ERIC-PCR genotypic characterization of Haemophilus parasuis isolated from Brazilian swine

Haemophilus parasuis infection, known as Glässer’s disease, is characterized by fibrinous polyserositis, arthritis and meningitis in piglets. Although traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, the molecular-based methods are alternatives for species-specific tests and epidemiologic study. The aim of this study was to characterize H. parasuis strains isolated from different states of Brazil by serotyping, PCR and ERIC-PCR. Serotyping revealed serovar 4 as the most prevalent (24 %), followed by serovars 14 (14 %), 5 (12 %), 13 (8 %) and 2 (2 %), whereas 40 % of the strains were considered as non-typeable. From 50 strains tested 43 (86%) were positive to Group 1 vtaA gene that have been related to virulent strains of H.parasuis. ERIC-PCR was able to type isolates tested among 23 different patterns, including non-typeable strains. ERIC-PCR patterns were very heterogeneous and presented high similarity between strains of the same animal or farm origin. The results indicated ERIC-PCR as a valuable tool for typing H. parasuis isolates collected in Brazil.This study was sponsored by FAPESP (process: 07/04893-9)

Molecular epidemiology of Listeria monocytogenes isolated from different sources in Brazil

Listeria monocytogenes is an important foodborne pathogen that primarily affects pregnant women, neonates, the elderly and immune-compromised individuals, and it may cause abortion, septicemia, and meningitis. From the 13 capsular groups described, serotypes 4b, 1/2b and 1/2a are most closely related to human infection. For this reason, serotyping has limited value as an epidemiological tool; thus, improved discriminatory typing methods are required to enhance knowledge of L. monocytogenes contamination and infection. The aim of this study was to characterize the genetic diversity of L. monocytogenes isolates in the pork processing industry in Sao Paulo, Brazil and human infection isolates by ERICPCR and single enzyme AFLP. Serotypes 1/2c and 4b were frequent among isolates from pork and slaughterhouse/market environments, whereas serotypes 4b and 1/2a were observed among human isolates. ERIC-PCR and AFLP revealed 34 and 31 distinct profiles, respectively, which had tendencies of separation according to serogroup and isolate origin. The genetic profiles from slaughterhouse and market environments suggest the possibility of different sources of Listeria contamination in the environment, although in certain cases, continuous contamination caused by the persistence of clonal strains is also a possibility.Listeria monocytogenes é um importante patógeno de origem alimentar que afeta principalmente grávidas, neonatos, idosos e indivíduos imunocomprometidos, e pode causar abortamento, septicemia e meningite. Dos 13 grupos capsulares descritos, os sorotipos 4b, 1/2b e 1/2a são os mais relacionados à infecção humana. Por esta razão, a sorotipagem possui valor limitado como ferramenta epidemiológica e, dessa forma, métodos mais discriminatórios são necessários para melhorar o conhecimento sobre a contaminação e a infecção por L. monocytogenes. O objetivo deste estudo foi caracterizar a diversidade genética de isolados de L. monocytogenes da indústria de processamento de carne suína no Estado de São Paulo, Brasil, e compará-los a isolados de casos de infecção humana através do ERIC-PCR e AFLP com uma única enzima. Os sorotipos 1/2c e 4b foram frequentes em carne suína e ambientes de abatedouros e mercados, enquanto os sorotipos 4b e 1/2a foram observados nos isolados de humanos. ERIC-PCR e AFLP resultaram em 34 e 31 perfis distintos, respectivamente, com uma tendência a separar de acordo com o sorogrupo e a origem do isolado. Os perfis genéticos de ambiente dos abatedouros e mercados sugerem a possibilidade de diferentes origens de contaminação por Listeria nos ambientes estudados, porém, em alguns casos, é possível que ocorra a persistência de cepas clonais causando contaminação contínua

Characterization of antibiotic resistance in Listeria spp. isolated from slaughterhouse environments, pork and human infections

Introduction: Listeria species are susceptible to most antibiotics. However, over the last decade, increasing reports of multidrug-resistant Listeria spp. from various sources have prompted public health concerns. The objective of this study was to characterize the antibiotic susceptibility of Listeria spp. and the genetic mechanisms that confer resistance. Methodology: Forty-six Listeria spp. isolates were studied, and their minimal inhibitory concentrations of antibiotics were determined by microdilution using Sensititre standard susceptibility MIC plates. The isolates were screened for the presence of gyrA, parC, lde, lsa(A), lnu(A), and mprF by PCR, and the amplified genes were sequenced. Results: All isolates were susceptible to penicillin, ampicillin, tetracycline, erythromycin, and carbapenems. Resistance to clindamycin, daptomycin, and oxacillin was found among L. monocytogenes and L. innocua, and all species possessed at least intermediate resistance to fluoroquinolones. GyrA, parC, and mprF were detected in all isolates; however, mutations were found only in gyrA sequences. A high daptomycin MIC, as reported previously, was observed, suggesting an intrinsic resistance of Listeria spp. to daptomycin. Conclusions: These results are consistent with reports of emerging resistance in Listeria spp. and emphasize the need for further genotypic characterization of antibiotic resistance in this genusFAPESP, 2010/19005-4CAPE

Phenotypic and Genotypic characterization of Arcobacter spp. strains from swine

Dentre as espécies conhecidas do gênero Arcobacter, as espécies A. butzleri, A. cryaerophilus e A. skirrowii são consideradas potecialmente zoonóticas, podendo ser transmitidas por alimentos de origem animal. O presente estudo teve como objetivos isolar e caracterizar fenotipica e genotipicamente cepas de Arcobacter spp. provenientes de carcaças de suínos e amostras de ambiente de abatedouro localizado no Estado de São Paulo. As cepas isoladas foram submetidas a reação em cadeia pela polimerase para a identificação e detecção de um grupo de genes de virulência. A concentração inibitória mínima frente a nove antimicrobianos usados para o controle da infecção pelo agente foi determinada e as cepas foram analisadas através do PFGE e pelo AFLP. Dentre as 30 carcaças avaliadas, 25 foram positivas para o agente e 70 cepas foram selecionadas e identificadas como Arcobacter spp. As espécies isoladas foram A. butzleri (n=61), A. cryaerophilus (n=7) e A. skirrowii (n=2). A frequência dos possíveis genes de virulência encontrada variou de 71,4% a 100% para os genes tlyA, pldA, cj1349, ciaB, cadF e mviN. Não foram detectados os genes hecA, hecB e irgA. O perfil de virulência ciaB/ cj1349/ mviN/ cadF/ pldA/ tlyA foi o mais frequente e detectado em 66% das cepas. Todas as cepas foram sensíveis à gentamicina e tetraciclina e 77,1% foram multirresistentes, dentre estas o perfil mais frequente foi de resistência a azitromicina/ florfenicol/ ácido nalidíxico/ telitromicina/ clindamicina Houve grande diversidade genotípica entre as cepas através do PFGE e do AFLP, e ambas a técnicas apresentaram o mesmo poder discriminatório na análise das cepas isoladas.Among the known species of the genus Arcobacter, the species A. butzleri, A. cryaerophilus and A. skirrowii are considered potentially zoonotic and can be transmitted by food of animal origin. This study aimed to isolate and characterize phenotypically and genotypically strains of Arcobacter spp from swine carcasses and slaughterhouse environment samples located in the State of São Paulo. The isolated strains were subjected to polymerase chain reaction for identification and detection of a group of putative virulence genes. The minimum inhibitory concentration was determined against nine antimicrobials indicated for the control of infection by the agent and the strains were analyzed by PFGE and by AFLP. Among the 30 carcasses evaluated, 25 were positive for the agent and 70 strains were selected and identified as Arcobacter spp. The isolated species were A. butzleri (n = 61), A. cryaerophilus (n = 7) and A. skirrowii (n = 2). The frequency of virulence genes found ranged from 71.4 % to 100 % for genes tlyA , pldA , cj1349 , ciaB , cadF and mviN . The genes hecA, hecB and irgA were not detected. The virulence profile ciaB/ cj1349/ mviN/ cadF/ pldA/ tlyA was the most frequent and detected in 66 % of the strains. All strains were susceptible to gentamicin and tetracycline and 77.1% were multirresistant, among these the most common profile of resistance was azithromycin/ florfenicol/ nalidixic acid/ telithromycin/ clindamycin There were large genotypic diversity among strains by PFGE and AFLP and both techniques showed the same discriminatory power in the analysis of the isolated strains

Isolation and characterization of Arcobacter spp. strains from intensive swine productions and slaughterhouses

As espécies do gênero Arcobacter eram classificadas até a década de 1990 como pertencentes ao gênero Campylobacter. Atualmente três das cinco espécies deste gênero são consideradas potencialmente zoonóticas, podendo ser transmitidas por alimentos de origem animal. O presente estudo teve como objetivo isolar e caracterizar geneticamente amostras de Arcobacter spp., isoladas a partir de 120 amostras de carcaças, 120 amostras de fezes e 24 de amostras de músculo suíno coletadas em dois abatedouros localizados no Estado de São Paulo. Os isolados obtidos foram submetidos ao PCR-Multiplex para a determinação das espécies do gênero e analisados através da eletroforese em campo pulsado. O agente foi isolado de 71,6% das carcaças, 4,16% das amostras de fezes e 8,3% das amostras de músculo. As espécies mais prevalentes foram A. butzleri e A. cryaerophilus. A análise dos dados obtidos no PFGE (SmaI) revelou 51 perfis distintos, com um índice discriminatório de 0,98 e grande diversidade genotípica entre as amostras . O sítio de isolamento mais frequente para o agente foram as carcaças e o PFGE mostrou-se um bom instrumento para a caracterização e discriminação de cepas deste gêneroThe species of Arcobacter spp. genus have been classified until 1990 as species belonging to the genus Campylobacter. Nowadays, three of five species of this genus are consider as potencially zoonotic, and can be transmitted from food of animal origin. The present study goal was to isolate and characterize genetically strains of Arcobacter spp. isolated from 120 carcasses, 120 faeces and 24 swine muscle sampled in two swine slaughterhouses located in São Paulo State. Isolates were submitted to multiplex-PCR to identify species of the genus and analyzed by pulsed field gel electrophoresis. The agent was isolated from 71,6% of carcasses, 4,16% of faeces and 8,3% of muscles samples. A. butzleri and A. cryaerophilus were the most prevalent species. The analysis of data obtained in PFGE showed 51 distinct profiles, the discriminatory index of 0,98 and it demonstrated large genotipic diversity among the strains. The main isolation site were the carcasses and the PFGE was a good tool to characterize and discriminate Arcobacter spp. strains

Epidemiologia molecular de Listeria monocytogenes isoladas de diferentes fontes no Brasil

Listeria monocytogenes é um importante patógeno de origem alimentar que afeta principalmente grávidas, neonatos, idosos e indivíduos imunocomprometidos, e pode causar abortamento, septicemia e meningite. Dos 13 grupos capsulares descritos, os sorotipos 4b, 1/2b e 1/2a são os mais relacionados à infecção humana. Por esta razão, a sorotipagem possui valor limitado como ferramenta epidemiológica e, dessa forma, métodos mais discriminatórios são necessários para melhorar o conhecimento sobre a contaminação e a infecção por L. monocytogenes. O objetivo deste estudo foi caracterizar a diversidade genética de isolados de L. monocytogenes da indústria de processamento de carne suína no Estado de São Paulo, Brasil, e compará-los a isolados de casos de infecção humana através do ERIC-PCR e AFLP com uma única enzima. Os sorotipos 1/2c e 4b foram frequentes em carne suína e ambientes de abatedouros e mercados, enquanto os sorotipos 4b e 1/2a foram observados nos isolados de humanos. ERIC-PCR e AFLP resultaram em 34 e 31 perfis distintos, respectivamente, com uma tendência a separar de acordo com o sorogrupo e a origem do isolado. Os perfis genéticos de ambiente dos abatedouros e mercados sugerem a possibilidade de diferentes origens de contaminação por Listeria nos ambientes estudados, porém, em alguns casos, é possível que ocorra a persistência de cepas clonais causando contaminação contínua

Virulence Genes and Antimicrobial Resistance Profiles of Pasteurella multocida Strains Isolated from Rabbits in Brazil

Pasteurella multocida is responsible for a wide range of diseases in domestic animals. In rabbits, the agent is related to nasal discharge, pneumonia, otitis media, pyometra, orchitis, abscess, and septicemia. One hundred and forty rabbits with respiratory diseases from four rabbitries in São Paulo State, Brazil were evaluated for the detection of P. multocida in their nasal cavities. A total of twenty-nine animals were positive to P. multocida isolation, and 46 strains were selected and characterized by means of biochemical tests and PCR. P. multocida strains were tested for capsular type, virulence genes, and resistance profile. A total of 45.6% (21/46) of isolates belonged to capsular type A, and 54.34% (25/46) of the isolates were untypeable. None of the strains harboured toxA or pfhA genes. The frequency of the other twenty genes tested was variable, and the data generated was used to build a dendrogram, showing the relatedness of strains, which were clustered according to origin. Resistance revealed to be more common against sulfonamides and cotrimoxazole, followed by erythromycin, penicillin, and amoxicillin

Genotypic Characterization of Yersinia enterocolitica Biotype 4/O:3 Isolates from Pigs and Slaughterhouses Using SE-AFLP, ERIC-PCR, and PFGE

Made available in DSpace on 2015-09-21T17:25:33Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) christiane_reis_etal_IOC_2013.pdf: 9747161 bytes, checksum: 0b937f29eb74ff98a20ce4ac00bacd5e (MD5) Previous issue date: 2013Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil / Faculdades Metropolitanas Unidas (FMU). Faculdade de Medicina Veterinária. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil / Universidade de São Paulo. Faculdade de Saúde Pública. Laboratório de Saúde Pública. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil / Faculdades Metropolitanas Unidas (FMU). Faculdade de Medicina Veterinária. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Faculdade de Saúde Pública. Laboratório de Saúde Pública. São Paulo, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Yersinia enterocolitica is a foodborne pathogen that causes illness in humans and animals. The biotype 4/O:3 has been commonly associated with yersiniosis and is characterized by the presence of chromosomal and extra-chromosomal virulence genes.Molecular typing methods have been successfully used to characterize Y. enterocolitica genetic heterogeneity and to study the epidemiology of the bacteria from different origins. In this study, 320 Y. enterocolitica biotype 4/O:3 isolates originating in pigs and slaughterhouses were characterized according to the virulence profile, and 61 isolates were typified through SE-AFLP, ERIC-PCR, and PFGE techniques. The majority of the isolates originated from pigs, and the predominant virulence profile was ail+ virF+ rfbC+ ystA+, representing 83.4% of the tested isolates. All of the Y. enterocolitica 4/O:3 isolates were positive for at least ystA gene.The SE-AFLP and ERIC-PCR patterns were highly homogeneous. The SE-AFLP was more discriminative than the ERIC-PCR and tended to cluster isolates according to the slaughterhouse. Despite the limited genetic diversity of Y. enterocolitica 4/O:3, PFGE was shown to be the most discriminative technique considering one band of difference. Fattening pigs proved to be an important reservoir of Y. enterocolitica biotype 4/O:3 carrying virulence genes

Characterization of Yersinia enterocolitica Biotype 1A Strains Isolated from Swine Slaughterhouses and Markets

Made available in DSpace on 2015-09-21T17:25:48Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) ernesto_hoffer2_etal_IOC_2013.pdf: 1270140 bytes, checksum: 0c580a57faf3d58caa921b6084dbb9f0 (MD5) Previous issue date: 2013Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil / Faculdades Metropolitanas Unidas (FMU). Faculdade de Medicina Veterinária. . São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Saúde Pública. Laboratório de Saúde Pública. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Faculdade de Saúde Pública. Laboratório de Saúde Pública. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal. São Paulo, SP, Brasil.Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in22distinct pulsotypes, varying from50%to84%of genetic similarity.Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A