlos
Listeria
Carne bovina
PFGE
Listeria
Beef
PFGE
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::INSPECAO DE PRODUTOS DE ORIGEM ANIMAL
title_short Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
title_full Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
title_fullStr Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
title_full_unstemmed Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
title_sort Caracterização fenotípica e molecular de isolados de Listeria monocytogenes obtidos em uma planta de processamento de carne bovina
author Camargo, Anderson Carlos
author_facet Camargo, Anderson Carlos
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/6707579945973222
dc.contributor.author.fl_str_mv Camargo, Anderson Carlos
dc.contributor.advisor-co1.fl_str_mv Pinto, Paulo Sérgio de Arruda
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793537Y3
dc.contributor.advisor1.fl_str_mv Nero, Luís Augusto
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763792E2
dc.contributor.referee1.fl_str_mv Pinto, José Paes de Almeida Nogueira
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/9581073011963766
contributor_str_mv Pinto, Paulo Sérgio de Arruda
Nero, Luís Augusto
Pinto, José Paes de Almeida Nogueira
dc.subject.por.fl_str_mv Listeria
Carne bovina
PFGE
topic Listeria
Carne bovina
PFGE
Listeria
Beef
PFGE
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::INSPECAO DE PRODUTOS DE ORIGEM ANIMAL
dc.subject.eng.fl_str_mv Listeria
Beef
PFGE
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::INSPECAO DE PRODUTOS DE ORIGEM ANIMAL
description Listeria monocytogenes is a foodborne pathogen often associated to beef cuts and meat products. Its presence in beef cuts is usually related to cross contamination during processing, once L. monocytogenes is naturally part of its environmental microbiota. The consumption of food contaminated with this pathogen, mainly ready-to-eat products, can determine listeriosis, a severe disease specially to risk groups such as pregnant women, children, elderly, and immune-compromised adults, leading to mortality rates of 20 to 30% in cases of systemic infection. Despite listeriosis being considered as an infrequent disease, its high severity and mortality and consequent economic losses determine a wide range of studies focusing on L. monocytogenes. The present study aimed the assessment of the occurrence of Listeria spp. and L. monocytogenes in a beef processing plant and beef cuts, and the characterization of the obtained isolates by phenotypic and molecular methodologies. A total of 272 environmental, utensils and beef cuts samples was collected between 2009 and 2012 in a slaughterhouse located in Minas Gerais state, Brazil. Samples were obtained by surface swabbing (400 cm²) and subjected to Listeria spp. and L. monocytogenes detection according protocols ISO 11290-1 and ISO 11290-2. Suspect isolates were subjected to biochemical identification, molecular serotyping, sequencing of atypical PCR products, pulsed field gel electrophoresis (PFGE), detection of virulence genes by PCR (inlA, inlB, inlC, inlJ, plcA, hlyA, actA e iap), and phenotypic tests of antimicrobials resistance. Listeria spp. was identified in 61 samples (22.4%), being L. monocytogenes in 23 (8.5%), L. innocua in 46 (16.9%), and L. seeligeri in 1 (0.37%). A total of 231 isolates was obtained and identified as L. monocytogenes (96), L. innocua (129), and L. seeligeri (6). Among L. monocytogenes isolates, 1/2c or 3c serotype was the most prevalent (74/96), being present in 21 of the 23 positive samples; 15 isolates were identified as serotype 4b atypical and 7 as serotype 1/2b, 3b or 7. PFGE demonstrated the genetic diversity of the isolates and indicated possible contamination routes in the beef processing environment. The isolates were clustered in 6 major groups and 20 pulsetypes after macro-restriction by ApaI and AscI. Isolates with distinct serotypes were grouped in same groups, but never in a same pulsetype. Isolates obtained from distinct samples presented identical genetic profile, as observed in isolates obtained from distinct sampling visits. All tested virulence genes were identified in all L. monocytogenes isolates. Based on PFGE, 20 isolates were selected and tested by phenotypic tests to identify their resistance profiles against 15 antimicrobials; the isolates presented sensitivity to all tested antimicrobials, but 19 isolates were resistant to sulfamethoxazole and 2 also to trimethoprim. Other 2 isolates presented multidrug resistance (gentamicin, tobramycin, clindamycin, trimethoprim, and sulfamethoxazole) and 8 isolates presented intermediary resistance to erythromycin. The obtained data indicate the persistence of L. monocytogenes strains in the beef environment processing of the studied slaughterhouse, possible contamination routes, and the occurrence of multi-drug resistant isolates, to antimicrobials usually considered in the listeriosis treatment.
publishDate 2013
dc.date.available.fl_str_mv 2013-11-29
2015-03-26T13:47:16Z
dc.date.issued.fl_str_mv 2013-07-18
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dc.identifier.citation.fl_str_mv CAMARGO, Anderson Carlos. Phenotypic and molecular characterization of Listeria monocytogenes isolates obtained from a beef processing facility. 2013. 85 f. Dissertação (Mestrado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2013.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/5150
identifier_str_mv CAMARGO, Anderson Carlos. Phenotypic and molecular characterization of Listeria monocytogenes isolates obtained from a beef processing facility. 2013. 85 f. Dissertação (Mestrado em Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de) - Universidade Federal de Viçosa, Viçosa, 2013.
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